Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways

Gavard, Julie; Lambert, Mireille; Grosheva, Inna; Marthiens, Véronique; Irinopoulou, Théano; Riou, Jean-François; Bershadsky, Alexander D.; Mège, René-Marc

doi:10.1242/jcs.00857

Cited by 122 publications

(137 citation statements)

References 54 publications

(57 reference statements)

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“…On Ecad-Fc-functionalized glass, cells were very flat and round, with large focal adhesion-like cadherin plaques at the end of thick radial F-actin bundles around the periphery ( Fig. 1E), similar to observations in previous studies (34)(35)(36)(37). These prominent F-actin and E-cadherin structures were very different from the organization of these proteins in cells adhered to Ecad-Fc PA gels.…”

Section: Resultssupporting

confidence: 88%

“…The elastic moduli of the two gel formulations used in this study were verified by atomic force microscopy (AFM): 10%T, 1%C, 27.5-31.8 kPa (median: 29.07 kPa) and 10%T, 2.5% C, 52.6-67.2 kPa (median: 57.34 kPa) (Table S1). We also prepared glass coverslips (with an elastic modulus of ∼100 GPa) coated with Ecad-Fc, which have been used in most other studies (34)(35)(36)(37).…”

Section: Resultsmentioning

confidence: 99%

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Changes in E-cadherin rigidity sensing regulate cell adhesion

Collins

Denisin

Pruitt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of hightraction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42-and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cellcell adhesion.C ell adhesion is essential for tissue structure and function. Cells use specialized types of adhesions to interact with the surrounding environment, including integrin-based focal adhesions at cell-extracellular matrix (cell-ECM) contacts and cadherin-based adhesions at cell-cell contacts (1). Integrins bind to the ECM and intracellular proteins that link to the actin cytoskeleton and important signaling pathways (2). Similarly, cadherins regulate cellcell recognition and adhesion (3) and, through cytoplasmic adaptor proteins (catenins, vinculin) (4, 5), also link to the actin cytoskeleton and other proteins with signaling and scaffolding functions (6).Initiation of cell-cell adhesion requires significant reorganization of the actin cytoskeleton and is tightly controlled by the activities of actin nucleating proteins and Rho GTPases. Adhesion is initiated when filopodia from opposing cells come into contact with one another (7,8), and this process is regulated by Cdc42 activity (9, 10) and formin-dependent actin polymerization (11)(12)(13)(14). Intermediate stages of cell-cell contact formation involve lateral expansion of the contact by Rac1-induced and Arp2/3-dependent lamellipodial activity (15, 16). Finally, com...

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Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Changes in E-cadherin rigidity sensing regulate cell adhesion

Collins

Denisin

Pruitt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Subcellular fractionation, GST-pull down, immunoprecipitation and western blot Cells were subjected to sub-cellular fractionation between membrane-containing pellet and cytosol-containing supernatant as detailed in (Gavard et al, 2004). Rac activation was monitored by GST pull-downs, using the GST-PAK-CRIB (Cdc42/Rac Interacting Binding domain) bound to glutathione slurry resin (GE Healthcare, Saclay, France), as described in (Gavard and Gutkind, 2006).…”

Section: Ve-cadherin Internalization Assay and Immunofluorescencementioning

confidence: 99%

Remodeling of VE-cadherin junctions by the human herpes virus 8 G-protein coupled receptor

et al. 2010

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Kaposi Sarcoma (KS) are opportunistic tumors, associated with human herpes virus 8 (HHV8) infection. KS development is highly favored by immune-depression and remains the second most frequent tumor in acquired immune deficiency syndrome patients. Although it has been shown that experimental expression of the HHV8 G-proteincoupled receptor (vGPCR) in the endothelial compartment is alone sufficient to recapitulate the formation and progression of KS-like lesions, its functional effects on endothelial homeostasis are not fully understood. Here we show that vGPCR expression in endothelial cells induces an increase in paracellular permeability both in vivo and in vitro. By using pharmacological inhibitors and small interference RNA-based knockdown, we demonstrate an essential role for the PI(3)Kinase-c/Rac nexus in vGPCRmediated permeability. This was further accompanied by dramatic remodeling of VE-cadherin-dependent cell-cell junctions. Importantly, this in vitro vGPCR-initiated signaling signature was observed in a large panel of human KS. Altogether, our results support the hypothesis that endothelial vGPCR signaling is co-opted in KS, and unveil new key cellular targets for therapeutic intervention.

