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Divergent Signals and Cytoskeletal Assemblies Regulate Self-Organizing Polarity in Neutrophils
Abstract
Like neutrophilic leukocytes, differentiated HL-60 cells respond to chemoattractant by adopting a polarized morphology, with F-actin in a protruding pseudopod at the leading edge and contractile actin-myosin complexes at the back and sides. Experiments with pharmacological inhibitors, toxins, and mutant proteins show that this polarity depends on divergent, opposing "frontness" and "backness" signals generated by different receptor-activated trimeric G proteins. Frontness depends upon Gi-mediated production of 3'-phosphoinositol lipids (PI3Ps), the activated form of Rac, a small GTPase, and F-actin. G12 and G13 trigger backness signals, including activation of a second GTPase (Rho), a Rho-dependent kinase, and myosin II. Functional incompatibility causes the two resulting actin assemblies to aggregate into separate domains, making the leading edge more sensitive to attractant than the back. The latter effect explains both the neutrophil's ability to polarize in uniform concentrations of chemoattractant and its response to reversal of an attractant gradient by performing a U-turn.
... The polarization of an initially polymerization. Another clue into the intercellular coordination of the polarity pathway for 48 collective migration comes from chemotaxing neural crest cells [31]. As neural crest cells "chase" 49 placodal cells, before cell-cell contact, neural crest cells have high, localized Rac activity at the cell 50 front but after contact, junction proteins (N-cadherins) inhibit Rac localization. ...
... A steric repulsion is enforced between Rac and Rho 103 polarity molecules so that the two chemicals cannot cross paths at any moment in time. This 104 assumption is also well justified by biological data [47][48][49][50]. To minimize assumptions, the 105 reaction rates for both GTPases are identical. ...
... Notably, one limitation of our model comes back to the underlying single cell 589 polarization model: It is possible for our model to rely on other forms of feedback between the 590 biochemical and mechanical circuits or even solely one of the two circuits. For example, negative, 591 instead of positive, feedback between Rac and branched actin and Rho and actomyosin, respectively, 592 could do the job [18,48]. We also limited the dynamics of the model to the local chemical and 593 mechanical processes, but global mechanical effects, for example, membrane tension, could play an 594 important role in polarization of some cell types [60]. ...
Symmetry breaking, which is ubiquitous in biological cells, functionally enables directed cell movement and organized embryogenesis. Prior to movement, cells break symmetry to form a well-defined cell front and rear in a process called polarization. In developing and regenerating tissues, collective cell movement requires the coordination of the polarity of the migration machineries of neighboring cells. Though several works shed light on the molecular basis of polarity, fewer studies have focused on the regulation across the cell-cell junction required for collective polarization, thus limiting our ability to connect subcellular interactions to tissue-level dynamics. Here, we investigate how polarity signals are communicated from one cell to its neighbor to ensure coordinated front-to-rear symmetry breaking with the same orientation across the group. In a theoretical setting, we exhaustively search a variety of intercellular interactions and identify the conditions required for the Rho GTPase signaling module and/or cytoskeletal dynamics to achieve either co-alignment arrangement or supracellular arrangement of the polarity axes in a group of 2 and 4 cells. Our work shows that only asymmetric regulations are favorable – such interactions involve either up-regulation of the kinetic rate of complementary polarity components or opposite regulation of the kinetic rates of the same polarity components across the cell-cell junction. Surprisingly, our results hold if we further assume the presence of an external stimulus, intrinsic cellular variability, or larger group size. The results underline the potential of using quantitative models to probe the molecular interactions required for macroscopic biological phenomena. Lastly, we posit that asymmetric regulation is achieved through junction proteins and predict that in the absence of cytoplasmic tails of such linker proteins, the likeliness of doublet co-polarity is greatly diminished.
Author summary
Cells of the developing embryo undergo a highly complex chain of events that define their correct shape and positioning. Among these events, a crucial role belongs to coordinated cell movement of cells of different lineages over short and long distances to give rise to mature organs and organ systems. During collective movement, individual cells typically engage their autonomous polarity machinery, while being connected to their neighbors through adhesive cell-cell interactions. Despite advances in revealing the cell-cell interactions required for collective cell migration, a comprehensive picture of the molecular basis of intercellular communication for collective guidance is missing. To address this question, we devise a generalized mechanochemical model for cell polarity in a doublet and investigate how polarity signals are transmitted from one cell to another across seemingly symmetrical junctions. We have chosen to screen through all possible intercellular conditions of the Rho GTPase signaling circuit and/or cytoskeletal dynamics. Our systematic approach provides information on over 300 distinct conditions and reveals the intercellular regulation provided by junction proteins. In addition to predicting that only asymmetric interactions favor co-polarization, ensuring movement of the group in the same direction, our analysis also highlights the need for additional regulatory mechanisms for larger cell groups.
... Single cells move by extending a leading front that protrudes and a trailing rear that contracts and follows the front. These programs exhibit not only spatial compartmentalization of distinct intracellular signals to either the front or back of the cell (polarization) but also temporal coordination between these domains (Xu et al., 2003). Neutrophils are a type of innate immune cell that rely on properly oriented cell polarity to migrate to sites of injury where they hunt and kill invading pathogens (Lämmermann et al., 2013). ...
... At the back, the GTPase RhoA stimulates myosin-based contractility Hind et al., 2016;Tsai et al., 2019). These signaling domains are sustained by short-range positive feedback loops within the modules and are spatially separated by mutual antagonism between them (Xu et al., 2003;Ku et al., 2012). Coordination within and between the modules is critical for polarity maintenance during persistent migration (Tsai et al., 2019), but how this coordination is achieved is not fully understood. ...
... The migration phenotypes we observe for both kinase-dependent and kinase-independent arms of Rictor/mTORC2 could arise from its engagement with different portions of the migration cascade ( Figure 3A). A wide range of motile cells including neutrophils show a distinct front-back polarity and organize their protrusive fronts and contractile backs using Rac and RhoA/myosin signaling, respectively (Xu et al., 2003). We were interested in how mTORC2 regulates these polarity and cytoskeletal programs. ...
By acting both upstream and downstream of biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanosensitive mTORC2 programs in neutrophil polarity and motility. We find that the tension-based inhibition of leading edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the regulation of RhoA and Myosin II-based contractility at the trailing edge depend on mTORC2 kinase activity. mTORC2 is essential for spatial and temporal coordination of the front and back polarity programs for persistent migration under confinement. This mechanosensory pathway integrates multiple upstream signals, and we find that membrane stretch synergizes with biochemical co-input PIP 3 to robustly amplify mTORC2 activation. Our results suggest that different signalling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs for efficient coordination of neutrophil shape and movement.
[Media: see text] [Media: see text]
... Major targets include the small GTPases Cdc42 and Rac that act as master regulators at the cell front to organize polarity and promote protrusive behavior by mediating branched actin assembly 10 . At the cell rear, the small GTPase RhoA acts through the kinase ROCK and phosphorylation of myosin regulatory light chain to facilitate assembly of myosin II filaments and actomyosin contraction, which is responsible for the retraction of the cell's trailing edge 11 . Locally, the cell front (protruding) and rear (retracting) domains are self-reinforcing via positive feedback and are mutually antagonistic 6,[11][12][13] . ...
... At the cell rear, the small GTPase RhoA acts through the kinase ROCK and phosphorylation of myosin regulatory light chain to facilitate assembly of myosin II filaments and actomyosin contraction, which is responsible for the retraction of the cell's trailing edge 11 . Locally, the cell front (protruding) and rear (retracting) domains are self-reinforcing via positive feedback and are mutually antagonistic 6,[11][12][13] . Indeed, modeling has demonstrated that this signaling architecture is sufficient to generate stable cell polarization in response to external cues or even spontaneously by amplifying molecular noise [14][15][16][17] . ...
... Prior work has shown that myosin II contractile activity at the cell's rear contributes to the maintenance of polarity, while also inhibiting cell front polarity programs 11,[38][39][40] . We therefore sought to test the effects of altering myosin contractility in our optogenetic reversal assay. ...
Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback.
... (C) Interaction details of S1P with residues in the binding site indicated. (D and E) Comparison of pEC 50 values and E max of each variant. The representative dose-response curves from same-day experiment and membrane expression of S1PRs are shown in fig. ...
... S6D). Dual signaling via G i and G 12/13 has been linked to enhancements in directed cell migration (50) and might contribute to the efficacy of S1PR3 as a promigratory receptor (51,52). By contrast, the near selective G 13 coupling of S1PR2 may be critical for its ability to antagonize S1PR1-mediated G i responses and thereby promote lymphocyte retention in tissues (53). ...
... S1P-or FTY720-P-induced AP-TGF- release was calculated as described previously (30). AP-TGF- release signal over ligand concentration was fitted to a four-parameter logistic sigmoidal curve using Prism 9 software (GraphPad), from which EC 50 and E max values were obtained. For JTE-013 inhibition experiment, JTE-013 was serially diluted in HBSS with 5 mM Hepes (pH 7.4) and 0.01% fatty acid-free BSA in the presence or absence of 1 M FTY720-P. ...
Sphingosine-1-phosphate (S1P) regulates immune cell trafficking, angiogenesis, and vascular function via its five receptors. Inherited mutations in S1P receptor 2 (S1PR2) occur in individuals with hearing loss, and acquired mutations in S1PR2 and Gα13 occur in a malignant lymphoma. Here, we present the cryo-electron microscopy structure of S1P-bound S1PR2 coupled to the heterotrimeric G13. Interaction between S1PR2 intracellular loop 2 (ICL2) and transmembrane helix 4 confines ICL2 to engage the α5 helix of Gα13. Transforming growth factor-α shedding assays and cell migration assays support the key roles of the residues in S1PR2-Gα13 complex assembly. The structure illuminates the mechanism of receptor disruption by disease-associated mutations. Unexpectedly, we showed that FTY720-P, an agonist of the other four S1PRs, can trigger G13 activation via S1PR2. S1PR2F274I variant can increase the activity of G13 considerably with FTY720-P and S1P, thus revealing a basis for S1PR drug selectivity.
... During immune surveillance, neutrophils use chemotaxis to navigate through highly complex tissue environments to reach sites of infection and inflammation. This process involves integrating directional cues detected by G-protein coupled receptors (GPCRs), as well as physical constraints of the environment, into an underlying autonomous polarity circuitry (Prentice-Mott et al., 2013;Weiner et al., 2007;Xu et al., 2003). Upon encountering obstacles, neutrophils may be forced to split their single leading edge into multiple competing fronts. ...
... Chemoattractants, such as N-formyl-methionyl-leucyl-phenylalanine (fMLF), activate signaling via the heterotrimeric GTPase Giα, which orients and reinforces morphologically and chemically distinct cell front and rear domains, leading to directed migration (Xu et al., 2003). Rac and Cdc42 are well-established cell front polarity regulators, inducing branched actin polymerization at the leading edge via activation of the Arp2/3 complex (Etienne-Manneville and Hall, 2002). ...
... Rac and Cdc42 are well-established cell front polarity regulators, inducing branched actin polymerization at the leading edge via activation of the Arp2/3 complex (Etienne-Manneville and Hall, 2002). At the cell rear, RhoA activity activates the cascade of ROCK and phosphorylation of myosin regulatory light chain to facilitate assembly of active myosin II bipolar filaments and actomyosin contraction (Xu et al., 2003). Locally, the cell front and rear domains are selfreinforcing via positive feedback, and at the same time are mutually antagonistic (Hind et al., 2016;Wang et al., 2002Wang et al., , 2013Weiner et al., 2002;Xu et al., 2003). ...
As neutrophils navigate complex environments to reach sites of infection, they may encounter obstacles that force them to split their front into multiple leading edges, raising the question of how the cell selects which front to maintain and which front(s) to abandon. Here we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels, enabling us to probe the cell-intrinsic properties of their decision-making process. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the decision-making process, rather than amplifying early pre-existing asymmetries. Furthermore, we use optogenetic tools to show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, optogenetic attempts to reverse cell decisions reveal that, once an edge begins retracting, it commits to this fate, with the kinase ROCK limiting its sensitivity to inputs until the retraction is complete. Collectively our results suggest a "selective listening" model in which both actively protruding cell fronts and actively retracting cell rears have strong commitment to their current migratory program.
... One such protein was the RH family Rho-specific guanine nucleotide exchange factor (RhoGEF), PDZ-RhoGEF (PRG; also known as ARHGEF11). The role of PRG in the regulation of cell migration downstream of G i -coupled chemoattractant receptors is thought to be mediated primarily by their coupling to G 12/13 subunits (20,21). We demonstrated that PRG was an effector of active G i1 and G i3 but was poorly activated by G i2 , a highly homologous (~85% identical) G i family member. ...
... We decided to pursue PRG for several reasons. PRG has a role in regulation of neutrophil migration downstream of G i -coupled chemoattractant receptors (20,21). PRG localizes to the rear of migrating neutrophils and activates Rho and myosin-dependent tail retraction during migration in response to chemoattractants (21). ...
... To understand the physiological relevance of the G i -dependent mechanism for Rho regulation, we examined the role of G i signaling in human neutrophils. Of the multiple G-protein-activated RhoGEFs expressed in neutrophils, PRG mediates Rho-dependent polarized accumulation of phosphorylated (at Ser 19 )-myosin light chain (MLC) at the trailing edge of migrating neutrophils (20,21). BioID2-G i1 -QL, or BioID2-CaaX. ...
G protein–coupled receptors (GPCRs) that couple to the Gα i family of G proteins are key regulators of cell and tissue physiology. Our previous work has revealed new roles for Gα i in regulating the migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors. Here, we used an intact cell proximity–based labeling coupled to tandem mass tag (TMT)–based quantitative proteomics analysis to identify proteins that selectively interacted with the GTP-bound form of Gα i1 . Multiple targets were identified and validated with a BioID2-tagged, constitutively active Gα i1 mutant, suggesting a network of interactions for activated Gα I proteins in intact cells. We showed that active Gα i1 , but not Gα i2 , stimulated one candidate protein, PDZ-RhoGEF (PRG), despite more than 85% sequence identity between the G proteins. We also demonstrated in primary human neutrophils that active Gα i likely regulated the polarization of phosphorylated myosin light chain, a process critical for migration, through the activation of PRG. The identification and characterization of new targets directly or indirectly regulated by Gα i will aid in the investigation of the functional roles of Gα i -coupled GPCRs in multiple biological processes.
... I n leukocytes, chemotaxis is driven almost exclusively by G protein-coupled receptors (GPCRs) coupling to the G i α family of G proteins [1][2][3][4] . Through partially understood pathways, these receptors trigger activation of polarity and motility signaling driven by Rho-family GTPases, phospholipid signaling, and different actin assemblies. ...
... Through partially understood pathways, these receptors trigger activation of polarity and motility signaling driven by Rho-family GTPases, phospholipid signaling, and different actin assemblies. Rac, Cdc42, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), and branched actin coordinate the cell front, while RhoA and contractile actomyosin complexes define the cell rear 1,[5][6][7][8] . Accurate cell steering requires that the polarity programs receive and rapidly incorporate directional cues from receptors, enabling responses to differences in input strength across the cell. ...
... Indeed, Cdc42 activity can form traveling waves in neutrophil-like cells when actin is depolymerized 11 . Thus, many models for directional sensing in chemotaxis involve balancing of positive and negative feedback or feedforward loops that collaborate to restrict, but also amplify receptor-derived signals 2,3,5 . Nevertheless, the negative signaling mechanisms helping to maintain spatial information remain unclear. ...
