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Expression analysis and cloning of Cdc42/Rac family members and the evaluation of the efficacy of siRNAs. (A) RT-PCR analysis performed on MEF lysates demonstrating the relative expression levels of different Cdc42 and Rac family members in these cells. Plus and minus columns denote the inclusion and omission of reverse transcriptase during the single-stranded cDNA synthesis reaction. (B) Western blotting analysis demonstrating the efficiency of siRNA-mediated knockdown of endogenous Cdc42 (left), Rac1 (middle), and RhoG (right).
Source publication
Cdc42 and Rac family GTPases are important regulators of morphology, motility, and polarity in a variety of mammalian cell types. However, comprehensive analysis of their roles in the morphological and behavioral aspects of chemotaxis within a single experimental system is still lacking. Here we demonstrate using a direct viewing chemotaxis assay t...
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Context 1
... of individual Cdc42 and Rac family members in the PDGF-dependent migration of MEFs, siRNAs were used to specifically inhibit the expression of these proteins. Initially, RT-PCR analysis was performed using total cytoplasmic RNA isolated from MEF cultures in order to determine which Cdc42 and Rac family members were expressed in these cells (Fig. 4A). mRNA encoding Cdc42, Rac1, and RhoG could be readily amplified from MEF lysates and appeared to be abundant in these cells. Tc10, Tcl, Wrch1, and Rac3 mRNA could also be amplified, although less effi- ciently than Cdc42, Rac1, and RhoG mRNA, suggesting that these GTPases are less abundantly expressed in these cells. Chp and Rac2 mRNA ...
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... sequence all resulted in the loss of the same band of 20 kDa when transfected into MEFs (see Fig. S3 in the supplemental material), demonstrating that this band rep- resented RhoG. Western blotting analysis revealed that endog- enous levels of Cdc42, Rac1, and RhoG were almost undetect- able 48 h after transfection with their respective siRNAs (Fig. ...
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... after microinjection of recombinant C3 into both L-cell fibroblasts (32) and primary rat embryonic fibroblasts (13). Although C3 microinjection did have a profound effect on cell morphology, in the majority of cells observed this treatment had no signif- icant effect on the speed or the directional response of cells to the PDGF gradient (see Fig. S4B and C in the supplemental material). Although in a few cases microinjected cells retracted completely and detached from the substrate, a phenotype pre- viously observed after microinjection of high concentrations of C3 into fibroblasts, the majority of cells were motile and ex- hibited efficient chemotaxis despite their severely ...
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... contrast to Rac1, however, the basal level of RhoA ac- tivity was reduced in Cdc42 knockdown cells (Fig. 9B). This latter finding is intriguing, especially since similarities can be seen between the Cdc42 knockdown phenotype (Fig. 5A) and that of cells microinjected with C3 (see Fig. S4A in the sup- plemental material). We also note that stress fibers were often less abundant in those cells where morphology had been se- verely affected by Cdc42 knockdown (Fig. 6C and D). It is therefore tempting to postulate that the retracted cell pheno- type frequently observed after knockdown of Cdc42 could, in part, be due to ...
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Citations
... As we have shown above that Col/Fib presents a higher stiffness compared to collagen, which might directly activate PSCs, we were interested to analyze the activating effects of the fibrinogen component on PSCs in greater detail. For this, we treated PSCs cultured in a 2D monolayer with either fibrinogen or thrombin, the two components added to the commonly shared collagen matrix, and analyzed the expression of common PSC activation markers (alpha smooth muscle actin (ACTA2) and platelet-derived growth factor receptor (PDGFRβ) [18,35,36]), markers for the production of ECM proteins directly involved in the potential compression of blood vessels in PDAC (Collagen type 1 alpha 1 (COL1α1) and hyaluronic acid synthase 2 (HAS2) [1,4,37]) and a marker related to cell-ECM attachment (cell division cycle 42 (CDC42) [38,39]) (Fig. 3B). We found that fibrinogen was mainly inducing the expression of ACTA2, indicating PSC activation, and HAS2, while thrombin induced HAS2 and CDC42, displaying that both components play a crucial role in our model. ...