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“…It has been demonstrated that PI3K plays an essential role in the epidermal growth factor (EGF)-induced membrane protrusion by regulating not only Akt (Higuchi et al, 2001;Nishita et al, 2004;Hafizi et al, 2005) but also Rac and Ral GTPases (Tian et al, 2002;Gavard et al, 2004;Nishita et al, 2004;Takaya et al, 2004). However, the mechanism by which Akt regulates the cytoskeletal remodeling and the relationship to Ral and Rac has been still remains elusive.…”

Section: Introductionmentioning

confidence: 99%

“…PDK1 phosphorylates Akt at Thr 308 , which causes a charge-induced conformational change, allowing Akt for substrate binding and an increased rate of catalysis. Akt is further activated by the phosphorylation of Ser 473 , which can be catalyzed by various kinases, including PDK2, DNA-PK, mammalian target of rapamysin, rictor complex, integrin linked kinase, and protein kinase C␤II (Feng et al, 2004;Kawakami et al, 2004;Fayard et al, 2005;Sarbassov et al, 2005 It has been demonstrated that PI3K plays an essential role in the epidermal growth factor (EGF)-induced membrane protrusion by regulating not only Akt (Higuchi et al, 2001;Nishita et al, 2004;Hafizi et al, 2005) but also Rac and Ral GTPases (Tian et al, 2002;Gavard et al, 2004;Nishita et al, 2004;Takaya et al, 2004). However, the mechanism by which Akt regulates the cytoskeletal remodeling and the relationship to Ral and Rac has been still remains elusive.…”

mentioning

confidence: 99%

Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

Yoshizaki

Mochizuki

Gotoh

et al. 2007

MBoC

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We studied the spatiotemporal regulation of Akt (also called protein kinase B), phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P 2 ], and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] by using probes based on the principle of fluorescence resonance energy transfer. On epidermal growth factor (EGF) stimulation, the amount of PtdIns(3,4,5)P 3 was increased diffusely in the plasma membrane, whereas that of PtdIns(3,4)P 2 was increased more in the nascent lamellipodia than in the plasma membrane of the central region. The distribution and time course of Akt activation were similar to that of increased PtdIns(3,4)P 2 levels, which were most prominent in the nascent lamellipodia. Moreover, we found that upon EGF stimulation 3-phosphoinositide-dependent protein kinase-1 (PDK1) was also recruited to nascent lamellipodia in an Akt-dependent manner. Because PDK1 is known to activate Ral GTPase and because Ral is required for EGF-induced lamellipodial protrusion, we speculated that the PDK1-Akt complex may be indispensable for the induction of lamellipodia. In agreement with this idea, EGF-induced lamellipodia formation was promoted by the overexpression of Akt and inhibited by an Akt inhibitor or a Ral-binding domain of Sec5. These results identified the Akt-PDK1 complex as an upstream positive regulator of Ral GTPase in the induction of lamellipodial protrusion. INTRODUCTIONClass I phosphoinositide 3-kinase (PI3K) is a key mediator of intracellular signaling pathways that regulate actin cytoskeletal reorganization and polarized cell migration. Activated PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ] to generate phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ], which, in turn, activates a variety of pleckstrin homology (PH) domain-containing proteins such as Akt (also called protein kinase B) (Saltiel and Pessin, 2002;Sulis and Parsons, 2003). PtdIns(3,4,5)P 3 is dephosphorylated to yield PtdIns(4,5)P 2 by a tumor suppressor protein, phosphatase and tensin homolog deleted in chromosome 10 (PTEN), which is frequently mutated or deleted in various human cancers (Sulis and Parsons, 2003). The resulting PTEN deficiency causes accumulation of PtdIns(3,4,5)P 3 in cells, and, as a consequence, an increase in cell motility in fibroblasts (Liliental et al., 2000;Higuchi et al., 2001;Hafizi et al., 2005). Another dephosphorylated derivative of PtdIns (3,4,5)P 3 is PtdIns(3,4)P 2 produced by phosphoinositide 5-phosphatases, including Src homology 2-containing inositol-5-phosphatase (SHIP). This PtdIns(3,4)P 2 also binds to various PH domain-containing proteins, which overlap significantly to those bound to PtdIns(3,4,5)P 3 (Lemmon and Ferguson, 2000;Maffucci and Falasca, 2001).Among many PH domain-containing proteins, a serinethreonine kinase, Akt, has been studied most extensively. Akt has been implicated in the control of diverse cellular functions, including glucose metabolism, gene transcription, cell proliferation, and apoptosis (Brazil et al., 2004;Fayard et al., 2005)...

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Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways

Cited by 122 publications

References 54 publications

Changes in E-cadherin rigidity sensing regulate cell adhesion

Changes in E-cadherin rigidity sensing regulate cell adhesion

Remodeling of VE-cadherin junctions by the human herpes virus 8 G-protein coupled receptor

Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

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