During chemotaxis, neutrophils use cell surface G Protein Coupled Receptors to detect chemoattractant gradients. The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities. Positive feedback could promote rapid signal spreading, yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell. How the signal transduction network achieves signal amplification while preserving spatial information remains unclear. The GTPase Cdc42 is a cell-front polarity coordinator that is predictive of cell turning, suggesting an important role in spatial processing. Here we directly measure information flow from receptors to Cdc42 by pairing zebrafish parapinopsina, an optogenetic G Protein Coupled Receptor with reversible ON/OFF control, with a spectrally compatible red/far red Cdc42 Fluorescence Resonance Energy Transfer biosensor. Using this toolkit, we show that positive and negative signals downstream of G proteins shape a rapid, dose-dependent Cdc42 response. Furthermore, F-actin and Cdc42 itself provide two distinct negative signals that limit the duration and spatial spread of Cdc42 activation, maintaining output signals local to the originating receptors.
... Their directed migration is mediated by detection of chemoattractants such as N-formyl-methionyl-leucyl-phenylalanine (fMLF) via G protein-coupled receptors (GPCRs), fairly evenly distributed on the cell surface 1,2 . Ligand binding to the receptor activates signaling via G αi , leading to cell polarization and directional motility 3 . ...
... At the front, Cdc42 and Rac induce actin polymerization, whereas RhoA regulates myosin II contractility at the rear. The front and rear signaling modules are mutually exclusive 3 and are governed by positive feedback loops for selfamplification and polarity maintenance [5][6][7] . In addition, tension by the plasma membrane has been demonstrated to act as a longrange inhibitor, mechanically preventing the generation of multiple cell fronts 8 . ...
... Once established, front-rear polarity in migrating neutrophils is thought to be relatively stable, as evidenced by the observation that polarized neutrophils often steer their original front to make a U-turn instead of repolarizing, suggesting that neutrophils are more sensitive to chemoattractants towards their front as compared to their rear 1 . To examine the role of polarity in chemoattractant sensing, researchers have historically challenged neutrophils migrating on two-dimensional (2D) planar substrates using point sources of chemoattractant at different angles with respect to the original direction of migration 3,[9][10][11] . In these experiments, chemoattractant is typically delivered using a micropipette positioned near the cell, resulting in diffusion of the attractant over the entire cellular surface. ...
To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows. Here we investigate what is necessary to overwrite pre-existing front-rear polarity in neutrophil-like HL60 cells migrating inside straight microfluidic channels. Using subcellular optogenetic receptor activation, we show that receptor inputs can reorient weakly polarized cells, but the rear of strongly polarized cells is refractory to new inputs. Transient stimulation reveals a multi-step repolarization process, confirming that cell rear sensitivity to receptor input is the primary determinant of large-scale directional reversal. We demonstrate that the RhoA/ROCK/myosin II pathway limits the ability of receptor inputs to signal to Cdc42 and reorient migrating neutrophils. We discover that by tuning the phosphorylation of myosin regulatory light chain we can modulate the activity and localization of myosin II and thus the amenability of the cell rear to ‘listen’ to receptor inputs and respond to directional reprogramming.
... One of the proteins identified was the RH family RhoGEF, PDZ-RhoGEF (PRG), also known as ARHGEF11. The role of PRG in regulation of cell migration downstream of Gi-coupled chemoattractant receptors is well characterized, but it is thought to be mediated primarily by additional coupling of these receptors to G12/13 subunits [20,21]. We also demonstrate that PRG is an effector of active Gαi1 and Gi3 but is poorly activated by Gi2, a highly homologous (~85% identical) Gi family member. ...
... We decided to pursue PRG for several reasons; PRG was strongly enriched in the BioID2-Gi1-QL samples with a P-value <0.001, and PRG has an established role in regulation of neutrophil migration downstream of Gi-coupled chemoattractant receptors [20,21]. PRG localizes to the back of migrating neutrophils and activates Rho and myosin-dependent tail retraction during migration in response to chemoattractants [21]. ...
... Previous studies have shown that of the multiple G protein-activated RhoGEFs expressed in neutrophils, PRG mediates Rho-dependent polarized accumulation of phosphorylated (at serine 19) -myosin light chain (P-MLC) at the trailing edge of migrating neutrophils [20,21]. This has been proposed to result from FPR1-dependent Gα13 activation [20] in part because PTX treatment of HL60 cells, a neutrophil-like cell line, only partially inhibited fMLF-dependent Rho activation and asymmetric localization of P-MLC [20]. ...
G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our recent work has discovered novel roles for Gα i in migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors, but the molecular target(s) of Gα i in these processes remain to be identified. We adopted an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interact with the GTP-bound form of Gα i1 . Multiple targets were identified and validated for selective biotinylation by active BioID2-Gα i1 (Q204L), suggesting a previously unappreciated network of interactions for activated Gα i proteins in intact cells. Extensive characterization of one candidate protein, PDZ-RhoGEF (PRG), revealed that active-Gα i1 strongly activates PRG. Strikingly, large differences in the ability of Gα i1 , Gα i2 , and Gα i3 isoforms to activate PRG were observed despite over 85% sequence identity. We also demonstrate the functional relevance of the interaction between active Gα i and PRG ex vivo in primary human neutrophils. Identification and characterization of new targets regulated by Gα i both individually and in networks provide insights that will aid not only in investigation of diverse functional roles of Gi-coupled GPCRs in biology but also in the development of novel therapeutic approaches.
Summary
Proximity-based labeling approach was used to identify signaling networks and signaling mechanisms downstream of Gi-coupled receptors.
... Their directed migration is mediated by detection of chemoattractant such as fMLF via G protein-coupled receptors (GPCRs), fairly evenly distributed on the cell surface 1,2 . Ligand binding to the receptor activates signaling via G i , leading to cell polarization and directional motility 3 . ...
... At the front, Cdc42 and Rac1 induce actin polymerization, while RhoA regulates myosin II contractility at the rear. The front and rear signaling modules are mutually exclusive 3 and are governed by positive feedback loops for self-amplification and polarity maintenance [5][6][7] . In addition, tension by the plasma membrane has been demonstrated to act as a long-range inhibitor, mechanically preventing the generation of multiple cell fronts 8 . ...
... Once established, front-rear polarity in migrating neutrophils is thought to be relatively stable, as evidenced by the observation that polarized neutrophils often steer their original front to make a U-turn instead of repolarizing, suggesting that neutrophils are more sensitive to chemoattractants towards their front as compared to their rear 1 . To examine the role of polarity in chemoattractant sensing, researchers have historically challenged neutrophils migrating on 2-D planar substrates using point sources of chemoattractant at different angles with respect to the original direction of migration 3,[9][10][11] . In these experiments, chemoattractant is typically delivered using a micropipette positioned near the cell, resulting in diffusion of the attractant over the entire cellular surface. ...
To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows. Here we investigate what is necessary to overwrite pre-existing front/rear polarity in neutrophil-like HL60 cells migrating inside straight microfluidic channels. Using subcellular optogenetic receptor activation, we show that receptor inputs can reorient weakly polarized cells, but the rear of strongly polarized cells is refractory to new inputs. Transient stimulation reveals a multi-step repolarization process, confirming that cell rear sensitivity to receptor input is the primary determinant of large-scale directional reversal. We demonstrate that the RhoA/ROCK/myosin II pathway limits the ability of receptor inputs to signal to Cdc42 and reorient migrating neutrophils. We discover that by tuning the phosphorylation of myosin regulatory light chain we can modulate the activity and localization of myosin II and thus the amenability of the cell rear to "listen" to receptor inputs and respond to directional reprogramming.
... 10 The distinct actin structures define cell polarity, orienting cells with pseudopods at the leading edge and uropods at the rear for migration toward higher concentrations of stimuli. On the other hand, it is well known that uniformly applied stimuli also induce symmetry breaking (i.e., self-organizing polarity), 11 allowing polarized cells to move randomly, which is reminiscent of the random motility of T cells on immobilized chemokines. ...
... In this case, G12/G13 initiated the ''backness'' signal through RhoA, whereas Gi signaling transmits the ''frontness'' signal through the Rac-F-actin pathway. 11 Since different heterotrimeric G proteins transmit a chemoattractant receptor signaling to distinct Rho family members, inhibition of either of the two pathways results in the domination of the other. However, this is not the case with T cells, in which both ''frontness'' of the Rac signal and ''backness'' of the RhoA signal are initiated by the same Gi signaling and generation of cell polarity. ...
Lymphocyte trafficking requires fine-tuning of chemokine-mediated cell migration. This process depends on cytoskeletal dynamics and polarity, but its regulation remains elusive. We quantitatively measured cell polarity and revealed critical roles performed by integrin activator Rap1 in this process, independent of substrate adhesion. Rap1-deficient naive T cells exhibited impaired abilities to reorganize the actin cytoskeleton into pseudopods and actomyosin-rich uropods. Rap1-GTPase activating proteins (GAPs), Rasa3 and Sipa1, maintained an unpolarized shape; deletion of these GAPs spontaneously induced cell polarization, indicative of the polarizing effect of Rap1. Rap1 activation required F-actin scaffolds, and stimulated RhoA activation and actomyosin contractility at the rear. Furthermore, talin1 acted on Rap1 downstream effectors to promote actomyosin contractility in the uropod, which occurred independently of substrate adhesion and talin1 binding to integrins. These findings indicate that Rap1 signaling to RhoA and talin1 regulates chemokine-stimulated lymphocyte polarization and chemotaxis in a manner independent of adhesion.
... One challenge in investigating whether cortical waves can act as the mediators of EFs is that in many of the cell types that show a strong response to EFs, the wave area is comparable to the cell area. Furthermore, waves are generated at the leading edge of the cell during directed migration (Xu et al., 2003), so that wave dynamics are tightly coupled with cell dynamics. For instance, when a cell responds to an EF reversal, waves typically remain at the cell front as the cell turns. ...
... Thus, the response of migrating cells to a changing guidance cue can be predicted from the characteristics of the waves driving the migration process. Indeed, the U-turn behaviors of neutrophils and differentiated, single D. discoideum cells in response to EF reversal (Hind et al., 2016;Sato et al., 2007;Srinivasan et al., 2003;Xu et al., 2003), which are usually ascribed to stable cell polarity, may instead reflect the persistence and 2D turning behavior of cortical waves in these environments (Figure 3). ...
Electrotaxis, the directional migration of cells in a constant electric field, is important in regeneration, development, and wound healing. Electrotaxis has a slower response and a smaller dynamic range than guidance by other cues, suggesting that the mechanism of electrotaxis shares both similarities and differences with chemical-gradient-sensing pathways. We examine a mechanism centered on the excitable system consisting of cortical waves of biochemical signals coupled to cytoskeletal reorganization, which has been implicated in random cell motility. We use electro-fused giant Dictyostelium discoideum cells to decouple waves from cell motion and employ nanotopographic surfaces to limit wave dimensions and lifetimes. We demonstrate that wave propagation in these cells is guided by electric fields. The wave area and lifetime gradually increase in the first 10 min after an electric field is turned on, leading to more abundant and wider protrusions in the cell region nearest the cathode. The wave directions display ‘U-turn’ behavior upon field reversal, and this switch occurs more quickly on nanotopography. Our results suggest that electric fields guide cells by controlling waves of signal transduction and cytoskeletal activity, which underlie cellular protrusions. Whereas surface receptor occupancy triggers both rapid activation and slower polarization of signaling pathways, electric fields appear to act primarily on polarization, explaining why cells respond to electric fields more slowly than to other guidance cues.
... A large body of evidence suggests that the Gα12/13 family regulate motility in a wide variety of cells [21,35,[72][73][74][75][76][77]. ...
... Defects in cell migration during development were described in animal models bearing a genetic ablation of the Gα13-coding gene (also accompanied by ablation of the Gα12-coding gene), such as Drosophila [73], zebrafish [72], and Xenopus [55], as well as in a mouse model displaying a conditional knockdown of Gα12-/13coding genes in the nervous system [86]. Furthermore, the absence of Gα13 was also associated with an impairment of either GPCR-driven chemokinesis, as shown by embryonic fibroblasts derived from Gα13-knockout mice [21], or cell polarity and chemotaxis, as exhibited by Gα13-defective immune cells [74,87]. On the other hand, upregulation of Gα13-related pathways was also correlated with tumor invasion in different types of cancer [15,76,77,88]. ...
Microglia are the resident immune cells of the CNS that are activated in response to a variety of stimuli. This phenotypical change is aimed to maintain the local homeostasis, also by containing the insults and repair the damages. All these processes are tightly regulated and coordinated and a failure in restoring homeostasis by microglia can result in the development of neuroinflammation that can facilitate the progression of pathological conditions. Indeed, chronic microglia activation is commonly recognized as a hallmark of many neurological disorders, especially at an early stage. Many complex pathways, including cytoskeletal remodeling, are involved in the control of the microglial phenotypical and morphological changes that occur during activation. In this work, we focused on the small GTPase Gα13 and its role at the crossroad between RhoA and Rac1 signaling when microglia is exposed to pro-inflammatory stimulation. We propose the direct involvement of Gα13 in the cytoskeletal rearrangements mediated by FAK, LIMK/cofilin, and Rac1 during microglia activation. In fact, we show that Gα13 knockdown significantly inhibited LPS-induced microglial cell activation, in terms of both changes in morphology and migration, through the modulation of FAK and one of its downstream effectors, Rac1. In conclusion, we propose Gα13 as a critical factor in the regulation of morphological and functional properties of microglia during activation, which might become a target of intervention for the control of microglia inflammation.
... Neutrophil recruitment to the damage site results from the coordination of gradient sensing, protrusive motility, and cell polarity involving cytoskeletal reorganization. Members of the Rho GTPase family, including Rho, Rac, and Cdc42, are key regulators of chemotaxis in diverse systems [16,17]. We have previously reported that Cdc42 regulates the long axis polarization state of neutrophils through its effector WASp protein [6]. ...
... While Rac1 functions as a regulator of lamellipodium and gradient sensing at the leading front of migrating neutrophils and was found important for migration of neutrophils into the lungs [18]. Rho A localizes towards uropod /back in migrating neutrophils and regulates myosin II regulatory light chain (p-MLC) mediated contraction at back [16]. Deficiency of myosin light-chain kinase (MYLK) leads to defective neutrophil adhesion and recruitment to inflamed tissue [19]. ...
Neutrophils, the early responders of the immune system, eliminate intruders, but their over-activation can also instigate tissue damage leading to various autoimmune and inflammatory disease conditions. As approaches causing neutropenia are associated with immunodeficiency, targeting aberrant neutrophil infiltration offers an attractive strategy in neutrophil-centered diseases including acute lung injury. Rho GTPase family proteins Rho, Rac and Cdc42 play important role as regulators of chemotaxis in diverse systems. Rho inhibitors protected against lung injuries, while genetic Rho-deficiency exhibited neutrophil hyperactivity and exacerbated lung injury. These differential outcomes might be due to distinct effects on different cell types or activation/ inhibition of specific signaling pathways responsible for neutrophil polarity, migration and functions. In this study, we explored neutrophil centric effects of Rho signaling mitigation. Consistent with previous reports, Rho signaling inhibitor Y-27632 provided protection against acute lung injury, but without regulating LPS mediated systemic increase of neutrophils in the circulation. Interestingly, the adoptive transfer approach identified a specific defect in neutrophil migration capacity after Rho signaling mitigation. These defects were associated with loss of polarity and altered actin dynamics identified using time-lapse in vitro studies. Further analysis revealed a rescue of stimulation-dependent L-selectin shedding on neutrophils with Rho signaling inhibitor. Surprisingly, functional blocking of L-selectin (CD62L) led to defective recruitment of neutrophils into inflamed lungs. Further, single-cell level analyses identified MAPK signaling as downstream mechanism of Rho signaling and L-selectin mediated effects. p-AKT levels were diminished in detergent resistance membrane-associated signalosome upon Rho signaling inhibition and blockade of selectin. Moreover, inhibition of AKT signaling as well as selectin blocking led to defects in neutrophil polarity. Together, this study identified Rho-dependent distinct L-selectin and AKT signaling mediated regulation of neutrophil recruitment to inflamed lung tissue.
