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LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from the Trans-Golgi Network
Abstract and Figures
The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here, we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in Madin-Darby canine kidney cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 small interfering RNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans-Golgi network (TGN) of the apical PM marker p75-green fluorescent protein (GFP) but did not interfere with the apical PM marker glycosyl phosphatidylinositol-YFP or the basolateral PM marker neural cell adhesion molecule-GFP. High-resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynamin-syndapin-cortactin-dependent generation and/or fission of precursors to p75 transporters.
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... In polarized epithelial cells (e.g., MDCK cells), LIMK1 and PKD1 are two additional elements of the membrane fission machinery acting at the TGN that regulate the exit of membrane proteins aimed to the apical or basolateral membrane, respectively [43,44]. For example, a LIMK1-actin-cortactin-dynamin-dependent fission machinery mediates exit from the GA of p75 NTR -containing carriers aimed to the apical surface [43]. ...
... In polarized epithelial cells (e.g., MDCK cells), LIMK1 and PKD1 are two additional elements of the membrane fission machinery acting at the TGN that regulate the exit of membrane proteins aimed to the apical or basolateral membrane, respectively [43,44]. For example, a LIMK1-actin-cortactin-dynamin-dependent fission machinery mediates exit from the GA of p75 NTR -containing carriers aimed to the apical surface [43]. LIMK1 and PKD are also expressed in neurons, localized to the GA, and engaged in the dynamin-mediated fission of Golgi-derived tubules and the generation of Golgi outposts [10,13,14]. ...
Neurons are highly polarized cells requiring precise regulation of trafficking and targeting of membrane proteins to generate and maintain different and specialized compartments, such as axons and dendrites. Disruption of the Golgi apparatus (GA) secretory pathway in developing neurons alters axon/dendritic formation. Therefore, detailed knowledge of the mechanisms underlying vesicles exiting from the GA is crucial for understanding neuronal polarity. In this study, we analyzed the role of Brefeldin A-Ribosylated Substrate (CtBP1-S/BARS), a member of the C-terminal-binding protein family, in the regulation of neuronal morphological polarization and the exit of membrane proteins from the Trans Golgi Network. Here, we show that BARS is expressed during neuronal development in vitro and that RNAi suppression of BARS inhibits axonal and dendritic elongation in hippocampal neuronal cultures as well as largely perturbed neuronal migration and multipolar-to-bipolar transition during cortical development in situ. In addition, using plasma membrane (PM) proteins fused to GFP and engineered with reversible aggregation domains, we observed that expression of fission dominant-negative BARS delays the exit of dendritic and axonal membrane protein-containing carriers from the GA. Taken together, these data provide the first set of evidence suggesting a role for BARS in neuronal development by regulating post-Golgi membrane trafficking.
... Transport along these routes is directed by apical signals such as N-glycans (Scheiffele et al., 1995), O-glycans (Yeaman et al., 1997) and GPI anchors (Lisanti et al., 1989;Powell et al., 1991), and basolateral signals that resemble tyrosine and di-leucine endocytic motifs in structure (Lipardi et al., 2002). A variety of molecules have been postulated to mediate these trafficking processes, such as clathrin (Deborde et al., 2008), the clathrin adaptor AP-1A and AP-1B complexes (Cancino et al., 2007;Fölsch et al., 1999;Gan et al., 2002;Gonzalez and Rodriguez-Boulan, 2009;Gravotta et al., 2007;, microtubule motors (Jaulin et al., 2007;Perez Bay et al., 2013;Weisz and Rodriguez-Boulan, 2009) and regulators of the actin cytoskeleton Gupta et al., 2016;Salvarezza et al., 2009). ...
