Kris A. DeMali

Integrin signaling to the actin cytoskeleton

Integrin engagement stimulates the activity of numerous signaling molecules, including the Rho family of GTPases, tyrosine phosphatases, cAMP-dependent protein kinase and protein kinase C, and stimulates production of PtdIns(4,5)P2. Integrins promote actin assembly via the recruitment of molecules that directly activate the actin polymerization machinery or physically link it to sites of cell adhesion.

Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion

Cell migration involves many steps, including membrane protrusion and the development of new adhesions. Here we have investigated whether there is a link between actin polymerization and integrin engagement. In response to signals that trigger membrane protrusion, the actin-related protein (Arp)2/3 complex transiently binds to vinculin, an integrin-associated protein. The interaction is regulated, requiring phosphatidylinositol-4,5-bisphosphate and Rac1 activation, and is sufficient to recruit the Arp2/3 complex to new sites of integrin aggregation. Binding of the Arp2/3 complex to vinculin is direct and does not depend on the ability of vinculin to associate with actin. We have mapped the binding site for the Arp2/3 complex to the hinge region of vinculin, and a point mutation in this region selectively blocks binding to the Arp2/3 complex. Compared with WT vinculin, expression of this mutant in vinculin-null cells results in diminished lamellipodial protrusion and spreading on fibronectin. The recruitment of the Arp2/3 complex to vinculin may be one mechanism through which actin polymerization and membrane protrusion are coupled to integrin-mediated adhesion.

Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to “myosin II” has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.

Coupling membrane protrusion and cell adhesion

The ability of cells to extend cell membranes is central to numerous biological processes, including cell migration, cadherin-mediated junction formation and phagocytosis. Much attention has been focused on understanding the signals that trigger membrane protrusion and the architecture of the resulting extension. Similarly, cell adhesion has been extensively studied, yielding a wealth of information about the proteins involved and how they signal to the cytoplasm. Although we have learned much about membrane protrusion and cell adhesion, we know less about how these two processes are coupled. Traditionally it has been thought that they are linked by the signaling pathways they employ – for example, those involving Rho family GTPases. However, there are also physical links between the cellular machineries that mediate cell adhesion and membrane protrusion, such as vinculin.

Vinculin regulates cell-surface E-cadherin expression by binding to β-catenin

Vinculin was identified as a component of adherens junctions 30 years ago, yet its function there remains elusive. Deletion studies are consistent with the idea that vinculin is important for the organization of cell-cell junctions. However, this approach removes vinculin from both cell-matrix and cell-cell adhesions, making it impossible to distinguish its contribution at each site. To define the role of vinculin in cell-cell junctions, we established a powerful short hairpin-RNA-based knockdown/substitution model system that perturbs vinculin preferentially at sites of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology was altered, and cadherin-dependent adhesion was reduced. These defects resulted from impaired E-cadherin cell-surface expression. We have investigated the mechanism for the effects of vinculin and found that the reduced surface E-cadherin expression could be rescued by introduction of vinculin, but not of a vinculin A50I substitution mutant that is defective for β-catenin binding. These findings suggest that an interaction between β-catenin and vinculin is crucial for stabilizing E-cadherin at the cell surface. This was confirmed by analyzing a β-catenin mutant that fails to bind vinculin. Thus, our study identifies vinculin as a novel regulator of E-cadherin function and provides important new insight into the dynamic regulation of adherens junctions.

New Insights into Vinculin Function and Regulation

Vinculin is a cytoplasmic actin-binding protein enriched in focal adhesions and adherens junctions that is essential for embryonic development. Much is now known regarding the role of vinculin in governing cell-matrix adhesion. In the past decade that the crystal structure of vinculin and the molecular details for how vinculin regulates adhesion events have emerged. The recent data suggests a critical function for vinculin in regulating integrin clustering, force generation, and strength of adhesion. In addition to an important role in cell-matrix adhesion, vinculin is also emerging as a regulator of apoptosis, Shigella entry into host cells, and cadherin-based cell-cell adhesion. A close inspection of this work reveals that there are similarities between vinculins role in focal adhesions and these processes and also some intriguing differences.