... Junctions become leaky to facilitate diapedesis, and specific endothelial cell-neutrophil surface interactions allow the cell to pass between junctions. On the opposite side of the neutrophil, actin-myosin contractile interactions propel the neutrophil towards the gradient [32,33]. Neutrophils maintain polarity (i.e., a side facing the chemotactic gradient and the opposite side propelling the cell along the vessel wall) through myosin-actin interactions. ...
... Rac is prominent in lamellipodia to promote F-actin polymerization by activating the Wiskott-Aldrich Syndrome protein (WASP) family [36]. RhoA is the key factor stimulating phosphorylation of myosin light chain (MLC) kinase through Rho associated protein kinase to promote contraction [32]. Activation of Rac is primarily dependent on PIP3, whereas RhoA is activated by PIP2, the downstream regulators of PI3K [37]. ...
Neutrophils are key effector cells of the innate immune system, serving as a first line of defense in the response to injury and playing essential roles in the wound healing process. Following myocardial infarction (MI), neutrophils infiltrate into the infarct region to propagate inflammation and begin the initial phase of cardiac wound repair. Pro-inflammatory neutrophils release proteases to degrade extracellular matrix (ECM), a necessary step for the removal of necrotic myocytes as a prelude for scar formation. Neutrophils transition their phenotype over time to regulate MI inflammation resolution and stabilize scar formation. Neutrophils contribute to the evolution from inflammation to resolution and scar formation by serving anti-inflammatory and repair functions. As anti-inflammatory cells, neutrophils contribute ECM proteins during scar formation, in particular fibronectin, galectin-3, and vimentin. The diverse and polarizing functions that contribute to MI wound repair make this innate immune cell a viable target to improve MI outcomes. Thus, understanding the signaling involved in neutrophil physiology in the context of MI may help to identify novel therapeutic targets.
... k 0 is the dissociation rate at ∂ x v = 0, and k 1 tells us how the dissociation rate is affected by the stretching effect of the cytoplasmic flow. Besides being regulated by the force on the cell ends against actin polymerizations, actin polymerization at the cell ends also depends on the distribution of actin activators [19][20][21][22]. In the presence of environmental cues, a gradient of actin activator concentration within the cell is established, and actin polymerization is polarized due to this concentration gradient. ...
The one-dimensional crawling movement of a cell is considered in this theoretical study. Our active gel model shows that a moving cell with weakly mechanosensitive adhesion complexes tends to move at constant velocity. As the mechanosensitivity of the adhesion complexes increases, a cell with sufficiently strong myosin contractile or high actin polymerization rate can exhibit stick-slip motion. Finally, a cell with highly mechanosensitive adhesion complexes exhibits periodic back-and-forth migration. A simplified model that assumes that the cell crawling dynamics are controlled by the evolution of the myosin density dipole and the asymmetry of adhesion complex distribution captures the motility behaviors of crawling cells qualitatively. It suggests that complex cell crawling behaviors could result from the interplay between the distribution of contractile force and mechanosensitive bonds.
... The copolarization between ERM proteins and MCAM in our system resembles other cellular features involved in migration. Uropods are knob-like structures described in Dictyostelium discoideum and normal and malignant leukocytes (Sánchez-Madrid and Del Pozo, 1999;Borset et al., 2000;Xu et al., 2003;Van Haastert and Devreotes, 2004;Dampmann et al., 2020;Bein et al., 2022), as well as in the Walker 256 carcinosarcoma (Rossy et al., 2007), 11A squamous cell carcinoma (Faber et al., 2013), and MDA-MB-231 breast cancer cell lines (Poincloux et al., 2011). Uropods are stably polarized and include adhesion proteins, ERM proteins, myosin-II, pMLC, and PI(4,5)P 2 (Rosenman et al., 1993;Del Pozo et al., 1995;Serrador et al., 1997;Sánchez-Madrid and Del Pozo, 1999;Eddy et al., 2000;Fais and Malorni, 2003;Lee et al., 2004;Lacalle et al., 2007;Lokuta et al., 2007;Sánchez-Madrid and Serrador, 2009;Martinelli et al., 2013;Liu et al., 2015;Hind et al., 2016;García-Ortiz and Serrador, 2020). ...
The WRAMP structure is a protein network associated with tail-end actomyosin contractility, membrane retraction, and directional persistence during cell migration. A marker of WRAMP structures is melanoma cell adhesion molecule (MCAM) which dynamically polarizes to the cell rear. However, factors that mediate MCAM polarization are still unknown. In this study, BioID using MCAM as bait identifies the ERM family proteins, moesin, ezrin, and radixin, as WRAMP structure components. We also present a novel image analysis pipeline, Protein Polarity by Percentile (“3P”), which classifies protein polarization using machine learning and facilitates quantitative analysis. Using 3P, we find that depletion of moesin, and to a lesser extent ezrin, decreases the proportion of cells with polarized MCAM. Furthermore, although co-polarized MCAM and ERM proteins show high spatial overlap, 3P identifies subpopulations with ERM proteins closer to the cell periphery. Live-cell imaging confirms that MCAM and ERM protein polarization is tightly coordinated, but ERM proteins enrich at the cell edge first. Finally, deletion of a juxtamembrane segment in MCAM previously shown to promote ERM protein interactions impedes MCAM polarization. Our findings highlight the requirement for ERM proteins in recruitment of MCAM to WRAMP structures and an advanced computational tool to characterize protein polarization.
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... HL-60 cells are differentiated into a granulocytic lineage by DMSO and are used as a model for neutrophils [19,27,28]. To thoroughly investigate the role of LRRK2 in chemotaxis, we analyzed dHL-60 cells. ...
Background
Neutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2 -deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N -formyl-Met-Leu-Phe ( f MLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells.
Methods
Lrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2.
Results
f MLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo . Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced f MLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon f MLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon f MLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to f MLP.
Conclusions
LRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon f MLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on f MLP-induced chemotactic activity in dHL-60 cells.
... All cell lines have been tested with negative Mycoplasma. For experiments, HL60 cells were differentiated by adding 1.3% DMSO into the medium for 7 days (Xu et al., 2003). To establish the stable RNAi cell lines, we transfected shRNA into HEK293T cells. ...
Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis , and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.
... In the further course of the signaling cascade, PIK3 converts PIP 2 to PIP 3 , which accumulates in specific areas of the cell on the side where the chemoattractant has bound to the receptors. This restricted localization is critical for directional migration along the chemotactic gradient [42,43]. This restricted localization is maintained by PTEN in Dictyostelium and by SHIP1 in mice [44,45], which may be of interest for further studies to better understand the molecular action of LAs upon PMNs. ...
Various functions of polymorphonuclear neutrophils (PMNs) are related to diseases and postoperative plasma changes. The influence of some local anesthetics (LAs) on PMNs obtained by conventional isolation methods and their functions has already been demonstrated. This study investigates the effect of selected LAs on PMNs, comparing a new isolation method with conventional ones. To obtain the PMNs, we performed either gelafundin sedimentation, hypotonic lysis or density gradient centrifugation. Subsequently, PMNs were mixed with different concentrations of bupivacaine, levobupivacaine, lidocaine or ropivacaine. Live cell imaging and flow cytometry were performed to quantify the migration, ROS production, NETosis and antigen expression of PMNs. We found the inhibition of chemotaxis and ROS production by LAs. PMNs showed a strong reduction in time to half maximal NETosis in response to bupivacaine and lidocaine, but not to levobupivacaine and ropivacaine. We also found distinct differences in survival time and migration duration between the isolation methods. This suggests that the careful selection of LAs has a short-term impact on in vitro PMNs.
... Cell culture, transfection, and isolation of human and mouse neutrophils NCTC, HEK-293, U937 cells and HUVECs were cultured in DMEM supplemented with 10% fetal bovine serum and HL60 cells were cultured in RPMI 1640 with 10% fetal bovine serum. For experiments, HL60 cells were differentiated by adding 1.3% DMSO into the medium for 7 days (Xu et al., 2003). To establish the stable RNAi cell lines, we transfected shRNA into HEK293T cells. ...
Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.
... All cell lines have been tested with negative Mycoplasma. For experiments, HL60 cells were differentiated by adding 1.3% DMSO into the medium for 7 days (Xu et al., 2003). To establish the stable RNAi cell lines, we transfected shRNA into HEK293T cells. ...
Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.
... All cell lines have been tested with negative Mycoplasma. For experiments, HL60 cells were differentiated by adding 1.3% DMSO into the medium for 7 days (Xu et al., 2003). To establish the stable RNAi cell lines, we transfected shRNA into HEK293T cells. ...
ELife digest
Every day, the immune cells clears the remains of billions of old and damaged cells that have undergone a controlled form of death. Removing them quickly helps to prevent inflammation or the development of autoimmune diseases.
While immune cells called neutrophils are generally tasked with removing invading bacteria, macrophages are thought to be responsible for clearing dead cells. However, in healthy tissue, the process occurs so efficiently that it can be difficult to confirm which cells are responsible.
To take a closer look, Cao et al. focused on the liver by staining human samples to identify both immune and dead cells. Unexpectedly, there were large numbers of neutrophils visible inside dead liver cells. Further experiments in mice revealed that after entering the dead cells, neutrophils engulfed the contents and digested the dead cell from the inside out. This was a surprising finding because not only are neutrophils not usually associated with dead cells, but immune cells usually engulf cells and bacteria from the outside rather than burrowing inside them.
The importance of this neutrophil behaviour was shown when Cao et al. studied samples from patients with an autoimmune disease where immune cells attack the liver. In this case, very few dead liver cells contained neutrophils, and the neutrophils themselves did not seem capable of removing the dead cells, leading to inflammation. This suggests that defective neutrophil function could be a key contributor to this autoimmune disease.
The findings identify a new role for neutrophils in maintaining healthy functioning of the liver and reveal a new target in the treatment of autoimmune diseases. In the future, Cao et al. plan to explore whether compounds that enhance clearance of dead cells by neutrophils can be used to treat autoimmune liver disease in mouse models of the disease.
... Directed migration towards a gradient of chemoattractant is crucial for the neutrophils. While RHOA expression is reduced in the leading edge, activated RHOA promotes myosin cytoskeleton reorganization at the rear and regulates actomyosin contraction [43][44][45][46]. Its downstream effector, ROCK, regulates cell spreading and uropod formation [47]. ...
Primarily identified as an important regulator of cytoskeletal dynamics, the small GTPase Ras homolog gene family member A (RHOA) has been implicated in the transduction of signals regulating a broad range of cellular functions such as cell survival, migration, adhesion and proliferation. Deregulated activity of RHOA has been linked to the growth, progression and metastasis of various cancer types. Recent cancer genome-wide sequencing studies have unveiled both RHOA gain and loss-of-function mutations in primary leukemia/lymphoma, suggesting that this GTPase may exert tumor-promoting or tumor-suppressive functions depending on the cellular context. Based on these observations, RHOA signaling represents an attractive therapeutic target for the development of selective anticancer strategies. In this review, we will summarize the molecular mechanisms underlying RHOA GTPase functions in immune regulation and in the development of hematological neoplasms and will discuss the current strategies aimed at modulating RHOA functions in these diseases.
... The idea of using 2-D pattern formation to describe polarization is not new, and much work has been conducted in this area [13,21]. The Turing-type diffusion reaction equations are widely used to form polarization patterns in these works, which consist of both simulations and experiments [22,23]. Specifically, polarization activators are represented by the active form of either cytosolic or cytoskeletal proteins, while polarization inhibitors by the inactive form [24,25]. Thus, it is the interplay between the active and inactive form that shapes the stable regions in the Turing-type diffusion field. ...
The RBL-2H3 mast cell immunological synapse dynamics is often simulated with reaction–diffusion and Fokker–Planck equations. The equations focus on how the cell synapse captures receptors following an immune response, where the receptor capture at the immunological site appears to be a delayed process. This article investigates the physical nature and mathematics behind such time-dependent delays. Using signal processing methods, convolution and cross-correlation-type delay capture simulations give a χ-squared range of 22 to 60, in good agreement with experimental results. The cell polarization event is offered as a possible explanation for these capture delays, where polarizing rates measure how fast the cell polarization event occurs. In the case of RBL-2H3 mast cells, polarization appears to be associated with cytoskeletal rearrangement; thus, both cytoskeletal and diffusional components are considered. From these simulations, a maximum polarizing rate ranging from 0.0057 s−2 to 0.031 s−2 is obtained. These results indicate that RBL-2H3 mast cells possess both temporal and spatial memory, and cell polarization is possibly linked to a Turing-type pattern formation.
... The effects of Blebbistatin on PMNs have been extensively studied and closely resemble the effects of H. pylori infection, suggesting that this pathogen may also inhibit myosin IIA activity to disrupt chemotaxis. Specific shared features include elongated uropods decorated with actin filaments and spikes, trapped nuclear lobes, diminished leading edge size, small actin puncta throughout the cytosol that mediate actin flow, elevated RLC S19 phosphorylation, and diminished migration speed and directionality (9,10,14,15,47,64,65). Of note, maximum cell elongation and near total ablation of uropod retraction was achieved when infection was combined with Blebbistatin (Supplementary Video 10), which many indicate that H. pylori and Blebbistatin inhibit myosin IIA to different extents or at different stages of its activity cycle. ...
Helicobacter pylori is a major human pathogen that colonizes the gastric mucosa and plays a causative role in development of peptic ulcers and gastric cancer. Neutrophils are heavily infected with this organism in vivo and play a prominent role in tissue destruction and disease. Recently, we demonstrated that H. pylori exploits neutrophil plasticity as part of its virulence strategy eliciting N1-like subtype differentiation that is notable for profound nuclear hypersegmentation. We undertook this study to test the hypothesis that hypersegmentation may enhance neutrophil migratory capacity. However, EZ-TAXIScan™ video imaging revealed a previously unappreciated and progressive chemotaxis defect that was apparent prior to hypersegmentation onset. Cell speed and directionality were significantly impaired to fMLF as well as C5a and IL-8. Infected cells oriented normally in chemotactic gradients, but speed and direction were impaired because of a uropod retraction defect that led to cell elongation, nuclear lobe trapping in the contracted rear and progressive narrowing of the leading edge. In contrast, chemotactic receptor abundance, adhesion, phagocytosis and other aspects of cell function were unchanged. At the molecular level, H. pylori phenocopied the effects of Blebbistatin as indicated by aberrant accumulation of F-actin and actin spikes at the uropod together with enhanced ROCKII-mediated phosphorylation of myosin IIA regulatory light chains at S19. At the same time, RhoA and ROCKII disappeared from the cell rear and accumulated at the leading edge whereas myosin IIA was enriched at both cell poles. These data suggest that H. pylori inhibits the dynamic changes in myosin IIA contractility and front-to-back polarity that are essential for chemotaxis. Taken together, our data advance understanding of PMN plasticity and H. pylori pathogenesis.