... In particular ACTA2 displayed a 1.3 times upregulation, indicating the activation of PSCs. Furthermore, HAS2, a gene involved in the production of hyaluronic acid, which is a protein that forms a crucial part in vessel compression in PDAC due to its capability to swell when in aqueous solution [9], and CDC42, a gene directly related to cell-ECM attachment and related migration/contraction [38,39], displayed a 2 and 1.9 times upregulation, respectively, which might be crucial for the potential contraction of blood vessel in our model. While the upregulation of ACTA2, HAS2 and CDC42 might be related to pure presence of fibrinogen and thrombin in this culture, COL1α1 and PDGFRβ might be induced by the increased mechanical stiffness of the material. ...
Fibrotic tumors, such as pancreatic ductal adenocarcinoma (PDAC), are characterized for high desmoplastic reaction, which results in high intra-tumoral solid stress leading to the compression of blood vessels. These microarchitectural alterations cause loss of blood flow and poor intra-tumoral delivery of therapeutics. Currently, there is a lack of relevant in vitro models capable of replicating these mechanical characteristics and to test anti-desmoplastic compounds. Here, a multi-layered vascularized 3D PDAC model consisting of primary human pancreatic stellate cells (PSCs) embedded in collagen/fibrinogen (Col/Fib), mimicking tumor tissue within adjunct healthy tissue, is presented to study the fibrosis-induced compression of vasculature in PDAC. It is demonstrated how the mechanical and biological stimulation induce PSC activation, extracellular matrix production and eventually vessel compression. The clinical relevance is confirmed by correlating with patient transcriptomic data. Furthermore, the effects of gradual vessel compression on the fluid dynamics occurring within the channel is evaluated in silico. Finally, it is demonstrated how cancer-associated fibroblast (CAF)-modulatory therapeutics can inhibit the cell-mediated compression of blood vessels in PDAC in vitro, in silico and in vivo. It is envisioned that this 3D model is used to improve the understanding of mechanical characteristics in tumors and for evaluating novel anti-desmoplastic therapeutics.
... Actomyosin-dependent contractility generates the tension necessary for the retraction of the cell´s rear-end, the maintenance of membrane tension and cell shape (76). Finally, emphasizing the key importance of actin organization and its molecular regulators, 2D motility is inhibited upon disruption of F-actin organization with pharmacological inhibitors (100, 101) or upon perturbation of some of molecular regulators of actin, including Rac1 and Rac2 (71,102), dedicator of cytokinesis (DOCK2), a GEF for Rac (103,104), Cdc42 (71), RhoA (65), GEF-H1, a GEF for RhoA/Rac (105), ROCK (51, 101), MyoII (68, 101), mDia (106), WASP (107,108), the Arp2/3 complex (88), cofilin (105), Slinshot (105), and profilin (105). Interestingly, in cells that migrate in 3D environments, inhibition of some of aforementioned actin regulators, including RhoA (109), ROCK (80,100,109,110), MyoII (80,110), mDia1 (111), WAVE (112), also lead to impairment of the motility. ...
... Rac, Cdc42, RhoG, Transient depletion of any of these three small GTPases, which are regulators of the actin cytoskeleton, in mouse embryo fibroblasts (MEFs), resulted in a reduction of the migratory speed of these cells; but not their ability to detect the direction of the chemoattractant platelet-derived growth factor (PDGF) (receptor PDGF-R) (71). Therefore, in MEFs the small GTPases Rac, Cdc42, Rhogovern motility, but not PDGF-Rcontrolled directional sensing. ...
... We analyzed whether a set of signaling molecules that relay signals from both types of receptors (see Cdc42, WASP, Myo II, and Rac1 in Tables 3, 4), could affect GPCR-and RTK-mediated directional sensing and motility. Only Cdc42, which regulates motility, but not directional sensing, exerts a similar effect downstream of both types of receptors (71,142). However, Rac1, WASP and Myo II, play opposite roles downstream of GPCRs and RTKs. ...