The homologous P-type copper-ATPases (Cu-ATPases) ATP7A and ATP7B are the key regulators of copper homeostasis in mammalian cells. In polarized epithelia, upon copper treatment, ATP7A and ATP7B traffic from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively. We characterized the sorting pathways of Cu-ATPases between TGN and the plasma membrane and identified the machinery involved. ATP7A and ATP7B reside on distinct domains of TGN in limiting copper conditions, and in high copper, ATP7A traffics to basolateral membrane, whereas ATP7B traverses common recycling, apical sorting and apical recycling endosomes en route to apical membrane. Mass spectrometry identified regulatory partners of ATP7A and ATP7B that include the adaptor protein-1 complex. Upon knocking out pan-AP-1, sorting of both Cu-ATPases is disrupted. ATP7A loses its trafficking polarity and localizes on both apical and basolateral surfaces in high copper. By contrast, ATP7B loses TGN retention but retained its trafficking polarity to the apical domain, which became copper independent. Using isoform-specific knockouts, we found that the AP-1A complex provides directionality and TGN retention for both Cu-ATPases, whereas the AP-1B complex governs copper-independent trafficking of ATP7B solely. Trafficking phenotypes of Wilson disease-causing ATP7B mutants that disrupts putative ATP7B–AP1 interaction further substantiates the role of AP-1 in apical sorting of ATP7B.
... Alternatively, GOPs could emerge post somatic Golgi fragmentation due to heightened neuronal activity, with remnants of the degraded Golgi potentially serving as blueprints for the subsequent assembly of satellite Golgi arrays [34]. Formation of GOPs might also ensue from the primary somatic GA, prompting inquiries into the participation of Golgi fission machinery components such as LIMK1 [35] and Protein Kinase D1 (PKD1) [36], and the potential involvement of their proximal regulators and distal effectors. Notably, evidence underscores the potential influence of a RhoA-Rho kinase (Rock) signaling cascade in the orchestration of polarized GOPs during neuronal morphogenesis. ...
This article critically evaluates the multifunctional role of the Golgi apparatus within neurological paradigms. We succinctly highlight its influence on neuronal plasticity, development, and the vital trafficking and sorting mechanisms for proteins and lipids. The discourse further navigates to its regulatory prominence in neurogenesis and its implications in Alzheimer's Disease pathogenesis. The emerging nexus between the Golgi apparatus and SARS-CoV-2 underscores its potential in viral replication processes. This consolidation accentuates the Golgi apparatus's centrality in neurobiology and its intersections with both neurodegenerative and viral pathologies. In essence, understanding the Golgi's multifaceted functions harbors profound implications for future therapeutic innovations in neurological and viral afflictions.
... The overexpression of a kinase dead LIMK1 mutant, a constitutively activated cofilin, or the use of LIMK1 siRNA, selectively slowed down this exit from the TGN. These authors also showed that in p75 carrier vesicles, LIMK1 cooperates with dynamin 2, and cortactin and syndapin, two dynamin-interacting proteins for the fission processes of the vesicle from the TGN [91]. To our knowledge, the role of LIMK1 in membrane trafficking was only documented by these two papers. ...
LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the only two members of the LIM kinase family. They play a crucial role in the regulation of cytoskeleton dynamics by controlling actin filaments and microtubule turnover, especially through the phosphorylation of cofilin, an actin depolymerising factor. Thus, they are involved in many biological processes, such as cell cycle, cell migration, and neuronal differentiation. Consequently, they are also part of numerous pathological mechanisms, especially in cancer, where their involvement has been reported for a few years and has led to the development of a wide range of inhibitors. LIMK1 and LIMK2 are known to be part of the Rho family GTPase signal transduction pathways, but many more partners have been discovered over the decades, and both LIMKs are suspected to be part of an extended and various range of regulation pathways. In this review, we propose to consider the different molecular mechanisms involving LIM kinases and their associated signalling pathways, and to offer a better understanding of their variety of actions within the physiology and physiopathology of the cell.
... Cortactin may in uence mucin secretion in the following ways. First, cortactin is involved in the processes of membrane remodeling; it strengthens the stability of F-actin and modulates lament organization [49,50] , and it can reduce the depolymerization of F-actin by promoting and stabilizing the branched chained F-actin networks [51] . Second, cortactin contributes to the Ca 2+ -dependent formation of F-actin; it regulates exocytotic events depending on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases but independent of actin polymerization [32] . ...