... There are two pools of actin structures within neuronal growth cones: the dynamic F-actin filaments with rapid disassembly and assembly, and the stable F-actin bundles coexisting with myosin (actomyosin) and with slower kinetics (Schaefer et al., 2002). PIP 3 could either promote the assembly of dynamic F-actin or interfere with the formation of the actomyosin structures (Xu et al., 2003). Our observation of the interaction between actin and PTEN suggests that PIP 3 and PTEN could form either a negative feedback loop involving reorganization of dynamic F-actin structures or a double negative (which is, in many ways, similar to a positive) feedback loop involving formation and function of actomyosin complexes (Fig. 5A). ...
Axon guidance during neural wiring involves a series of precisely controlled chemotactic events by the motile axonal tip, the growth cone. A fundamental question is how neuronal growth cones make directional decisions in response to extremely shallow gradients of guidance cues with exquisite sensitivity. Here we report that nerve growth cones possess a signal amplification mechanism during gradient sensing process. In neuronal growth cones of Xenopus spinal neurons, phosphatidylinositol-3,4,5-trisphosphate (PIP3), an important signaling molecule in chemotaxis, was actively recruited to the up-gradient side in response to an external gradient of brain-derived neurotrophic factor (BDNF), resulting in an intracellular gradient with approximate 30-fold amplification of the input. Furthermore, a reverse gradient of phosphatase and tensin homolog (PTEN) was induced by BDNF within the growth cone and the increased PTEN activity at the down-gradient side is required for the amplification of PIP3 signals. Mechanistically, the establishment of both positive PIP3 and reverse PTEN gradients depends on the filamentous actin network. Together with computational modeling, our results revealed a double negative feedback loop among PTEN, PIP3 and actomyosin for signal amplification, which is essential for gradient sensing of neuronal growth cones in response to diffusible cues.
... Dictyostelium cells are reported to experience a switch-like, in-plane reorientation of their polarity when the source of chemoattractant cAMP is suddenly moved from facing the front of the cells to the back of the cells [68,69]. Meanwhile, HL-60 cells and neutrophils have been observed maintaining their polarity and instead executing U-turns to follow the reversal cues [70,71]. The type of reversal and repolarization can also depend on the amplitude and timescale of the changing signal. ...
Groups of eukaryotic cells can coordinate their crawling motion to follow cues more effectively, stay together, or invade new areas. This collective cell migration depends on cell-cell interactions, which are often studied by colliding pairs of cells together. Can the outcome of these collisions be predicted? Recent experiments on trains of colliding epithelial cells suggest that cells with a smaller contact angle to the surface or larger speeds are more likely to maintain their direction (“win”) upon collision. When should we expect shape or speed to correlate with the outcome of a collision? We build a model for two-cell collisions within the phase field approach, which treats cells as deformable objects. We can reproduce the observation that cells with high speed and small contact angles are more likely to win with two different assumptions for how cells interact: (1) velocity-aligning, in which we hypothesize that cells sense their own velocity and align to it over a finite timescale, and (2) front-front contact repolarization, where cells polarize away from cell-cell contact, akin to contact inhibition of locomotion. Surprisingly, though we simulate collisions between cells with widely varying properties, in each case, the probability of a cell winning is completely captured by a single summary variable: its relative speed (in the velocity-aligning model) or its relative contact angle (in the contact repolarization model). Both models are currently consistent with reported experimental results, but they can be distinguished by varying cell contact angle and speed through orthogonal perturbations.
... The first step in neutrophil activation is rapid F-actin polymerization at the leading edge, polarizing the cell 21 . Many signaling molecules are involved in this step, and a great deal of work has been performed to fully understand the mechanism of neutrophil activation and directional migration [22][23][24] . External temperature can also affect F-actin polymerization 25 , the diffusion rate of signaling molecules and integrins in neutrophils 26 , cell membrane fluidity 27,28 and many other processes involved in directed neutrophil migration. ...
Cell migration plays an essential role in wound healing and inflammatory processes inside the human body. Peripheral blood neutrophils, a type of polymorphonuclear leukocyte (PMN), are the first cells to be activated during inflammation and subsequently migrate toward an injured tissue or infection site. This response is dependent on both biochemical signaling and the extracellular environment, one aspect of which includes increased temperature in the tissues surrounding the inflammation site. In our study, we analyzed temperature-dependent neutrophil migration using differentiated HL-60 cells. The migration speed of differentiated HL-60 cells was found to correlate positively with temperature from 30 to 42 °C, with higher temperatures inducing a concomitant increase in cell detachment. The migration persistence time of differentiated HL-60 cells was higher at lower temperatures (30–33 °C), while the migration persistence length stayed constant throughout the temperature range. Coupled with the increased speed observed at high temperatures, this suggests that neutrophils are primed to migrate more effectively at the elevated temperatures characteristic of inflammation. Temperature gradients exist on both cell and tissue scales. Taking this into consideration, we also investigated the ability of differentiated HL-60 cells to sense and react to the presence of temperature gradients, a process known as thermotaxis. Using a two-dimensional temperature gradient chamber with a range of 27–43 °C, we observed a migration bias parallel to the gradient, resulting in both positive and negative thermotaxis. To better mimic the extracellular matrix (ECM) environment in vivo, a three-dimensional collagen temperature gradient chamber was constructed, allowing observation of biased neutrophil-like differentiated HL-60 migration toward the heat source.
... The exposure to uniform fields of chemokines and chemoattractants is already sufficient to induce neutrophil self-polarization and random movement (Zigmond, 1981). Studies with HL-60 cells identified the fundamental mechanisms of neutrophil self-polarization, revealing crucial roles of PIP3 signaling, small Rho GTPases and regulators of branched actin networks for establishing neutrophil polarity (Xu et al., 2003;Saha et al., 2018;Tsai et al., 2019). It is well established that both soluble and surface-bound attractants support such undirected movement patterns of neutrophil chemokinesis or haptokinesis, respectively. ...
Neutrophils are key cells of our innate immune response with essential roles for eliminating bacteria and fungi from tissues. They are also the prototype of an amoeboid migrating leukocyte. As one of the first blood-recruited immune cell types during inflammation and infection, these cells can invade almost any tissue compartment. Once in the tissue, neutrophils undergo rapid shape changes and migrate at speeds higher than most other immune cells. They move in a substrate-independent manner in interstitial spaces and do not follow predetermined tissue paths. Instead, neutrophil navigation is largely shaped by the chemokine and chemoattractant milieu around them. This highlights the decisive role of attractant-sensing G-protein coupled receptors (GPCRs) and downstream molecular pathways for controlling amoeboid neutrophil movement in tissues. A diverse repertoire of cell-surface expressed GPCRs makes neutrophils the perfect sentinel cell type to sense and detect danger-associated signals released from wounds, inflamed interstitium, dying cells, complement factors or directly from tissue-invading microbes. Moreover, neutrophils release attractants themselves, which allows communication and coordination between individual cells of a neutrophil population. GPCR-mediated positive feedback mechanisms were shown to underlie neutrophil swarming, a population response that amplifies the recruitment of amoeboid migrating neutrophils to sites of tissue injury and infection. Here we discuss recent findings and current concepts that counteract excessive neutrophil accumulation and swarm formation. In particular, we will focus on negative feedback control mechanisms that terminate neutrophil swarming to maintain the delicate balance between tissue surveillance, host protection and tissue destruction.
... This study also proposed the existence of two feedback pathways, namely between the RAN-GTP gradient and the Arp2/3 complex, and the suppressing crosstalk between the Arp2/3 actin network and the myosin II network ( Figure 2) [27]. This hypothesis has been supported by findings of other studies [28][29][30][31][32], but it is still not fully understood how the RAN-GTP mechanism can change the association or the activity state of factors required for cytoskeletal rearrangements. Further research is necessary to elucidate the multifaceted relationships between Arp2/3 and the actin network [27]. ...
Human female fertility and reproductive lifespan decrease significantly with age, resulting in an extended post-reproductive period. The central dogma in human female reproduction contains two important aspects. One is the pool of oocytes in the human ovary (the ovarian reserve; approximately 106 at birth), which diminishes throughout life until menopause around the age of 50 (approximately 103 oocytes) in women. The second is the quality of oocytes, including the correctness of meiotic divisions, among other factors. Notably, the increased rate of sub- and infertility, aneuploidy, miscarriages, and birth defects are associated with advanced maternal age, especially in women above 35 years of age. This postponement is also relevant for human evolution; decades ago, the female aging-related fertility drop was not as important as it is today because women were having their children at a younger age. Spindle assembly is crucial for chromosome segregation during each cell division and oocyte maturation, making it an important event for euploidy. Consequently, aberrations in this segregation process, especially during the first meiotic division in human eggs, can lead to implantation failure or spontaneous abortion. Today, human reproductive medicine is also facing a high prevalence of aneuploidy, even in young females. However, the shift in the reproductive phase of humans and the strong increase in errors make the problem much more dramatic at later stages of the female reproductive phase. Aneuploidy in human eggs could be the result of the non-disjunction of entire chromosomes or sister chromatids during oocyte meiosis, but partial or segmental aneuploidies are also relevant. In this review, we intend to describe the relevance of the spindle apparatus during oocyte maturation for proper chromosome segregation in the context of maternal aging and the female reproductive lifespan.
... Unpolarized cells have a homogeneous distribution of inactive GDP-Cdc42, and polarity establishment involves the asymmetric accumulation of active GTP-Cdc42 at a region on the cell cortex to generate a cell's "front" (Fig. 1B). Many cells also generate a specialized "back", and polarity can be reinforced by mutually inhibitory interactions between "front" and "back" specific factors (119). Active Cdc42 is recognized by proteins called "effectors", which are recruited to the polarity region and transduce Cdc42 localization into orientation of the cytoskeleton towards the front, leading to growth or migration in that direction. ...
Accurate decoding of spatial chemical landscapes is critical for many cell functions. Eukaryotic cells decode local chemical gradients to orient growth or movement in productive directions. Recent work on yeast model systems, whose gradient sensing pathways display much less complexity than those in animal cells, has suggested new paradigms for how these very small cells successfully exploit information in noisy and dynamic pheromone gradients to identify their mates. Pheromone receptors regulate a polarity circuit centered on the conserved Rho-family GTPase, Cdc42. The polarity circuit contains both positive and negative feedback pathways, allowing spontaneous symmetry breaking and also polarity site disassembly and relocation. Cdc42 orients the actin cytoskeleton, leading to focused vesicle traffic that promotes movement of the polarity site and also reshapes the cortical distribution of receptors at the cell surface. In this article, we review the advances from work on yeasts and compare them with the excitable signaling pathways that have been revealed in chemotactic animal cells.
Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Because of the lack of DREAM inhibitors, we used shRNA to knock down the KCNIP3 gene encoding DREAM in HL-60 cells, which have similar morphological and functional characteristics to human neutrophils after differentiation (Xu et al., 2003). In control experiments, we confirmed that isolated human blood neutrophils expressed DREAM mRNA and protein (Fig. S2 B;and Fig. 3,I and J). ...
The interaction between neutrophils and endothelial cells is critical for the pathogenesis of vascular inflammation. However, the regulation of neutrophil adhesive function remains not fully understood. Intravital microscopy demonstrates that neutrophil DREAM promotes neutrophil recruitment to sites of inflammation induced by TNF-α but not MIP-2 or fMLP. We observe that neutrophil DREAM represses expression of A20, a negative regulator of NF-κB activity, and enhances expression of pro-inflammatory molecules and phosphorylation of IκB kinase (IKK) after TNF-α stimulation. Studies using genetic and pharmacologic approaches reveal that DREAM deficiency and IKKβ inhibition significantly diminish the ligand-binding activity of β2 integrins in TNF-α-stimulated neutrophils or neutrophil-like HL-60 cells. Neutrophil DREAM promotes degranulation through IKKβ-mediated SNAP-23 phosphorylation. Using sickle cell disease mice lacking DREAM, we show that hematopoietic DREAM promotes vaso-occlusive events in microvessels following TNF-α challenge. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases.
... The opposite localisation of the two antagonistic proteins leads to the formation of the PIP3 gradient, essential for cell polarisation . Similar finding have also been found during neutrophils migration where increased level of PI(3,4,5)P3 at the cell leading edge activates Rac GTPases and therefore F-actin polymerisation (Xu, Wang et al. 2003). During epithelial morphogenesis, many organs require the formation of tubes and this process partially depends on PTEN. ...
Cell migration is crucial during morphogenesis and also in the adult where it participates in tissue renewal, immune response, wound healing as well as in cancer invasion and metastasis. In certain cases, cells move as individuals while some other processes require collective cell migration (Rorth 2012). In order to migrate collectively, cells need to establish and maintain a front-rear polarity axis, redistribute proteins in a polarised fashion and form cell-cell and cell-matrix interaction. Collective cell migration is crucial for many physiological processes, from embryonic development where it is involved in gastrulation and morphogenesis to the adult where it participates in wound healing, tissue renewal and immune responses. Many pathologies have been linked with aberrant collective cell migration, the first of all being cancer spreading (Rorth 2009, Friedl, Sahai et al. 2012, Te Boekhorst, Preziosi et al. 2016). During my PhD, I focused on the PTEN dependent mechanisms controlling collective cell migration. A wide number of genes are altered during oncogenesis including inactivation of tumour suppressors such as p53, p16 and retinoblastoma (Rb) and overexpression of gene encoding epidermal growth factor (EGF) (Tamura, Gu et al. 1999). PTEN is one such tumour suppressor gene which is frequently mutated or deleted in a wide range of human cancers, from glioblastomas to prostate, breast, kidney, lung, testes and thyroid cancers. In particular, PTEN`s function is altered in more than 60% of glioblastomas. It is altered mostly in high-grade invasive glioblastomas but not in low-grade gliomas suggesting an important correlation between PTEN absence and invasive properties of the cancer cells (Rasheed, Stenzel et al. 1997, Dey, Crosswell et al. 2008). In addition, PTEN is known to regulate several cellular functions including cell migration and lots of the mechanisms involved in single cell migration have been extensively studied (Davies, Gibbs et al. 1999, Iijima and Devreotes 2002, Gerisch, Schroth-Diez et al. 2012). Glioblastoma form the most common and lethal primary intracerebral tumours (Davis, Kupelian et al. 2001). Tumour spreading in the brain parenchyma is largely responsible for the resistance of gliomas to cancer treatment and yet no therapeutic treatment has been found to prevent tumour infiltration. The mechanisms by which cells invade the central nervous system have not yet been directly observed and for some aspects they still remain elusive (Davies, Gibbs et al. 1999). Glioblastoma can arise from astrocytes or their precursors and they have an incidence of approximately 5 cases per 100.000 inhabitants (Furnari, Fenton et al. 2007). Astrocytes are the main glial cells of the central nervous system. They participate in the regulation of brain homeostasis and in the formation of the blood-brain barrier (Kimelberg and Nedergaard 2010). Astrocyte migrate in a collective fashion during development (Gnanaguru, Bachay et al. 2013) and in the adult brain, they have been shown to undergo astrogliosis in response to inflammation or trauma. Here they are able to elongate, polarise and eventually migrate toward the site of interest in order to create a glial scar (Sofroniew 2014). For these many reasons, in the lab we use primary rat astrocyte as preferential model to study the mechanism of collective cell migration (Etienne-Manneville 2006) [...]