Chemoattraction, defined as the migration of a cell toward a source of a chemical gradient, is controlled by chemoattractant receptors. Chemoattraction involves two basic activities, namely, directional sensing, a molecular mechanism that detects the direction of a source of chemoattractant, and actin-based motility, which allows the migration of a cell towards it. Current models assume first, that chemoattractant receptors govern both directional sensing and motility (most commonly inducing an increase in the migratory speed of the cells, i.e. chemokinesis), and, second, that the signaling pathways controlling both activities are intertwined. We performed a meta-analysis to reassess these two points. From this study emerge two main findings. First, although many chemoattractant receptors govern directional sensing, there are also receptors that do not regulate cell motility, suggesting that is the ability to control directional sensing, not motility, that best defines a chemoattractant receptor. Second, multiple experimental data suggest that receptor-controlled directional sensing and motility can be controlled independently. We hypothesize that this independence may be based on the existence of separated signalling modules that selectively govern directional sensing and motility in chemotactic cells. Together, the information gathered can be useful to update current models representing the signalling from chemoattractant receptors. The new models may facilitate the development of strategies for a more effective pharmacological modulation of chemoattractant receptor-controlled chemoattraction in health and disease.
... However, there was a strong trend for the reduced proliferation of CAFs in the pl-PDGFB KO mice and it is possible that such a difference could lead to a reduced number of CAFs over time. Considering the potent chemotactic effect PDGFB exerts on fibroblasts in vitro [31][32][33], it is also fully possible that the reduced amount of CAFs is caused by impaired recruitment. Moreover, we could not detect any compensatory upregulation of other PDGF isoforms in the TME of pl-PDGFB KO mice. ...
Simple Summary
The aim of this study was to investigate the relative contribution of PDGFB derived specifically from platelets to the remodeling of the extracellular matrix (ECM) in tumors. Platelets are a major source of growth factors, which are released from the platelet granules upon activation. Platelets are continuously activated in the tumor microenvironment, due to their similarities to a wound. However, the role of platelet-derived factors in ECM remodeling has not been fully addressed. To this end, we made use of a mouse model with conditional deletion of PDGFB in platelets, which was crossbred to the RIP1-Tag2 model for pancreatic neuroendocrine carcinoma. The amount of tumor-associated PDGFB protein showed a 10-fold reduction in mice lacking PDGFB in platelets. Moreover, ECM deposition, the amount of cancer-associated fibroblasts and TGFβ signaling were all reduced in tumors from mice with platelet-specific deletion of PDGFB, demonstrating the significant contribution of a platelet-derived factor to ECM remodeling in tumors.
Abstract
Platelets constitute a major reservoir of platelet-derived growth factor B (PDGFB) and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. To address the specific role of platelet-derived PDGFB in the tumor microenvironment, we have created a mouse model with conditional knockout of PDGFB in platelets (pl-PDGFB KO). Lack of PDGFB in platelets resulted in 10-fold lower PDGFB concentration in the tumor microenvironment, fewer cancer-associated fibroblasts and reduced deposition of the extracellular matrix (ECM) molecules fibronectin and collagen I in the orthotopic RIP1-Tag2 model for pancreatic neuroendocrine cancer. Myosin light chain phosphorylation, promoting cell contraction and, consequently, the mechano-induced release of active transforming growth factor (TGF) β from extracellular compartments, was reduced in tumors from pl-PDGFB KO mice. In agreement, TGFβ signaling, measured as phosphorylated Smad2, was significantly hampered in tumors from mice lacking PDGFB in their platelets, providing a plausible explanation for the reduced deposition of extracellular matrix. These findings indicate a major contribution of platelet-derived PDGFB to a malignant transformation of the tumor microenvironment and address for the first time the role of PDGFB released specifically from platelets in the remodeling of the ECM in tumors.