Purpose
Mucus secretion is excessively increased in airway epithelial cells in pathological states. This process is related to the cytoskeleton and the increase in exocytosis sites, but the movement of secreted molecules and how secretion increases remain unclear. In this study, we examined the potential role of myristoylated alanine rich C kinase substrate (MARCKS) and the cortical actin-binding protein cortactin in airway mucin secretion. Also we investigated the effect of activated Cdc42-associated kinase 1 (ACK1) in this process.
Methods
Human airway epithelial cells were treated with neutrophil elastase (NE) after treatment with siRNA to specifically knock down MARCKS, ACK1 and cortactin expression. The expression and localization of cortactin and MARCKS were observed by western blotting and immunofluorescence, and the phosphorylated forms of MARCKS, cortactin and ACK1 were detected. The interaction of cortactin and ACK1 was analyzed by coimmunoprecipitation. MUC5AC protein expression was measured by ELISAs.
Results
Phosphorylated cortactin was highly expressed, mainly at the cell membrane, after NE stimulation, and phosphorylated MARCKS was mainly expressed in the cytoplasm. Coimmunoprecipitation revealed that ACK1 and cortactin interacted with each other. Knockdown of MARCKS suppressed phosphorylation of cortactin, while cortactin siRNA had no significant effect on MARCKS activation. Knockdown of MARCKS, cortactin and ACK1 by siRNA attenuated the phosphorylation of cortactin and reduced MUC5AC secretion.
Conclusion
These results suggest that both cortactin and MARCKS are involved in MUC5AC secretion by increasing F-actin polymerization and translocation and that MARCKs and ACK1 play an important role in the activation of cortactin.
... The mechanisms by which PMVs are released involve LIMK, which is phosphorylating cofilin, an enzyme whose function is to cleave filamentous actin molecules, allowing for the assembly and accumulation of actin filaments necessary for the budding of MVs from cell surfaces [199]. ARF6 is another small GTP-binding protein and member of the RAS superfamily whose function is to mediate cytoskeletal remodeling and intracellular vesicle trafficking to the plasma membrane. ...
In addition to being biological barriers where the internalization or release of biomolecules is decided, cell membranes are contact structures between the interior and exterior of the cell. Here, the processes of cell signaling mediated by receptors, ions, hormones, cytokines, enzymes, growth factors, extracellular matrix (ECM), and vesicles begin. They triggering several responses from the cell membrane that include rearranging its components according to the immediate needs of the cell, for example, in the membrane of platelets, the formation of filopodia and lamellipodia as a tissue repair response. In cancer, the cancer cells must adapt to the new tumor microenvironment (TME) and acquire capacities in the cell membrane to transform their shape, such as in the case of epithelial−mesenchymal transition (EMT) in the metastatic process. The cancer cells must also attract allies in this challenging process, such as platelets, fibroblasts associated with cancer (CAF), stromal cells, adipocytes, and the extracellular matrix itself, which limits tumor growth. The platelets are enucleated cells with fairly interesting growth factors, proangiogenic factors, cytokines, mRNA, and proteins, which support the development of a tumor microenvironment and support the metastatic process. This review will discuss the different actions that platelet membranes and cancer cell membranes carry out during their relationship in the tumor microenvironment and metastasis.
According to modern ideas, the basis of intellectual problems in neurological brain damage is active forgetting, regulated by Rac and Rho small GTPases-dependent signal stages of actin remodeling. The key enzyme of these cascades is LIM kinase 1 (LIMK1). Changes in limk1 gene expression lead to neurocognitive pathologies. Rapid screening and testing of targeted therapeutic agents modifying protein-protein interactions of GTPases and components of signaling cascades requires the development and validation of simple animal models. Such an opportunity is provided by Drosophila, the mutant strains of which allow you to identify the nodal moments of intersection of biochemical and neural networks, accompanying active forgetting.