... In leukocytes, membrane protrusions are often found associated with cell polarity and migration, which can be a protruding structure at the leading edge (a pseudopod) or a contractile structure at the rear (a uropod). The two structures differ in cytoskeletal composition; pseudopods are enriched with newly synthesized actin, and uropods are composed of contractile actin-myosin complexes (Sánchez-Madrid and Serrador, 2009;Xu et al., 2003). While studies on HSC morphologies are relatively few, researchers have described the microspikes or protrusions of mouse HSCs or human HPCs with various terms (e.g., magnupodia, tenupodia, proteopodia, and uropods), which resemble morphological and functional features of pseudopods and/or uropods (Francis et al., 1998;Frimberger et al., 2001;Wagner et al., 2005). ...
Membrane-bound factors expressed by niche stromal cells constitute a unique class of localized cues and regulate the long-term functions of adult stem cells, yet little is known about the underlying mechanisms. Here, we used a supported lipid bilayer (SLB) to recapitulate the membrane-bound interactions between hematopoietic stem cells (HSCs) and niche stromal cells. HSCs cluster membrane-bound stem cell factor (mSCF) at the HSC-SLB interface. They further form a polarized morphology with aggregated mSCF under a large protrusion through a synergy with VCAM-1 on the bilayer, which drastically enhances HSC adhesion. These features are unique to mSCF and HSCs among the factors and hematopoietic populations we examined. The mSCF–VCAM-1 synergy and the polarized HSC morphology require PI3K signaling and cytoskeletal reorganization. The synergy also enhances nuclear retention of FOXO3a, a crucial factor for HSC maintenance, and minimizes its loss induced by soluble SCF. Our work thus reveals a unique role and signaling mechanism of membrane-bound factors in regulating stem cell morphology and function.
... In this context, a mechanism including different small GTPases, namely Rho, Rac, and Cdc42 at the rear and front edge as well as feedback loops has been described [51]. Actin polymerization and Rac and Cdc42 activity is prominent at the leading edge and affects downregulation of RhoA, while contractile actin-myosin filaments are associated with high RhoA activity at the trailing edge and affect downregulation of Cdc42 and Rac [85][86][87]. Feedback loops reinforce the spatially different expression of the small GTPases, generating gradients of RhoA, Rac, and Cdc42 activity from between the rear and front edge, and thus sustain the polarity of neutrophils [87]. However, this complex signaling network is not completely understood yet, and while all three GTPases definitely play some part in the extent and mechanisms by which these small GTPases regulate neutrophil motility, the signaling network is still under discussion [13,51,87]. ...
Chronic inflammation, the activation of immune cells and their cross-talk with cardiomyocytes in the pathogenesis and progression of heart diseases has long been overlooked. However, with the latest research developments, it is increasingly accepted that a vicious cycle exists where cardiomyocytes release cardiocrine signaling molecules that spiral down to immune cell activation and chronic state of low-level inflammation. For example, cardiocrine molecules released from injured or stressed cardiomyocytes can stimulate macrophages, dendritic cells, neutrophils and even T-cells, which then subsequently increase cardiac inflammation by co-stimulation and positive feedback loops. One of the key proteins involved in stress-mediated cardiomyocyte signal transduction is a small GTPase RhoA. Importantly, the regulation of RhoA activation is critical for effective immune cell response and is being considered as one of the potential therapeutic targets in many immune-cell-mediated inflammatory diseases. In this review we provide an update on the role of RhoA at the juncture of immune cell activation, inflammation and cardiac disease.
... In innate immunity, Rho GTPases are involved in phagocytosis and regulation of leukocyte chemotaxis and motility [25][26][27][28]. Rho GTPases also play an important role in adaptive immunity by regulating the activation and migration of T and B cells and forming immunological synapses between dendritic cells and antigen-specific T cells, which are prerequisites for inducing an adaptive T-cell response [29][30][31][32]. ...
Patient: Female, 10-year-old (5-year-old at diagnosis)
Final Diagnosis: Combined immunodeficiency
Symptoms: Infection
Medication: —
Clinical Procedure: —
Specialty: Genetics • Immunology
Objective
Rare co-existance of disease or pathology
Background
X-linked intellectual disabilities constitute a group of clinically and genetically heterogeneous disorders that are divided into syndromic and nonsyndromic forms. PAK3 mutations are associated with X-linked nonsyndromic forms of intellectual disability, with the most common clinical features being cognitive deficit, large ears, oral motor hypotonia, and neurobehavioral abnormalities. These mutations have been reported to be associated with either loss of the PAK3 protein or loss of its kinase activity. We report a case with the novel PAK3 variant c.685C>T p.(Pro229Ser), which has not been previously described.
Case Report
We report the first case of a PAK3 mutation to present with the common clinical features along with immuno-deficiency resembling common variable immune deficiency. Our patient was a 10-year-old girl who had experienced septic shock with a rapidly deteriorating course when she was 5-years-old. The initial immune work-up showed lymphopenia affecting all cell lines, but preferentially the B-cell compartment. Further work-up of this patient revealed low levels of immunoglobulin (Ig) G, undetectable IgA, reduced IgG1 and IgG2 subclasses, and poor response to the diphtheria/tetanus vaccine. Lymphocyte function, tested as the response to the mitogen phytohemagglutinin, was low and fluctuated between 9% and 22% compared with control samples. The patient experienced recurrent respiratory tract infections, and she responded well to regular intravenous Ig treatment and antibiotic prophylaxis.
Conclusions
The current case might provide a new insight into PAK3 gene function. Although further evidence is needed, it is worth considering that immunological abnormalities may be associated with PAK3 gene mutations.
... Moreover, depletion of CAV1 led to AMPK activation followed by a p53-dependent G1 cell-cycle arrest and autophagy, suggesting that elevated CAV1 may contribute to the ATP generation (Ha et al., 2012). This is already a common view that there is mutual inhibition between the lamellipodia formation in cell frontness and the contractile stress fiber formation in cell backness (Li et al., 2003;Meili and Firtel, 2003;Xu et al., 2003) during persistent migration. Thus, we propose that CAV-1 depletion disequilibrates the balance between RhoA-mediated actomyosin bundles and Rac1-mediated lamellipodia formation, which potentiates the interpretation of the cell migration defects. ...
The actin cytoskeleton and membrane-associated caveolae contribute to active processes, such as cell morphogenesis and motility. How these two systems interact and control directional cell migration is an outstanding question but remains understudied. Here we identified a negative feedback between contractile actin assemblies and phosphorylated caveolin-1 (CAV-1) in migrating cells. Cytoplasmic CAV-1 vesicles display actin-associated motilities by sliding along actin filaments or/and coupling to do retrograde flow with actomyosin bundles. Inhibition of contractile stress fibers, but not Arp2/3-dependent branched actin filaments, diminished the phosphorylation of CAV-1 on site Tyr14, and resulted in substantially increased size and decreased motility of cytoplasmic CAV-1 vesicles. Reciprocally, both the CAV-1 phospho-deficient mutation on site Tyr14 and CAV-1 knockout resulted in dramatic AMPK phosphorylation, further causing reduced active level of RhoA-myosin II and increased active level of Rac1-PAK1-Cofilin, consequently led to disordered contractile stress fibers and prominent lamellipodia. As a result, cells displayed depolarized morphology and compromised directional migration. Collectively, we propose a model in which feedback-driven regulation between actin and CAV-1 instructs persistent cell migration.
... Tethers observed in control cells were also typically much shorter and thicker than in the Cdc42-KO line. Interestingly, inhibition of myosin-II with blebbistatin can cause cytoplasmic tethers HL-60 cells 3 , and local Cdc42 activation can induce a longdistance myosin response 29 . Collectively, these findings suggest that Cdc42 cell front activity may mediate long-range regulation of protrusion-inhibiting, cell rear polarity signals. ...
During chemotaxis, neutrophils use cell surface G-Protein Coupled Receptors (GPCRs) to detect chemoattractant gradients. The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities. Positive feedback could promote rapid signal spreading, yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell. How the signal transduction network achieves signal amplification while preserving spatial information remains unclear. The GTPase Cdc42 is a cell-front polarity coordinator that is predictive of cell turning, suggesting an important role in spatial processing. To directly measure information flow from receptors to Cdc42, we paired zebrafish parapinopsina, an optogenetic GPCR that allows reversible ON/OFF receptor control with a spectrally compatible red/far red Cdc42 FRET biosensor. Using this new toolkit, we show that positive and negative signals downstream of G-proteins shape a rapid, dose-dependent Cdc42 response. Furthermore, F-actin and Cdc42 itself provide two distinct negative signals that limit the duration and spatial spread of Cdc42 activation, maintaining output signals local to the originating receptors.
... The high local microtubule depolymerization rate at the uropod is causally connected to retraction of the cell's back demonstrating that microtubules are able to regulate local cellular retraction events (Kopf et al., 2020). Two signaling modules have been identified, which act in concert to balance morphological frontness and backness in migrating cells: RhoA-and actomyosin dependent contractility stimulate cellular backness, while F actin-rich protrusions at the cell front are regulated by a trimeric G protein, the small GTPase Rac, and 3 phosphoinositides such as PIP3 (Niggli, 2003;Xu et al., 2003;Yoo et al., 2010). Microtubules link RhoA and actomyosin activation by release of the RhoA-specific guanine nucleotide exchange factor (GEF)-H1 (Ren et al., 1998;Krendel et al., 2002;Chang et al., 2008). ...
The organization of microtubule arrays in immune cells is critically important for a properly operating immune system. Leukocytes are white blood cells of hematopoietic origin, which exert effector functions of innate and adaptive immune responses. During these processes the microtubule cytoskeleton plays a crucial role for establishing cell polarization and directed migration, targeted secretion of vesicles for T cell activation and cellular cytotoxicity as well as the maintenance of cell integrity. Considering this large spectrum of distinct effector functions, leukocytes require flexible microtubule arrays, which timely and spatially reorganize allowing the cells to accommodate their specific tasks. In contrast to other specialized cell types, which typically nucleate microtubule filaments from non-centrosomal microtubule organizing centers (MTOCs), leukocytes mainly utilize centrosomes for sites of microtubule nucleation. Yet, MTOC localization as well as microtubule organization and dynamics are highly plastic in leukocytes thus allowing the cells to adapt to different environmental constraints. Here we summarize our current knowledge on microtubule organization and dynamics during immune processes and how these microtubule arrays affect immune cell effector functions. We particularly highlight emerging concepts of microtubule involvement during maintenance of cell shape and physical coherence.
... Broadly speaking, pseudopod formation requires activation of the Rho-family GTPases Rac and Cdc42 to drive F-actin polymerization into protrusions that drive forward motion (Kraynov et al., 2000;Itoh et al., 2002;Ridley et al., 2003;Willard and Devreotes, 2006;Yang et al., 2016). Conversely, the sides and uropod contain active RhoA, myosin light chain kinase, and ezrin-radixin-moesin (ERM) protein scaffolding to support actomyosin-based contraction of the trailing edge and stabilize adhesion to the endothelium during extravasation (Yoshinaga-Ohara et al., 2002;Xu et al., 2003;Lee et al., 2004a;Hind et al., 2016). ...
The dynamic re-organization of cellular membranes in response to extracellular stimuli is fundamental to the cell physiology of myeloid and lymphoid cells of the immune system. In addition to maintaining cellular homeostatic functions, remodeling of the plasmalemma and endomembranes endow leukocytes with the potential to relay extracellular signals across their biological membranes to promote rolling adhesion and diapedesis, migration into the tissue parenchyma, and to ingest foreign particles and effete cells. Phosphoinositides, signaling lipids that control the interface of biological membranes with the external environment, are pivotal to this wealth of functions. Here, we highlight the complex metabolic transitions that occur to phosphoinositides during several stages of the leukocyte lifecycle, namely diapedesis, migration, and phagocytosis. We describe classical and recently developed tools that have aided our understanding of these complex lipids. Finally, major downstream effectors of inositides are highlighted including the cytoskeleton, emphasizing the importance of these rare lipids in immunity and disease.
... Metabolism of myo-inositol leads to the generation of the signaling molecules phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ). While the latter product, Ins(1,3,4,5)P 4 , suppresses the function of pleckstrin homology (PH) domain-containing protein activity, such as Akt serine/threonine kinase 1, the former product, PtdIns(3,4,5)P 3 , binds to PH domain-containing proteins (Supplementary Figure S8) and as a result activates downstream cellular signaling pathways, which modulate various cellular functions, including the regulation of membrane potential, phagocytosis, and superoxide species production [84][85][86]. Marshall et al. determined that pseudopodia extension and phagocytic particle engulfment was dependent upon type I phosphoinositide 3-kinase (PI3K) activity and the formation of PtdIns(3,4,5)P 3 , which rapidly accumulates at the site of phagocytosis and ensures membrane delivery to distending pseudopods [87]. Moreover, p47 Phox , an essential subunit of reduced nicotinamide adenine dinucleotide phosphate oxidase, has been shown to be recruited to type I PI3K signals [88]. ...
Macrophages (MΦs) are prevalent innate immune cells, present throughout human bodily tissues where they orchestrate innate and adaptive immune responses to maintain cellular homeostasis. MΦs have the capacity to display a wide array of functional phenotypes due to different microenvironmental cues, particularly soluble bacterial secretory products. Recent evidence has emerged demonstrating that metabolism supports MΦ function and plasticity, in addition to energy and biomolecular precursor production. In this study, 1D 1H-NMR-based metabolomics was used to identify the metabolic pathways that are differentially altered following primary human monocyte-derived MΦ exposure to P. aeruginosa planktonic- and biofilm-conditioned media (PCM and BCM). Metabolic profiling of PCM- and BCM-exposed MΦs indicated a significant increase in glycolytic metabolism, purine biosynthesis, and inositol phosphate metabolism. In addition, these metabolic patterns suggested that BCM-exposed MΦs exhibit a hyperinflammatory metabolic profile with reduced glycerol metabolism and elevated catabolism of lactate and amino acids, relative to PCM-exposed MΦs. Altogether, our study reveals novel findings concerning the metabolic modulation of human MΦs after exposure to secretory microbial products and contributes additional knowledge to the field of immunometabolism in MΦs.
The role and function of neutrophils are well known, but we still have incomplete understanding of the mechanisms by which neutrophils migrate from blood vessels to inflammatory sites. Neutrophil migration is a complex process that involves several distinct steps. To resist the blood flow and maintain their rolling, neutrophils employ tether and sling formation. They also polarize and form pseudopods and uropods, guided by hierarchical chemotactic agents that enable precise directional movement. Meanwhile, chemotactic agents secreted by neutrophils, such as CXCL1, CXCL8, LTB4, and C5a, can recruit more neutrophils and amplify their response. In the context of diapedesis neutrophils traverse the endothelial cells via two pathways: the transmigratory cup and the lateral border recycling department. These structures aid in overcoming the narrow pore size of the endothelial barrier, resulting in more efficient transmembrane migration. Interestingly, neutrophils exhibit a preference for the paracellular pathway over the transcellular pathway, likely due to the former’s lower resistance. In this review, we will delve into the intricate process of neutrophil migration by focusing on critical structures that underpins this process.