... Flowvers are paper pumps that can drive steady, tunable flows and mitigate bubbles in microfluidic devices, including gradient generators During live imaging of fibroblast chemotaxis, chemoattractant gradients should persist for at least 6 h to allow appreciable translocation and changes in the direction of the slow-moving cells. 42,[45][46][47] Therefore, gradient stability is paramount. In microfluidic gradient generators, two primary causes of gradient instability are pressure imbalances and the introduction of bubbles. ...
Microfluidics approaches have gained popularity in the field of directed cell migration, enabling control of the extracellular environment and integration with live-cell microscopy; however, technical hurdles remain. Among the challenges are the stability and predictability of the environment, which are especially critical for the observation of fibroblasts and other slow-moving cells. Such experiments require several hours and are typically plagued by the introduction of bubbles and other disturbances that naturally arise in standard microfluidics protocols. Here, we report on the development of a passive pumping strategy, driven by the high capillary pressure and evaporative capacity of paper, and its application to study fibroblast chemotaxis. The paper pumps—flowvers (flow + clover)—are inexpensive, compact, and scalable, and they allow nearly bubble-free operation, with a predictable volumetric flow rate on the order of μl/min, for several hours. To demonstrate the utility of this approach, we combined the flowver pumping strategy with a Y-junction microfluidic device to generate a chemoattractant gradient landscape that is both stable (6+ h) and predictable (by finite-element modeling calculations). Integrated with fluorescence microscopy, we were able to recapitulate previous, live-cell imaging studies of fibroblast chemotaxis to platelet derived growth factor (PDGF), with an order-of-magnitude gain in throughput. The increased throughput of single-cell analysis allowed us to more precisely define PDGF gradient conditions conducive for chemotaxis; we were also able to interpret how the orientation of signaling through the phosphoinositide 3-kinase pathway affects the cells’ sensing of and response to conducive gradients.
... After molecules have been transferred to the outer well, it diffuses radially to the inner well, while cells remain positioned over the annular bridge (because it is 20 m below the chamber surface). 158 Since the first report in 1991, it has become a popular tool for guidance studies, architectures of guidance cues. 168,169 Several different kinds of micro-architectures can be formed to achieve either continuous or gradient profiles of guidance molecules. ...
A deeper understanding of the brain and its function remains one of the most significant scientific challenges. It is not only required to find cures for a plethora of brain-related diseases and injuries, but it can also open up possibilities of achieving technological wonders such as brain-machine-interface and highly energy-efficient computing devices. Central to the brain's function is its basic functioning unit, i.e., the neuron. There has been a tremendous effort to understand the underlying mechanisms of neuronal growth on both biochemical and biophysical levels. In the past decade, this increased understanding has led to the possibility of controlling and modulating neuronal growth in vitro through external chemical and physical methods. In this review, we provide a detailed overview of the most fundamental aspects of neuronal growth and discuss how researchers are using interdisciplinary ideas to engineer neuronal networks in vitro. We first discuss the biochemical and biophysical mechanisms of neuronal growth as we stress the fact that the biochemical or biophysical processes during 2 neuronal growth are not independent of each other but are rather complementary. Next, we discuss how utilizing these fundamental mechanisms can enable control over neuronal growth for advanced neuroengineering and biomedical applications. At the end of this review, we discuss some of the open questions and our perspectives on the challenges and possibilities related to controlling and engineering the growth of neuronal networks, specifically in relation to the materials, substrates, model systems, modulation techniques, data science, and artificial intelligence.
... Cdc42 was considered as a critical regulator of actin dynamics which plays an crucial role in controlling the speed and direction of cell movement. 26,27 To further investigate the role of Cdc42 in L-lactate-mediated IEC migration, we treated Mode-K cells with or without L-lactate in the presence or absence of ZCL278, a specific inhibitor of Cdc42, 28 and recorded the cell movement every 15 minutes for 24 hours by using a video microscopy. Addition of ZCL278 ablated L-lactated-mediated IEC migration ( Figure 3A-C). ...