Ubiquitination of substrates usually have two fates: one is degraded by 26S proteasome, and the other is non-degradative ubiquitination modification which is associated with cell cycle regulation, chromosome inactivation, protein transportation, tumorigenesis, achondroplasia, and neurological diseases. Cullin3 (CUL3), a scaffold protein, binding with the Bric-a-Brac-Tramtrack-Broad-complex (BTB) domain of substrates recognition adaptor and RING-finger protein 1 (RBX1) form ubiquitin ligases (E3). Based on the current researches, this review has summarized the functions and effects of CUL3-E3 ligases mediated non-degradative ubiquitination.
Liver fibrosis, a condition characterized by extensive deposition and cross-linking of extracellular matrix (ECM) proteins, is idiosyncratic in cases of chronic liver injury. The dysregulation of ECM remodeling by hepatic stellate cells (HSCs), the main mediators of fibrosis, results in an elevated ECM stiffness that drives the development of chronic liver disease such as cirrhosis and hepatocellular carcinoma. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a key element in the regulation of ECM remodeling, which modulates the degradation and turnover of ECM components. We have previously reported that a rigid, fibrotic-like substrate can impact TIMP-1 expression at the protein level in HSCs without altering its mRNA expression. While HSCs are known to be highly susceptible to mechanical stimuli, the mechanisms through which mechanical cues regulate TIMP-1 at the post-translational level remain unclear. Here, we show a mechanism of regulation of plasma membrane tension by matrix stiffness. We found that this effect is orchestrated by the β1 integrin/RhoA axis and results in elevated exocytosis and secretion of TIMP-1 in a caveolin-1- and dynamin-2-dependent manner. We then show that TIMP-1 and caveolin-1 expression increases in cirrhosis and hepatocellular carcinoma. These conditions are associated with fibrosis, and this effect can be recapitulated in 3D fibrosis models consisting of hepatic stellate cells encapsulated in a self-assembling polypeptide hydrogel. This work positions stiffness-dependent membrane tension as a key regulator of enzyme secretion and function and a potential target for therapeutic strategies that aim at modulating ECM remodeling in chronic liver disease.
The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization.
We have identified and characterized a COOH-terminal motor domain-type kinesin superfamily protein (KIFC), KIFC3, in the kidney. KIFC3 is a minus end-directed microtubule motor protein, therefore it accumulates in regions where minus ends of microtubules assemble. In polarized epithelial cells, KIFC3 is localized on membrane organelles immediately beneath the apical plasma membrane of renal tubular epithelial cells in vivo and polarized MDCK II cells in vitro. Flotation assay, coupled with detergent extraction, demonstrated that KIFC3 is associated with Triton X-100-insoluble membrane organelles, and that it overlaps with apically transported TGN-derived vesicles. This was confirmed by immunoprecipitation and by GST pulldown experiments showing the specific colocalization of KIFC3 and annexin XIIIb, a previously characterized membrane protein for apically transported vesicles (Lafont, F., S. Lecat, P. Verkade, and K. Simons. 1998. J. Cell Biol. 142:1413-1427). Furthermore, we proved that the apical transport of both influenza hemagglutinin and annexin XIIIb was partially inhibited or accelerated by overexpression of motor-domainless (dominant negative) or full-length KIFC3, respectively. Absence of cytoplasmic dynein on these annexin XIIIb-associated vesicles and distinct distribution of the two motors on the EM level verified the existence of KIFC3-transport in epithelial cells.
The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization.