Groups of eukaryotic cells can coordinate their crawling motion to follow cues more effectively, stay together, or invade new areas. This collective cell migration depends on cell-cell interactions, which are often studied by colliding pairs of cells together. Can the outcome of these collisions be predicted? Recent experiments on trains of colliding epithelial cells suggest that cells with a smaller contact angle to the surface or larger speeds are more likely to maintain their direction (“win”) upon collision. When should we expect shape or speed to correlate with the outcome of a collision? To investigate this question, we build a model for two-cell collisions within the phase field framework, which allows for cell shape changes. We can reproduce the observation that cells with high speed and small contact angles are more likely to win with two different assumptions for how cells interact: (1) velocity aligning, in which we hypothesize that cells sense their own velocity and align to it over a finite timescale, and (2) front-front contact repolarization, where cells polarize away from cell-cell contact, akin to contact inhibition of locomotion. Surprisingly, though we simulate collisions between cells with widely varying properties, in each case, the probability of a cell winning is completely captured by a single summary variable: its relative speed (in the velocity-aligning model) or its relative contact angle (in the contact repolarization model). Both models are currently consistent with reported experimental results, but they can be distinguished by varying cell contact angle and speed through orthogonal perturbations.
A considerable amount is known about how eukaryotic cells move toward an attractant, and the mechanisms are conserved from Dictyostelium discoideum to human neutrophils. Relatively little is known about chemorepulsion, where cells move away from a repellent signal. We previously identified pathways mediating chemorepulsion in Dictyostelium, and here we show that these pathways, including Ras, Rac, protein kinase C, PTEN, and ERK1 and 2, are required for human neutrophil chemorepulsion, and, as with Dictyostelium chemorepulsion, PI3K and phospholipase C are not necessary, suggesting that eukaryotic chemorepulsion mechanisms are conserved. Surprisingly, there were differences between male and female neutrophils. Inhibition of Rho-associated kinases or Cdc42 caused male neutrophils to be more repelled by a chemorepellent and female neutrophils to be attracted to the chemorepellent. In the presence of a chemorepellent, compared with male neutrophils, female neutrophils showed a reduced percentage of repelled neutrophils, greater persistence of movement, more adhesion, less accumulation of PI(3,4,5)P3, and less polymerization of actin. Five proteins associated with chemorepulsion pathways are differentially abundant, with three of the five showing sex dimorphism in protein localization in unstimulated male and female neutrophils. Together, this indicates a fundamental difference in a motility mechanism in the innate immune system in men and women.
Optogenetic protein dimerization systems are powerful tools to investigate the biochemical networks that cells use to make decisions and coordinate their activities. These tools, including the improved Light-Inducible Dimer (iLID) system, offer the ability to selectively recruit components to subcellular locations, such as micron-scale regions of the plasma membrane. In this way, the role of individual proteins within signaling networks can be examined with high spatiotemporal resolution. Currently, consistent recruitment is limited by heterogeneous optogenetic component expression, and spatial precision is diminished by protein diffusion, especially over long time scales. Here, we address these challenges within the iLID system with alternative membrane anchoring domains and fusion configurations. Using live cell imaging and mathematical modeling, we demonstrate that the anchoring strategy affects both component expression and diffusion, which in turn impact recruitment strength, kinetics, and spatial dynamics. Compared to the commonly used C-terminal iLID fusion, fusion proteins with large N-terminal anchors show stronger local recruitment, slower diffusion of recruited components, efficient recruitment over wider gene expression ranges, and improved spatial control over signaling outputs. We also define guidelines for component expression regimes for optimal recruitment for both cell-wide and subcellular recruitment strategies. Our findings highlight key sources of imprecision within light-inducible dimer systems and provide tools that allow greater control of subcellular protein localization across diverse cell biological applications.
Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin’s affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.
We studied the effects of glucosylation of RhoA, Rac1, and Cdc42 at threonine-35 and-37 by Clostridium difficile toxin B on nucleotide binding, GTPase activity, and effector coupling and compared these results with the ADP ribosylation of RhoA at asparagine-41 catalyzed by Clostridium botulinum C3 transferase. Whereas glucosylation and ADP ribosylation had no major effects on GDP release from RhoA, Rac1, and Cdc42, the rate of GTPγS release from Rho proteins was increased 3-6-fold by glucosylation. ADP ribosylation decreased the rate of GTPγS release by about 50%. Glucosylation reduced the intrinsic activities of the GTPases by 3-7-fold and completely blocked GTPase stimulation by Rho-GAP. In contrast, ADP ribosylation slightly increased GTPase activity (∼2-fold) and had no major effect on GAP stimulation of GTPase. Whereas ADP ribosylation did not affect the interaction of RhoA with the binding domain of protein kinase N, glucosylation inhibited this interaction. Glucosylation of Rac1 markedly diminished its ability to support the activation of the superoxide-generating NADPH oxidase of phagocytes. Glucosylated Rac1 did not interfere with NADPH oxidase activation by unmodified Rac1, even when present in marked molar excess, indicating that it was incapable of competing for a common effector. The data indicate that the functional inactivation of small GTPases by glucosylation is mainly caused by inhibition of GTPase-effector protein interaction.
Redistribution of specialized molecules in migrating cells
develops asymmetry between two opposite cell poles, the leading edge
and the uropod. We show that acquisition of a motile phenotype in T
lymphocytes results in the asymmetric redistribution of ganglioside
GM3- and GM1-enriched raft domains to the leading edge and to the
uropod, respectively. This segregation to each cell pole parallels the
specific redistribution of membrane proteins associated to each raft
subfraction. Our data suggest that raft partitioning is a major
determinant for protein redistribution in polarized T cells, as ectopic
expression of raft-associated proteins results in their asymmetric
redistribution, whereas non-raft-partitioned mutants of these proteins
are distributed homogeneously in the polarized cell membrane. Both
acquisition of a migratory phenotype and SDF-1α-induced chemotaxis
are cholesterol depletion-sensitive. Finally, GM3 and GM1 raft
redistribution requires an intact actin cytoskeleton, but is
insensitive to microtubule disruption. We propose that membrane protein
segregation not only between raft and nonraft domains but also between
distinct raft subdomains may be an organizational principle that
mediates redistribution of specialized molecules needed for T cell
migration.
One of the elementary processes in morphogenesis is the formation of a spatial pattern of tissue structures, starting from almost homogeneous tissue. It will be shown that relatively simple molecular mechanisms based on auto- and cross catalysis can account for a primary pattern of morphogens to determine pattern formation of the tissue. The theory is based on short range activation, long range inhibition, and a distinction between activator and inhibitor concentrations on one hand, and the densities of their sources on the other. While source density is expected to change slowly, e.g. as an effect of cell differentiation, the concentration of activators and inhibitors can change rapidly to establish the primary pattern; this results from auto- and cross catalytic effects on the sources, spreading by diffusion or other mechanisms, and degradation.Employing an approximative equation, a criterium is derived for models, which lead to a striking pattern, starting from an even distribution of morphogens, and assuming a shallow source gradient. The polarity of the pattern depends on the direction of the source gradient, but can be rather independent of other features of source distribution. Models are proposed which explain size regulation (constant proportion of the parts of the pattern irrespective of total size). Depending on the choice of constants, aperiodic patterns, implying a one-to-one correlation between morphogen concentration and position in the tissue, or nearly periodic patterns can be obtained. The theory can be applied not only to multicellular tissues, but also to intracellular differentiation, e.g. of polar cells.The theory permits various molecular interpretations. One of the simplest models involves bimolecular activation and monomolecular inhibition. Source gradients may be substituted by, or added to, sink gradients, e.g. of degrading enzymes. Inhibitors can be substituted by substances required for, and depleted by activation.Sources may be either synthesizing systems or particulate structures releasing activators and inhibitors.Calculations by computer are presented to exemplify the main features of the theory proposed. The theory is applied to quantitative data on hydra — a suitable one-dimensional model for pattern formation — and is shown to account for activation and inhibition of secondary head formation.
On treatment with chemoattractant, the neutrophil plasma membrane becomes organized into detergent-resistant membrane domains (DRMs), the distribution of which is intimately correlated with cell polarization. Plasma membrane at the front of polarized cells is susceptible to extraction by cold Triton X-100, whereas membrane at the rear is resistant to extraction. After cold Triton X-100 extraction, DRM components, including the transmembrane proteins CD44 and CD43, the GPI-linked CD16, and the lipid analog, DiIC16, are retained within uropods and cell bodies. Furthermore, CD44 and CD43 interact concomitantly with DRMs and with the F-actin cytoskeleton, suggesting a mechanism for the formation and stabilization of DRMs. By tracking the distribution of DRMs during polarization, we demonstrate that DRMs progress from a uniform distribution in unstimulated cells to small, discrete patches immediately after activation. Within 1 min, DRMs form a large cap comprising the cell body and uropod. This process is dependent on myosin in that an inhibitor of myosin light chain kinase can arrest DRM reorganization and cell polarization. Colabeling DRMs and F-actin revealed a correlation between DRM distribution and F-actin remodeling, suggesting that plasma membrane organization may orient signaling events that control cytoskeletal rearrangements and, consequently, cell polarity.
Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.
Chemotactic factors stimulate a rapid increase in the cytosolic concentration of intracellular calcium ions ([Ca2+]in) in human polymorphonuclear leukocytes (PMNL), which may be an event that is critical to the expression of chemotaxis and other PMNL functions. Treatment of PMNL with pertussis toxin catalyzes ADP-ribosylation of a protein similar or identical to the inhibiting regulatory protein of adenylate cyclase, Gi, and suppresses the increase in [Ca2+]in elicited by leukotriene B4(LTB4) and formyl-methionyl-leucyl-phenylalanine. Chemotactic migration and lysosomal enzyme release elicited by chemotactic factors were inhibited by pertussis toxin with a concentration-dependence similar to that for inhibition of the increase in [Ca2+]in, without an effect on lysosomal enzyme release induced by the ionophore A23187 and phorbol myristate acetate. Activated pertussis toxin catalyzed the [32P]ADP-ribosylation of a 41 kD protein in homogenates of PMNL. The extent of [32P]ADP-ribosylation of this protein was reduced 59% by pretreatment of intact PMNL with pertussis toxin. Pertussis toxin selectively decreased the number of high-affinity receptors for LTB4 on PMNL by 60% without altering the number or binding properties of the low-affinity subset of receptors. Pertussis toxin modification of a membrane protein of PMNL analogous to Gi thus simultaneously alters chemotactic receptors and attenuates the changes in cytosolic calcium concentration and PMNL function caused by chemotactic factors.
Mechanical tension influences tissue morphogenesis and the synthetic, mitotic, and motile behavior of cells. To determine the effects of tension on epithelial motility and cytoskeletal organization, small, motile clusters of epidermal cells were artificially extended with a micromanipulated needle. Protrusive activity perpendicular to the axis of tension was dramatically suppressed. To determine the ultrastructural basis for this phenomenon, cells whose exact locomotive behavior was recorded cinemicrographically were examined by transmission electron microscopy. In untensed, forward-moving lamellar protrusions, microfilaments appear disorganized and anisotropically oriented. But in cytoplasm held under tension by micromanipulation or by the locomotive activity of other cells within the epithelium, microfilaments are aligned parallel to the tension. In non-spreading regions of the epithelial margin, microfilaments lie in tight bundles parallel to apparent lines of tension. Thus, it appears that tension causes alignment of microfilaments. In contrast, intermediate filaments are excluded from motile protrusions, being confined to the thicker, more central part of the cell. They roughly follow the contours of the cell, but are not aligned relative to tension even when microfilaments in the same cell are. This suggests that the organization of intermediate filaments is relatively resistant to physical distortion and the intermediate filaments may act as passive structural support within the cell. The alignment of microfilaments under tension suggests a mechanism by which tension suppresses protrusive activity: microfilaments aligned by forces exerted through filament-surface or filament-filament interconnections cannot reorient against such force and so cannot easily extend protrusions in directions not parallel to tension.
Model calculations are presented for various problems of development on the basis of a theory of primary pattern formation which we previously proposed. The theory involves short-range autocatalytic activation and longer-range inhibition (lateral inhibition). When a certain criterion is satisfied, self-regulating patterns are generated.
The autocatalytic features of the theory are demonstrated by simulations of the determination of polarity in the Xenopus retina. General conditions for marginal and internal activation, and corresponding effects of symmetry are discussed. Special molecular mechanisms of pattern formation are proposed in which activator is chemically converted into inhibitor, or an activator precursor is depleted by conversion into activator.
The (slow) effects of primary patterns on differentiation can be included into the formalism in a straightforward manner. In conjunction with growth, this can lead to asymmetric steady states of cell types, cell differentiation and proliferation as found, for instance, in growing and budding hydra. In 2 dimensions, 2 different types of patterns can be obtained. Under some assumptions, a single pattern-forming system produces a ‘bristle’ type pattern of peaks of activity with rather regular spacings on a surface. Budding of hydra is treated on this basis. If, however, gradients develop under the influence of a weak external or marginal asymmetry, a monotonic gradient can be formed across the entire field, and 2 such gradient-forming systems can specify ‘positional information’ in 2 dimensions.
If inhibitor equilibrates slowly, a spatial pattern may oscillate, as observed with regard to the intracellular activation of cellular slime moulds.
The applications are intended to demonstrate the ability of the proposed theory to explain properties frequently encountered in developing systems.
Two toxins, latrunculins A and B, which contain a new class of 16- and 14-membered marine macrolides attached to the rare
2-thiazolidinone moiety, were purified recently from the Red Sea sponge Latrunculia magnifica. The effects of these toxins
on cultured mouse neuroblastoma and fibroblast cells have been evaluated. In both types of cells, submicromolar toxin concentrations
rapidly induce striking changes in cell morphology that are reversible upon removal of the toxin. Immunofluorescence studies
with antibodies specific for cytoskeletal proteins reveal that the toxins cause major alterations in the organization of microfilaments
without obvious effects on the organization of the microtubular system.
Locomoting polymorphonuclear leukocytes (PMNs) exhibit a morphological polarity. We demonstrate that they also exhibit a behavioral polarity in their responsiveness to chemotactic factor stimulation. This is demonstrated by (a) the pattern of their locomotion in a homogeneous concentration of chemotactic factors, (b) their responses to increases in the homogeneous concentration of chemotactic factors, and (c) their responses to changes in the direction of a chemotactic gradient. The behavioral polarity is not a function of the rate of locomotion of the particular stimulant used to orient the cells, but may reflect an asymmetric distribution of chemotactic receptors or the motile machinery. The polar behavior affects the chemotactic ability of PMNs. The data are discussed in relation to possible mechanisms of sensing a chemotactic gradient.
Phosphatidylinositol (PtdIns) 3-kinase is an enzyme implicated in growth factor signal transduction by associating with receptor and nonreceptor tyrosine kinases, including the platelet-derived growth factor receptor. Inhibitors of PtdIns 3-kinase could potentially give a better understanding of the function and regulatory mechanisms of the enzyme. Quercetin, a naturally occurring bioflavinoid, was previously shown to inhibit PtdIns 3-kinase with an IC50 of 1.3 microgram/ml (3.8 microM); inhibition appeared to be directed at the ATP-binding site of the kinase. Analogs of quercetin were investigated as PtdIns 3-kinase inhibitors, with the most potent ones exhibiting IC50 values in the range of 1.7-8.4 micrograms/ml. In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 > 30 micrograms/ml). Since quercetin has also been shown to inhibit other PtdIns and protein kinases, other chromones were evaluated as inhibitors of PtdIns 3-kinase without affecting PtdIns 4-kinase or selected protein kinases. One such compound, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (also known as 2-(4-morpholinyl)-8-phenylchromone, LY294002), completely and specifically abolished PtdIns 3-kinase activity (IC50 = 0.43 microgram/ml; 1.40 microM) but did not inhibit PtdIns 4-kinase or tested protein and lipid kinases. Analogs of LY294002 demonstrated a very selective structure-activity relationship, with slight changes in structure causing marked decreases in inhibition. LY294002 was shown to completely abolish PtdIns 3-kinase activity in fMet-Leu-Phe-stimulated human neutrophils, as well as inhibit proliferation of smooth muscle cells in cultured rabbit aortic segments. Since PtdIns 3-kinase appears to be centrally involved with growth factor signal transduction, the development of specific inhibitors against the kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response.