... Interestingly, Cdc42 is also an essential factor in regulating cell direction persistence and cell speed. 26,52 Using video microscopy, we found that L-lactate treatment increased both cell speed and persistence F I G U R E 5 Mitochondrial ATP synthase mediates the effects of L-lactate on IEC migration. Mode-K cells were cultured in reduced serum (1% FBS) media, wounded, and treated with or without 5 mM L-lactate in the presence or absence of 2 nM oligomycin (n = 4 samples per group), images were taken 15 minutes apart for video microscopy (Video S2). ...
Lactate, one of the most common primary metabolites of bacteria and human cells, has been shown to play essential roles in the regulation of inflammatory diseases, including inflammatory bowel diseases. However, whether and how host‐derived lactate affects intestinal epithelial homeostasis is still not completely understood. Here, we investigated how L‐lactate, mainly produced by host cells, regulates intestinal epithelial cell (IEC) migration to promote intestinal wound healing. Using video microscopy and tracking individual cells, we found that L‐lactate enhanced IEC migration in direction persistence and speed. Mechanistically, L‐lactate promoted IEC mitochondrial ATP production. The mitochondrial ATP synthase inhibitor, oligomycin, significantly decreased IEC persistence and speed, which inhibited cell migration induced by L‐lactate. Furthermore, administering mice with L‐lactate suppressed colitis induced by dextran sulfate sodium. In conclusion, our study demonstrates that host‐derived L‐lactate promotes IEC mitochondrial ATP production to drive cell migration, promoting intestinal wound healing to alleviate intestinal inflammation.
... 2,4 Interestingly, PDGFR-α gene alterations have been associated with human birth defects associated to fusion failure, such as cleft palate 55 and NTDs (spina bifida and anencephaly 56,57 :. Prior to epithelial fusion, in which protuberances form a cellular bridge for initial contact, PDGF signaling can exert a dual function, operating as a chemoattractant in cell-cell recognition 58,59 and promoting Rac-1 translocation to the leading edge, thereby favoring the formation of lamellipodia and migration. 60,61 We observed an apical concentration of PDGF mRNA in the neural ectoderm of lesioned embryos. ...
Background
Neural tube (NT) closure is a complex developmental process that takes place in the early stages of embryogenesis and that is a key step in neurulation. In mammals, the process by which the neural plate generates the NT requires organized cell movements and tissue folding, and it terminates with the fusion of the apposed ends of the neural folds.
Results
Here we describe how almost identical cellular and molecular machinery is used to fuse the spinal neural folds as that involved in the repair of epithelial injury in the same area of the embryo. For both natural and wound activated closure of caudal neural tissue, hyaluronic acid and platelet‐derived growth factor signaling appear to be crucial for the final fusion step.
Conclusions
There seems to be no general wound healing machinery for all tissues but rather, a tissue‐specific epithelial fusion machinery that embryos activate when necessary after abnormal epithelial opening.
... This modulation can be achieved by spatially varying the rate of F-actin polymerization or the mechanical influence of Myosin II contractility [6,7]. In fibroblasts exposed to a steady PDGF gradient, many of the prominent signaling pathways that enhance the rate of F-actin polymerization have been found to be dispensable for chemotaxis [8][9][10], whereas regulation of Myosin IIA by phospholipase C (PLC)/protein kinase (PKC) signaling, a well-studied pathway activated by many receptors, is essential [11]. Another key finding in that study was that diacylglycerol (DAG), the lipid product of PLC that activates most PKC isoforms, is sharply concentrated in the fibroblasts' protrusions (lamellipodia) exposed to the highest concentration of PDGF [11]. ...