Syndapin I (SdpI) interacts with proteins involved in endocytosis and actin dynamics and was therefore proposed to be a molecular link between the machineries for synaptic vesicle recycling and cytoskeletal organization. We here report the identification and characterization of SdpII, a ubiquitously expressed isoform of the brain-specific SdpI. Certain splice variants of rat SdpII in other species were named FAP52 and PACSIN 2. SdpII binds dynamin I, synaptojanin, synapsin I, and the neural Wiskott-Aldrich syndrome protein (N-WASP), a stimulator of Arp2/3 induced actin filament nucleation. In neuroendocrine cells, SdpII colocalizes with dynamin, consistent with a role for syndapin in dynamin-mediated endocytic processes. The src homology 3 (SH3) domain of SdpI and -II inhibited receptor-mediated internalization of transferrin, demonstrating syndapin involvement in endocytosis in vivo. Overexpression of full-length syndapins, but not the NH2-terminal part or the SH3 domains alone, had a strong effect on cortical actin organization and induced filopodia. This syndapin overexpression phenotype appears to be mediated by the Arp2/3 complex at the cell periphery because it was completely suppressed by coexpression of a cytosolic COOH-terminal fragment of N-WASP. Consistent with a role in actin dynamics, syndapins localized to sites of high actin turnover, such as filopodia tips and lamellipodia. Our results strongly suggest that syndapins link endocytosis and actin dynamics.
The GTPases Rac and Cdc42Hs control diverse cellular functions. In addition to being mediators of intracellular signaling cascades, they have important roles in cell morphogenesis and mitogenesis. We have identified a novel PAK-related kinase, PAK4, as a new effector molecule for Cdc42Hs. PAK4 interacts only with the activated form of Cdc42Hs through its GTPase-binding domain (GBD). Co-expression of PAK4 and the constitutively active Cdc42HsV12 causes the redistribution of PAK4 to the brefeldin A-sensitive compartment of the Golgi membrane and the subsequent induction of filopodia and actin polymerization. Importantly, the reorganization of the actin cytoskeleton is dependent on PAK4 kinase activity and on its interaction with Cdc42Hs. Thus, unlike other members of the PAK family, PAK4 provides a novel link between Cdc42Hs and the actin cytoskeleton. The cellular locations of PAK4 and Cdc42Hs suggest a role for the Golgi in cell morphogenesis.
Cytoplasmic actin distributes between monomeric and filamentous phases in cells. As cells crawl, actin polymerizes near the plasma membrane of expanding peripheral cytoplasm and depolymerizes elsewhere. Thus, the finite actin filament lifetime, the diffusivity of actin monomer, and the distribution of actin between the polymer and monomer phases are key parameters in cell motility. The dynamics of cellular actin can be determined by following the evolution of fluorescence in the techniques of photoactivated fluorescence (PAF) or fluorescence recovery after photobleaching (FRAP) of microinjected actin derivatives. A mathematical model is discussed that measures monomer diffusion coefficients, filament turnover rates, and the fraction of actin polymerized from measurements of the evolution of fluorescence from a photoactivated band [Tardy et al. (1995) Biophys. J., 69:1674–1682; McGrath et al. (1998) Biophys. J., in press]. Applying this model to subconfluent endothelial cells shows that ∼40% of the actin is polymer and that these filaments turn over on average every 6 minutes. This report dicusses how PAF and FRAP can be combined with more traditional biochemistry to probe actin cytoskeleton remodeling in endothelial cells. Microsc. Res. Tech. 43:385–394 © 1998 Wiley-Liss, Inc.
Ubiquitous among eukaryotes, the ADF/cofilins are essential proteins responsible for the high turnover rates of actin filaments in vivo. In vertebrates, ADF and cofilin are products of different genes. Both bind to F-actin cooperatively and induce a twist in the actin filament that results in the loss of the phalloidin-binding site. This conformational change may be responsible for the enhancement of the off rate of subunits at the minus end of ADF/cofilin-decorated filaments and for the weak filament-severing activity. Binding of ADF/cofilin is competitive with tropomyosin. Other regulatory mechanisms in animal cells include binding of phosphoinositides, phosphorylation by LIM kinases on a single serine, and changes in pH. Although vertebrate ADF/cofilins contain a nuclear localization sequence, they are usually concentrated in regions containing dynamic actin pools, such as the leading edge of migrating cells and neuronal growth cones. ADF/cofilins are essential for cytokinesis, phagocytosis, fluid phase endocytosis, and other cellular processes dependent upon actin dynamics.