The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway.
G protein-coupled receptor agonists initiate a cascade of signaling events in neonatal rat ventricular myocytes that culminates in changes in gene expression and cell growth characteristic of hypertrophy. These responses have been previously shown to be dependent on Gq and Ras. Rho, a member of the Ras superfamily of GTPases, regulates cytoskeletal rearrangement and transcriptional activation of the c-fos serum response element. Immunofluorescence staining of cardiomyocytes shows that Rho is present and predominantly cytosolic. We used two inhibitors of Rho function, dominant negative N19RhoA and Clostridium botulinum C3 transferase, to examine the possible requirement for Rho in alpha1-adrenergic receptor-mediated hypertrophy. Both inhibitors markedly attenuated atrial natriuretic factor (ANF) reporter gene expression induced by alpha1-adrenergic receptor stimulation with phenylephrine, and virtually abolished the increase in ANF reporter gene expression induced by GTPase-deficient Galphaq. These effects were reproduced with the myosin light chain-2 reporter gene. Notably, N19RhoA did not block the ability of activated Ras to induce ANF and myosin light chain-2 reporter gene expression. Furthermore, activation of the extracellular signal-regulated kinase by phenylephrine was not blocked by N19RhoA, nor was it stimulated by an activated mutant of RhoA. Since activated RhoA and Ras produce a large synergistic effect on ANF-luciferase gene expression, we conclude that Rho functions in a pathway separate from but complementary to Ras. Our results provide direct evidence that Rho is an effector of Galphaq signaling and suggest for the first time that a low molecular weight GTPase other than Ras is involved in regulating myocardial cell growth and gene expression in response to heterotrimeric G protein-linked receptor activation.
Rho family GTPases have been assigned important roles in the formation of actin-based morphologies in nonneuronal cells. Here we show that microinjection of Cdc42Hs and Rac1 promoted formation of filopodia and lamellipodia in N1E-115 neuroblastoma growth cones and along neurites. These actin-containing structures were also induced by injection of Clostridium botulinum C3 exoenzyme, which abolishes RhoA-mediated functions such as neurite retraction. The C3 response was inhibited by coinjection with the dominant negative mutant Cdc42Hs(T17N), while the Cdc42Hs response could be competed by coinjection with RhoA. We also demonstrate that the neurotransmitter acetylcholine (ACh) can induce filopodia and lamellipodia on neuroblastoma growth cones via muscarinic ACh receptor activation, but only when applied in a concentration gradient. ACh-induced formation of filopodia and lamellipodia was inhibited by preinjection with the dominant negative mutants Cdc42Hs(T17N) and Rac1(T17N), respectively. Lysophosphatidic acid (LPA)-induced neurite retraction, which is mediated by RhoA, was inhibited by ACh, while C3 exoenzyme-mediated neurite outgrowth was inhibited by injection with Cdc42Hs(T17N) or Rac1(T17N). Together these results suggest that there is competition between the ACh- and LPA-induced morphological pathways mediated by Cdc42Hs and/or Rac1 and by RhoA, leading to either neurite development or collapse.
The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the alpha 6 beta 1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid- induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.
Lysophosphatidic acid (LPA) utilizes a G-protein-coupled receptor to activate the small GTP-binding protein Rho and to induce
rapid remodeling of the actin cytoskeleton. We studied the signal transduction from LPA receptors to Rho activation. Analysis
of the G-protein-coupling pattern of LPA receptors by labeling activated G-proteins with [α-32P]GTP azidoanilide revealed interaction with proteins of the Gq, Gi, and G12 subfamilies. We could show that in COS-7 cells, expression of GTPase-deficient mutants of Gα12 and Gα13 triggered Rho activation as measured by increased Rho-GTP levels. In Swiss 3T3 cells, incubation with LPA or microinjection
of constitutively active mutants of Gα12 and Gα13 induced formation of actin stress fibers and assembly of focal adhesions in a Rho-dependent manner. Interestingly, the LPA-dependent
cytoskeletal reorganization was suppressed by microinjected antibodies directed against Gα13, whereas Gα12-specific antibodies showed no inhibition. The tyrosine kinase inhibitor tyrphostin A 25 and the epidermal growth factor (EGF)
receptor-specific tyrphostin AG 1478 completely blocked actin stress fiber formation caused by LPA or activated Gα13 but not the effects of activated Gα12. Also, expression of the dominant negative EGF receptor mutant EGFR-CD533 markedly prevented the LPA- and Gα13-induced actin polymerization. Coexpression of EGFR-CD533 and activated Gα13 in COS-7 cells resulted in decreased Rho-GTP levels compared with expression of activated Gα13 alone. These data indicate that in Swiss 3T3 cells, G13 but not G12 is involved in the LPA-induced activation of Rho. Moreover, our results suggest an involvement of the EGF receptor in this
pathway.
Three members of the Rho family, Cdc42, Rac, and Rho are known to regulate the organization of actin-based cytoskeletal structures. In Bac1.2F5 macrophages, we have shown that Rho regulates cell contraction, whereas Rac and Cdc42 regulate the formation of lamellipodia and filopodia, respectively. We have now tested the roles of Cdc42, Rac, and Rho in colony stimulating factor-1 (CSF-1)–induced macrophage migration and chemotaxis using the Dunn chemotaxis chamber. Microinjection of constitutively activated RhoA, Rac1, or Cdc42 inhibited cell migration, presumably because the cells were unable to polarize significantly in response to CSF-1. Both Rho and Rac were required for CSF-1–induced migration, since migration speed was reduced to background levels in cells injected with C3 transferase, an inhibitor of Rho, or with the dominant-negative Rac mutant, N17Rac1. In contrast, cells injected with the dominant-negative Cdc42 mutant, N17Cdc42, were able to migrate but did not polarize in the direction of the gradient, and chemotaxis towards CSF-1 was abolished.
We conclude that Rho and Rac are required for the process of cell migration, whereas Cdc42 is required for cells to respond to a gradient of CSF-1 but is not essential for cell locomotion.
A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho-ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.
Directional sensing by eukaryotic cells does not require polarization of chemoattractant receptors. The translocation of the PH domain-containing protein CRAC in D. discoideum to binding sites on the inner face of the plasma membrane reflects activation of the G protein-linked signaling system. Increments in chemoattractant elicit a uniform response around the cell periphery. Yet when cells are exposed to a gradient, the activation occurs selectively at the stimulated edge, even in immobilized cells. We propose that such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G protein-linked chemotactic systems including those involving chemotactic cytokines in leukocytes.
Chemotaxis-competent cells respond to a variety of ligands by activating second messenger pathways leading to changes in the actin/myosin cytoskeleton and directed cell movement. We demonstrate that Dictyostelium Akt/PKB, a homologue of mammalian Akt/PKB, is very rapidly and transiently activated by the chemoattractant cAMP. This activation takes place through G protein-coupled chemoattractant receptors via a pathway that requires homologues of mammalian p110 phosphoinositide-3 kinase. pkbA null cells exhibit aggregation-stage defects that include aberrant chemotaxis, a failure to polarize properly in a chemoattractant gradient and aggregation at low densities. Mechanistically, we demonstrate that the PH domain of Akt/PKB fused to GFP transiently translocates to the plasma membrane in response to cAMP with kinetics similar to those of Akt/PKB kinase activation and is localized to the leading edge of chemotaxing cells in vivo. Our results indicate Akt/PKB is part of the regulatory network required for sensing and responding to the chemoattractant gradient that mediates chemotaxis and aggregation.
A major function of Rac2 in neutrophils is the regulation of oxidant production important in bacterial killing. Rac and the
related GTPase Cdc42 also regulate the dynamics of the actin cytoskeleton, necessary for leukocyte chemotaxis and phagocytosis
of microorganisms. Although these GTPases appear to be critical downstream components of chemoattractant receptor signaling
in human neutrophils, the pathways involved in direct control of Rac/Cdc42 activation remain to be determined. We describe
an assay that measures the formation of Rac-GTP and Cdc42-GTP based on their specific binding to the p21-binding domain of
p21-activated kinase 1. A p21-binding domain glutathione S-transferase fusion protein specifically binds Rac and Cdc42 in their GTP-bound forms both in vitro and in cell samples. Binding is selective for Rac and Cdc42 versusRhoA. Using this assay, we investigated Rac and Cdc42 activation in neutrophils and differentiated HL-60 cells. The chemoattractant
fMet-Leu-Phe and the phorbol ester phorbol myristate acetate stimulate formation of Rac-GTP and Cdc42-GTP with distinct time
courses that parallel cell activation. We also show that the signaling pathways leading to Rac and Cdc42 activation in HL-60
cells involve G proteins sensitive to pertussis toxin, as well as tyrosine kinase and phosphatidylinositol 3-kinase activities.
We have identified a Dictyostelium discoideum gene encoding a serine/threonine kinase, PAKa, a putative member of the Ste20/PAK family of p21-activated kinases, with a kinase domain and a long NH(2)-terminal regulatory domain containing an acidic segment, a polyproline domain, and a CRIB domain. PAKa colocalizes with myosin II to the cleavage furrow of dividing cells and the posterior of polarized, chemotaxing cells via its NH(2)-terminal domain. paka null cells are defective in completing cytokinesis in suspension. PAKa is also required for maintaining the direction of cell movement, suppressing lateral pseudopod extension, and proper retraction of the posterior of chemotaxing cells. paka null cells are defective in myosin II assembly, as the myosin II cap in the posterior of chemotaxing cells and myosin II assembly into cytoskeleton upon cAMP stimulation are absent in these cells, while constitutively active PAKa leads to an upregulation of myosin II assembly. PAKa kinase activity against histone 2B is transiently stimulated and PAKa incorporates into the cytoskeleton with kinetics similar to those of myosin II assembly in response to chemoattractant signaling. However, PAKa does not phosphorylate myosin II. We suggest that PAKa is a major regulator of myosin II assembly, but does so by negatively regulating myosin II heavy chain kinase.
Morphologic polarity is necessary for chemotaxis of mammalian cells. As a probe of intracellular signals responsible for this
asymmetry, the pleckstrin homology domain of the AKT protein kinase (or protein kinase B), tagged with the green fluorescent
protein (PHAKT-GFP), was expressed in neutrophils. Upon exposure of cells to chemoattractant, PHAKT-GFP is recruited selectively
to membrane at the cell's leading edge, indicating an internal signaling gradient that is much steeper than that of the chemoattractant.
Translocation of PHAKT-GFP is inhibited by toxin-B from Clostridium difficile, indicating that it requires activity of one or more Rho guanosine triphosphatases.
Buffering of intracellular Ca2+ transients in human neutrophils leads to reduced motility due to defective uropod detachment on fibronectin and vitronectin-coated surfaces. Since one potential target of a rise in [Ca2+]i is the activation of myosin II, we characterized the role of myosin II during motility. Treatment of neutrophils with a myosin inhibitor (2,3-butanedione monoxime), or myosin light chain kinase inhibitors (ML-7, ML-9, or KT5926) resulted in impaired uropod retraction and a dose-dependent decrease in chemokinesis following stimulation with N-formyl-Met-Leu-Phe (fMLP). Treatment with ML-9 resulted in a redistribution of F-actin and talin to the non-retracted uropods, mimicking the redistribution observed during [Ca2+]i buffering. Impairment of uropod retraction and redistribution of F-actin and talin by myosin II inhibition was only observed on adhesive substrates such as fibronectin and not on poorly adhesive substrates such as human serum-coated glass. At higher concentrations of ML-9, cell polarization was inhibited and pseudopod extension occurred radially. Using an antibody specific for serine 19-phosphorylated regulatory light chain of myosin II, regions of activated myosin II were found at the leading edge as well as the uropod in motile fMLP-stimulated cells. [Ca2+]i depletion caused a 50% decrease in the level of serine 19-phosphorylated myosin II suggesting that activation of myosin II by intracellular Ca2+ transients may be an essential step in establishing a polarized pseudopod and providing the force required for uropod retraction during PMN motility on adhesive surfaces.
Chemotactic factors stimulate a rapid increase in the cytosolic concentration of intracellular calcium ions ([Ca2+]in) in human polymorphonuclear leukocytes (PMNL), which may be an event that is critical to the expression of chemotaxis and other PMNL functions. Treatment of PMNL with pertussis toxin catalyzes ADP-ribosylation of a protein similar or identical to the inhibiting regulatory protein of adenylate cyclase, Gi, and suppresses the increase in [Ca2+]in elicited by leukotriene B4(LTB4) and formyl-methionyl-leucyl-phenylalanine. Chemotactic migration and lysosomal enzyme release elicited by chemotactic factors were inhibited by pertussis toxin with a concentration-dependence similar to that for inhibition of the increase in [Ca2+]in, without an effect on lysosomal enzyme release induced by the ionophore A23187 and phorbol myristate acetate. Activated pertussis toxin catalyzed the [32P]ADP-ribosylation of a 41 kD protein in homogenates of PMNL. The extent of [32P]ADP-ribosylation of this protein was reduced 59% by pretreatment of intact PMNL with pertussis toxin. Pertussis toxin selectively decreased the number of high-affinity receptors for LTB4 on PMNL by 60% without altering the number or binding properties of the low-affinity subset of receptors. Pertussis toxin modification of a membrane protein of PMNL analogous to Gi thus simultaneously alters chemotactic receptors and attenuates the changes in cytosolic calcium concentration and PMNL function caused by chemotactic factors.
Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control
cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide–binding proteins (G proteins) participate in
several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein α subunits, Gα12 and Gα13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Gα13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Gα12 inhibited stimulation by Gα13. Thus, p115 RhoGEF can directly link heterotrimeric G protein α subunits to regulation of Rho.
Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity
of the α subunits of certain heterotrimeric guanine nucleotide–binding proteins (G proteins). The guanine nucleotide exchange
factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and
a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the α subunits
of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gqsubfamilies of Gα proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.
The paper addresses the formation of striking patterns within originally near-homogenous tissue, the process prototypical for embryology, and represented in particularly puristic form by cut sections of hydra regenerating a complete animal with head and foot. Essential requirements are autocatalytic, self-enhancing activation, combined with inhibitory or depletion effects of wider range - “lateral inhibition”. Not only de-novo-pattern formation, but also well known, striking features of developmental regulation such as induction, inhibition, and proportion regulation can be explained on this basis. The theory provides a mathematical recipe for the construction of molecular models with criteria for the necessary non-linear interactions. It has since been widely applied to different developmental processes.