Chemotaxis of fibroblasts and other mesenchymal cells is critical for embryonic development and wound healing. Fibroblast chemotaxis directed by a gradient of platelet-derived growth factor (PDGF) requires signaling through the phospholipase C (PLC)/protein kinase C (PKC) pathway. Diacylglycerol (DAG), the lipid product of PLC that activates conventional PKCs, is focally enriched at the up-gradient leading edge of fibroblasts responding to a shallow gradient of PDGF, signifying polarization. To explain the underlying mechanisms, we formulated reaction-diffusion models including as many as three putative feedback loops based on known biochemistry. These include the previously analyzed mechanism of substrate-buffering by myristoylated alanine-rich C kinase substrate (MARCKS) and two newly considered feedback loops involving the lipid, phosphatidic acid (PA). DAG kinases and phospholipase D, the enzymes that produce PA, are identified as key regulators in the models. Paradoxically, increasing DAG kinase activity can enhance the robustness of DAG/active PKC polarization with respect to chemoattractant concentration while decreasing their whole-cell levels. Finally, in simulations of wound invasion, efficient collective migration is achieved with thresholds for chemotaxis matching those of polarization in the reaction-diffusion models. This multi-scale modeling framework offers testable predictions to guide further study of signal transduction and cell behavior that affect mesenchymal chemotaxis.
... Up to this date, the literature contains conflicting results concerning the relevance of the closest relatives of Rac GTPases in mammals, in particular RhoG, but to a certain extent also Cdc42. In spite of prominent studies establishing functions for RhoG in signalling complexes operating upstream of Rac [10][11][12], RhoG has also already been concluded to contribute to fibroblast migration independent of Rac activation [23], although it has remained unclear how that might occur mechanistically [24]. Of note, and again in full accordance with our previously published fibroblast data [6], our Rac1/2/3-deficient B16-F1 melanoma line failed entirely to form lamellipodia even upon expression of constitutively active RhoG (Figure 2(a,b)). ...
Cell migration frequently involves the formation of lamellipodial protrusions, the initiation of which requires Rac GTPases signalling to heteropentameric WAVE regulatory complex (WRC). While Rac-related RhoG and Cdc42 can potently stimulate lamellipodium formation, so far presumed to occur by upstream signalling to Rac activation, we show here that the latter can be bypassed by RhoG and Cdc42 given that WRC has been artificially activated. This evidence arises from generation of B16-F1 cells simultaneously lacking both Rac GTPases and WRC, followed by reconstitution of lamellipodia formation with specific Rho-GTPase and differentially active WRC variant combinations. We conclude that formation of canonical lamellipodia requires WRC activation through Rac, but can possibly be tuned, in addition, by WRC interactions with RhoG and Cdc42.
... Up to this date, the literature contains conflicting results concerning the relevance of the closest relatives of Rac GTPases in mammals, in particular RhoG, but to a certain extent also Cdc42. In spite of prominent studies establishing functions for RhoG in signalling complexes operating upstream of Rac [10][11][12], RhoG has also already been concluded to contribute to fibroblast migration independent of Rac activation [23], although it has remained unclear how that occur mechanistically [24]. Of note, and again in full accordance with our previously published fibroblast data [6], our Rac1/2/3-deficient B16-F1 melanoma line previously failed entirely to form lamellipodia even upon expression of constitutively active RhoG (Figure 2A and B). ...
Cell migration frequently involves the formation of lamellipodial protrusions, the initiation of which requires Rac GTPases signalling to heteropentameric WAVE regulatory complex (WRC). While Rac-related RhoG and Cdc42 can potently stimulate lamellipodium formation, so far presumed to occur by upstream signalling to Rac activation, we show here that the latter can be bypassed by RhoG and Cdc42 given that WRC has been artificially activated. This evidence arises from generation of B16-F1 cells simultaneously lacking both Rac GTPases and WRC, followed by reconstitution of lamellipodia formation with specific Rho-GTPase and differentially active WRC variant combinations. We conclude that formation of canonical lamellipodia requires WRC activation through Rac, but can possibly be tuned, in addition, by WRC interactions with RhoG and Cdc42.
