1H, 13C and 15N resonance assignments of the Cdc42-binding domain of TOCA1
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Abstract:
TOCA1 is a downstream effector protein of the small GTPase, Cdc42. It is a multi-domain protein that includes a membrane binding F-BAR domain, a homology region 1 (HR1) domain, which binds selectively to active Cdc42 and an SH3 domain. TOCA1 is involved in the regulation of actin dynamics in processes such as endocytosis, filopodia formation, neurite elongation, cell motility and invasion. Structural insight into the interaction between TOCA1 and Cdc42 will contribute to our understanding of the role of TOCA1 in actin dynamics. The (1)H, (15)N and (13)C NMR backbone and sidechain resonance assignment of the HR1 domain (12 kDa) presented here provides the foundation for structural studies of the domain and its interactions.Keywords:
CDC42
Filopodia
Small GTPase
Background information . The appropriate regulation of cell–cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E‐cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin‐mediated cell–cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E‐cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell–cell contacts has been inferred from the presence of filopodia, the typical F‐actin structure induced by Cdc42 activation, as cells approach each other to establish cell–cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret. Results . In the present study, we set out to address (a) whether Cdc42 is activated by new E‐cadherin cell–cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E‐cadherin clustering decreased Cdc42‐GTP levels. In the absence of serum‐ and migration‐induced Cdc42 activation, we demonstrated that cell–cell contacts do not induce filopodia or require Cdc42 function to assemble. Conclusion . We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re‐positioning or basolateral trafficking.
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(Cell Reports 11, 1577–1590; June 16, 2015) In the originally published version of this paper, Figure 7D contained an image inadvertently duplicated from a previously published paper. The figure has been updated with the relevant correct panel and now appears corrected with the paper online. The authors apologize for this error. NRP1 Regulates CDC42 Activation to Promote Filopodia Formation in Endothelial Tip CellsFantin et al.Cell ReportsJune 3, 2015In BriefDuring angiogenesis, new blood vessel sprouts are led by filopodia-studded tip cells to sense environmental signals and enable directional migration. NRP1 is a tip cell protein that senses angiogenic cues. Fantin et al. now show that NRP1 promotes tip cell function by enabling CDC42 activation for filopodia formation and actin remodeling. Full-Text PDF Open Access
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Cdc42, a member of the Rho family of GTPases, has been shown to play a role in cell adhesion, cytoskeletal arrangement, phagocytosis and cell motility and migration, in addition to a host of other diverse biological processes. The function of Rho-family GTPases in disease pathogenesis has been well established and identification of small, cell permeable molecules that selectively and reversibly regulate Rho GTPases is of high scientific and potentially therapeutic interest. There has been limited success in identifying inhibitors that specifically interact with small Rho family GTPases. The identified probe, ML141 (CID-2950007), is demonstrated to be a potent, selective and reversible non-competitive inhibitor of Cdc42 GTPase suitable for in vitro assays, with low micromolar potency and selectivity against other members of the Rho family of GTPases (Rac1, Rab2, Rab7). Given the highly complementary nature of the function of the Rho family GTPases, Cdc42 selective inhibitors such as those reported here should help untangle the roles of the proteins in this family.
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Tumor microenvironment is composed of biological, chemical and physical factors. Mechanical factors are more and more focused these years. Therefore, mimicking mechanical factors' contribution to cancer cell malignancy will greatly improve the advance in this field. Although the induced malignant behaviors are present under many stimuli such as growth or inflammatory factors, the cell key physical migration mechanisms are still missing. In this study, we identify that low shear stress significantly promotes the formation of needle-shaped membrane protrusions, which is called filopodia and important for the sense and interact of a cell with extracellular matrix in the tumor microenvironment. Under low shear stress, the migration is promoted while it is inhibited in the presence of ROCK inhibitor Y27632, which could abolish the F-actin network. Using cell imaging, we further unravel that key to these protrusions is Cell division cycle 42 (Cdc42) dependent. After Cdc42 activation, the filopodia is more and longer, acting as massagers to pass the information from a cell to the microenvironment for its malignant phenotype. In the Cdc42 inhibition, the filopodia is greatly reduced. Moreover, small GTPases Cdc42 rather than Rac1 and Rho directly controls the filopodia formation. Our work highlights that low shear stress and Cdc42 activation are sufficient to promote filopodia formation, it not only points out the novel structure for cancer progression but also provides the experimental physical basis for the efficient drug anti-cancer strategies.
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SUMMARY Compartmentalization of GTPase regulators into signaling nodules dictates the GTPase pathways selected. Rac and Cdc42 are synchronized at the cell edge for effective protrusion in motile cells but how their activity is coordinated remains elusive. Here, we discovered that ARHGAP39, a Rac and Cdc42 GTPase-activating protein, sequentially interacts with WAVE and mDia2 to control Rac/lamellipodia and Cdc42/filopodia protrusions, respectively. Mechanistically, ARHGAP39 binds to WAVE and, upon phosphorylation by Src kinase, inactivates Rac to promote Cdc42-induced filopodia formation. With our optimized FRET biosensor, we detected active Cdc42 at the filopodia tips that controls filopodia extension. ARHGAP39 is transported to filopodia tips by Myosin-X where it binds mDia2 and inactivates Cdc42 leading to filopodia retraction. Failure in lamellipodia to filopodia switch by defective ARHGAP39 impairs cell invasion and metastasis. Our study reveals that compartmentalization of ARHGAP39 within Rac/Cdc42 signaling nodules orchestrates the synchronization of lamellipodia/filopodia crosstalk and highlights the intricate regulation of leading edge dynamics in migrating cells.
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