Chemoattractants stimulate both cell locomotion and the orientation of this locomotion (chemotaxis) in polymorphonuclear leukocytes. Cell locomotion is a complex process which includes the coordinated protrusion of cell processes, formation of attachments to the substrate and contraction of the rear of the cell. To understand how chemoattractants regulate this process, it is helpful to dissect the process into components that can be examined separately. Comparison of these components in cells before and after stimulation with chemoattractant provides information about their regulation. In this review we focus on three components: how chemoattractants induce the development of cell polarity; how chemoattractants modulate cytoskeletal components (especially actin) to cause pseudopod protrusion; and how chemoattractant modulation of cell adhesions might contribute to cell locomotion. Spatial and temporal coordination of these and other components of locomotion result in efficient and directed cell movement. Our treatment of these questions is speculative and not comprehensive. We propose simple hypothetical models which can provide the reader with a conceptual framework that integrates the information available.
Treatment of rabbit neutrophils with pertussis toxin, but not cholera toxin, inhibits the increases produced by formylmethionyl-leucyl-phenylalanine, leukotriene B4 and the calcium ionophore A23187 in the amounts of actin associated with the cytoskeletons. The increase in the cytoskeletal actin produced by phorbol 12-myristate, 13-acetate on the other hand is not affected by pertussis toxin. Incubation of the neutrophils with cholera toxin, unlike pertussis toxin, did not inhibit the fMet-Leu-Phe induced rise in the intracellular concentration of free calcium, and caused only a shift to the right of the dose-response curve of N-acetyl-beta-glucosaminidase release. This shift was more marked in the presence of 1-methyl-3-isobutylxanthine. In addition, the stimulated breakdown of phosphatidylinositol 4,5 bis-phosphate was inhibited by pertussis toxin. These results suggest that pertussis toxin acts at an early step in the signal transduction and does not affect the sequence of reactions initiated by the activation of the protein kinase C. Furthermore, the guanine nucleotide regulatory protein Gi, but not Gs, is closely involved in signal transduction in these cells.
We used purified polyclonal antibodies to human cytoplasmic myosin-IIA and myosin-IIB directly labeled with fluorescent dyes to localize these myosin-II isozymes in HeLa cells, melanoma cells and blood cells. Both antibodies react strongly with myosin-II isozymes in HeLa cells, melanoma cells and blood eosinophils, but only anti-myosin-IIA antibodies stain platelets, lymphocytes, neutrophils and monocytes in smears of human blood. Both antibodies stain small spots along the stress fibers of interphase HeLa cells and melanoma cells, but double staining revealed that the detailed distributions of myosin-IIA and myosin-IIB differ. A low concentration of diffuse myosin-IIB is present in the cortex, both in lamellar regions around the periphery of the cell and over the free surface. Myosin-IIB is also concentrated in spots along perinuclear stress fibers. Myosin-IIA is absent from the cortex but is concentrated in spots along stress fibers located near the basal surface of cultured cells.
This population of peripheral stress fibers is highly enriched in myosin-IIA relative to myosin-IIB, but both are found together in centrally located stress fibers. In prophase and metaphase both isozymes are concentrated in the cortex in small spots less than 04.µm in size, similar to those in stress fibers. As the chromosomes begin the separate at anaphase, most of the myosin-II spots become concentrated in the outer 0.7 µm of the equatorial cortex in 100% of cells. This concentration of myosin-II isozymes in the cleavage furrow is maintained until the daughter cells separate. The superimposition of these small spots concentrated in the cleavage furrow produces the intense, uniform staining observed in conventional micrographs of whole cells.
Neutrophils contain a multicomponent NADPH oxidase system that is involved in the production of microbicidal oxidants. Stimulation of human neutrophils with the peptide FMLP activates this respiratory burst enzyme to produce superoxide and also has been shown to result in activation of phosphatidylinositol (Ptdlns) 3-kinase. Treatment of human neutrophils with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a potent and specific inhibitor of Ptdlns 3-kinase, resulted in complete inhibition of Ptdlns 3-kinase activity as well as in inhibition of superoxide production in FMLP-treated neutrophils in suspension; FMLP-stimulated oxidant production in adherent cells was also abolished. Treatment of human neutrophils with PMA resulted in production of superoxide without activation of Ptdlns 3-kinase; LY294002 did not block superoxide production in neutrophils exposed to PMA. In addition, LY294002 did not inhibit cellfree NADPH oxidase activation, CD11b-dependent adhesion, actin polymerization in response to FMLP, or FMLP-induced calcium flux. These results suggest that the signal transduction pathway of the FMLP-receptor involves activation of Ptdlns 3-kinase, which is required for subsequent superoxide production induced by the chemotactic peptide. Furthermore, Ptdlns 3-kinase may be located directly upstream of protein kinase C or other protein kinases, which in turn activate the NADPH oxidase system.
A new brain serine/threonine protein kinase may be a target for the p21ras-related proteins Cdc42 and Rac1. The kinase sequence is related to that of the yeast protein STE20, implicated in pheromone-response pathways. The kinase complexes specifically with activated (GTP-bound) p21, inhibiting p21 GTPase activity and leading to kinase autophosphorylation and activation. Autophosphorylated kinase has a decreased affinity for Cdc42/Rac, freeing the p21 for further stimulatory activities or downregulation by GTPase-activating proteins. This bimolecular interaction provides a model for studying p21 regulation of mammalian phosphorylation signalling pathways.
Motility is a complex process that depends on the coordination of many cellular functions, including the conversion of information from the environment into a series of coordinated responses that culminate in directed cell movement. Major advances have been made in the understanding of many functions involved in motility, such as transmembrane signaling events, leading to alterations in the actin cytoskeleton, and interactions between adhesion receptors and components of the cytoskeleton, providing a link between the extracellular and intracellular environments. Studies using yeast (Saccharomyces cerevisiae), slime molds (Dictyostelium discoideum) and nematodes (Caenorhabditis elegans) have advanced our understanding of the molecular biology of cytoskeletal proteins and have important implications for mammalian leukocyte motility.
Colchicine-induced stimulation of polymorphonuclear leukocyte (PMN) locomotion is an interesting model because extension of blebs at the front occurs at a rate (about 2.4 microns/s) which is far above that reported for growth of actin filaments. The following cytoskeletal changes were observed in colchicine-treated PMNs: (1) a small increase in cytoskeleton-associated actin was noted, as well as a somewhat more pronounced increase in cytoskeleton-associated alpha-actinin, as compared with untreated or DMSO-treated controls. There was, however, no measurable increase in F-actin as determined by NBD-phallacidin binding; (2) the values for the ratio (alpha-actinin/actin) are lower in PMNs treated with colchicine for 30 min, as compared with PMNs stimulated with fNLPNTL for 1 minute (non-polar ruffling cells) or 30 min (polarized locomoting cells); thus, this ratio may depend on the type of PMN motility; (3) in polarized PMNs F-actin was mainly located linearly all along the cell membrane; there was more intense staining at the front of the cells; (4) alpha-actinin appeared to colocalize with F-actin at the leading front, but not with F-actin at the tail of polarized cells; (5) myosin was preferentially found at the rear part of polarized cells but not or only to a small extent at the front. Our data indicate a close functional correlation between microtubules and microfilaments. We speculate that F-actin in combination with alpha-actinin promotes expansion of pseudopods, whereas myosin combined with F-actin promotes contraction.(ABSTRACT TRUNCATED AT 250 WORDS)
The lethal toxin (LT) from Clostridium sordellii belongs to the family of large clostridial cytotoxins causing morphological alterations in cultured cell lines accompanied by destruction of the actin cytoskeleton. C. sordellii LT exhibits 90% homology to Clostridium difficile toxin B, which has been recently identified as a monoglucosyltransferase (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). We report here that LT too is a glucosyltransferase, which uses UDP-glucose as cosubstrate to modify low molecular mass GTPases. LT selectively modifies Rac and Ras, whereas the substrate specificity of toxin B is confined to the Rho subfamily proteins Rho, Rac, and Cdc42, which participate in the regulation of the actin cytoskeleton. In Rac, both toxin B and LT share the same acceptor amino acid, threonine 35. Glucosylation of Ras by LT results in inhibition of the epidermal growth factor-stimulated p42/p44 MAP-kinase signal pathway. LT is the first bacterial toxin to inactivate Ras in intact cells.
Rho, Rac and Cdc42 are three Ras-related GTP-binding proteins that control the assembly and disassembly of the actin cytoskeleton in response to extracellular signals. During the past year, numerous candidate downstream targets for these GTPases have been identified using affinity chromatography and yeast two-hybrid techniques. These techniques have revealed that Rho regulates the myosin light chain phosphatase and that Rho and Rac control the synthesis of phosphatidylinositol 4,5-bisphosphate, two activities that might help to explain the effects of these GTPases on the actin cytoskeleton.
p160ROCK is a serine/threonine protein kinase that binds selectively to GTP-Rho and is activated by this binding. To identify its function, we transfected HeLa cells with wild type and mutants of p160ROCK and examined morphology of the transfected cells. Transfection with wild type and mutants containing the kinase domain and the coiled-coil forming region induced focal adhesions and stress fibers, while no induction was observed with a kinase-defective mutant or a mutant containing only the kinase domain. Furthermore, Rho-induced formation of focal adhesions and stress fibers was inhibited by co-expression of a mutant defective in both kinase and Rho-binding activities. Rho, however, still induced an increase in F-actin content in these cells. These results suggest that p160ROCK works downstream of Rho to induce formation of focal adhesions and that Rho-induced actin polymerization is mediated by other effector(s).
Abnormal smooth-muscle contractility may be a major cause of disease states such as hypertension, and a smooth-muscle relaxant that modulates this process would be useful therapeutically. Smooth-muscle contraction is regulated by the cytosolic Ca2+ concentration and by the Ca2+ sensitivity of myofilaments: the former activates myosin light-chain kinase and the latter is achieved partly by inhibition of myosin phosphatase. The small GTPase Rho and its target, Rho-associated kinase, participate in this latter mechanism in vitro, but their participation has not been demonstrated in intact muscles. Here we show that a pyridine derivative, Y-27632, selectively inhibits smooth-muscle contraction by inhibiting Ca2+ sensitization. We identified the Y-27632 target as a Rho-associated protein kinase, p160ROCK. Y-27632 consistently suppresses Rho-induced, p160ROCK-mediated formation of stress fibres in cultured cells and dramatically corrects hypertension in several hypertensive rat models. Our findings indicate that p160ROCK-mediated Ca2+ sensitization is involved in the pathophysiology of hypertension and suggest that compounds that inhibit this process might be useful therapeutically.
Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.
Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.
Directional cell motility implies the presence of a steering mechanism and a functional asymmetry between the front and rear of the cell. How this functional asymmetry arises and is maintained during cell locomotion is, however, unclear. Lamellar fragments of fish epidermal keratocytes, which lack nuclei, microtubules and most organelles, present a simplified, perhaps minimal, system for analyzing this problem because they consist of little other than the motile machinery enclosed by a membrane and yet can move with remarkable speed and persistence.
We have produced two types of cellular fragments: discoid stationary fragments and polarized fragments undergoing locomotion. The organization and dynamics of the actin-myosin II system were isotropic in stationary fragments and anisotropic in the moving fragments. To investigate whether the creation of asymmetry could result in locomotion, a transient mechanical stimulus was applied to stationary fragments. The stimulus induced localized contraction and the formation of an actin-myosin II bundle at one edge of the fragment. Remarkably, stimulated fragments started to undergo locomotion and the locomotion and associated anisotropic organization of the actin-myosin II system were sustained after withdrawal of the stimulus.
We propose a model in which lamellar cytoplasm is considered a dynamically bistable system capable of existing in a non-polarized or polarized state and interconvertible by mechanical stimulus. The model explains how the anisotropic organization of the lamellum is maintained in the process of locomotion. Polarized locomotion is sustained through a positive-feedback loop intrinsic to the actin-myosin II machinery: anisotropic organization of the machinery drives translocation, which then reinforces the asymmetry of the machinery, favoring further translocation.
Soluble factors from serum such as lysophosphatidic acid (LPA) are thought to activate the small GTP-binding protein Rho based on their ability to induce actin stress fibers and focal adhesions in a Rho-dependent manner. Cell adhesion to extracellular matrices (ECM) has also been proposed to activate Rho, but this point has been controversial due to the difficulty of distinguishing changes in Rho activity from the structural contributions of ECM to the formation of focal adhesions. To address these questions, we established an assay for GTP-bound cellular Rho. Plating Swiss 3T3 cells on fibronectin-coated dishes elicited a transient inhibition of Rho, followed by a phase of Rho activation. The activation phase was greatly enhanced by serum. In serum-starved adherent cells, LPA induced transient Rho activation, whereas in suspended cells Rho activation was sustained. Furthermore, suspended cells showed higher Rho activity than adherent cells in the presence of serum. These data indicate the existence of an adhesion-dependent negative-feedback loop. We also observed that both cytochalasin D and colchicine trigger Rho activation despite their opposite effects on stress fibers and focal adhesions. Our results show that ECM, cytoskeletal structures and soluble factors all contribute to regulation of Rho activity.
Substrate anchorage and cell locomotion entail the initiation and development of different classes of contact sites, which are associated with the different compartments of the actin cytoskeleton. The Rho-family GTPases are implicated in the signalling pathways that dictate contact initiation, maturation and turnover, but their individual roles in these processes remain to be defined.
We monitored the dynamics of peripheral, Rac-induced focal complexes in living cells in response to perturbations of Rac and Rho activity and myosin contractility. We show that focal complexes formed in response to Rac differentiated into focal contacts upon upregulation of Rho. Focal complexes were dissociated by inhibitors of myosin-II-dependent contractility but not by an inhibitor of Rho-kinase. The downregulation of Rac promoted the enlargement of focal contacts, whereas a block in the Rho pathway not only caused a dissolution of focal contacts but also stimulated membrane ruffling and formation of new focal complexes, which were associated with the advance of the cell front.
Rac functions to signal the creation of new substrate contacts at the cell front, which are associated with the induction of ruffling lamellipodia, whereas Rho serves in the maturation of existing contacts, with both contact types requiring contractility for their formation. The transition from a focal complex to a focal contact is associated with a switch to Rho-kinase dependence. Rac and Rho also influence the development of focal contacts and focal complexes, respectively, through mutually antagonistic pathways.
To explore the relative roles of protein-binding partners vs. lipid modifications in controlling membrane targeting of a typical peripheral membrane protein, Galpha(z), we directed its binding partner, betagamma, to mislocalize on mitochondria. Mislocalized betagamma directed wild-type Galpha(z) and a palmitate-lacking Galpha(z) mutant to mitochondria but did not alter localization of a Galpha(z) mutant lacking both myristate and palmitate. Thus, in this paradigm, a protein-protein interaction controls targeting of a peripheral membrane protein to the proper compartment, whereas lipid modifications stabilize interactions of proteins with membranes and with other proteins.
Activity of phosphatidylinositol (PI) 3-kinase is required for optimal migration of human neutrophils [Niggli and Keller (1999) Eur. J. Pharmacol. 335, 43-52]. We have tested the direct effect of a product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), on neutrophil migration. To this end, a membrane-permeant ester of PIP(3), dilauroyl phosphatidylinositol 3,4, 5-trisphosphate-heptakis-(acetooxymethyl)ester (PIP(3)/AM) was used. PIP(3)/AM (ED(50): 10-17 microM) induced development of polarity and accumulation of F-actin in the leading lamellae in up to 70% of the cells. These cells exhibited stimulated random migration, comparable to that observed in uniform concentrations of chemotactic peptide. Evidence is provided for a role of Rho-kinase and for activation of PI 3-kinase in a positive feedback loop in PIP(3)/AM-induced motility.