Identification of the receptor-associated signaling enzymes that are required for platelet-derived growth factor-AA-dependent chemotaxis and DNA synthesis.

Activation of the platelet-derived growth factor (PDGF) α receptor (αPDGFR) leads to cell migration and DNA synthesis. These events are preceded by the ligand-induced tyrosine phosphorylation of the receptor and its association with SH2-containing signaling enzymes including Src family members (Src), the phosphotyrosine phosphatase SHP-2, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ). In this study, we sought to systematically evaluate the relative roles of the signaling enzymes that are recruited to the αPDGFR for DNA synthesis and cell migration. Our approach was to generate and characterize tyrosine to phenylalanine αPDGFR mutants that failed to associate with one or more of the above listed signaling enzymes. In a 3T3-like cell line (Ph cells), PDGF-dependent DNA synthesis was strictly dependent on only one of the receptor-associated proteins, PI3K. In contrast, multiple signaling enzymes were required for maximal chemotaxis, as receptors unable to associate with either Src, PI3K, or PLCγ initiated chemotaxis to 4, 47, or 56% of the wild-type level, respectively. Furthermore, coexpression of mutant receptors revealed that these signaling enzymes do not need to be on the same receptor for a cell to respond chemotactically to PDGF. We conclude that for the αPDGFR, PI3K plays a major role in initiating DNA synthesis, whereas PI3K, PLCγ, and especially Src are required for chemotaxis.

Vinculin phosphorylation differentially regulates mechanotransduction at cell–cell and cell–matrix adhesions

Vinculin phosphorylation on residue Y822 is necessary for cell stiffening in response to tension on cadherins but not integrins.

Tetraspanin CD151 regulates RhoA activation and the dynamic stability of carcinoma cell-cell contacts.

Tetraspanins regulate integrin-dependent tumor cell interactions with the extracellular matrix. Here we show that tetraspanin CD151, which plays critical roles in regulating the adhesion and motility of individual tumor cells, is also an important regulator of collective tumor cell migration. Near total silencing of CD151 destabilizes E-cadherin-dependent carcinoma cell-cell junctions and enhances the collective migration of intact tumor cell sheets. This effect does not depend on reduced E-cadherin cell-surface expression or intrinsic adhesivity, or on obvious disruptions in the E-cadherin regulatory complex. Instead, the loss of CD151 causes excessive RhoA activation, loss of actin organization at cell-cell junctions, and increased actin stress fibers at the basal cell surface. Cell-cell contacts within CD151-silenced monolayers display a nearly threefold increase in remodeling rate and a significant reduction in lifespan as compared to cell-cell contacts within wild-type monolayers. CD151 re-expression restores junctional stability, as does acute treatment of CD151-silenced cells with a cell-permeable RhoA inhibitor. However, a CD151 mutant with impaired association with α3β1 integrin fails to restore junctional organization. These data reveal that, in addition to its roles in regulating tumor cell-substrate interactions, CD151 is also an important regulator of the stability of tumor cell-cell interactions, potentially through its interaction with α3β1 integrin. This could help to explain the phenotypes in human patients and mice lacking CD151.

Activation of Src Family Members Is Not Required for the Platelet-Derived Growth Factor β Receptor To Initiate Mitogenesis

ABSTRACT The basal activity of Src family kinases is readily detectable throughout the cell cycle and increases by two- to fivefold upon acute stimulation of cells with growth factors such as platelet-derived growth factor. Previous reports have demonstrated a requirement for Src activity for the G1/S and G2/M transitions. With a chimeric α-β PDGF receptor (PDGFR) expressed in fibroblasts, we have investigated the importance of the PDGF-mediated increase in Src activity at the G0/G1 transition for subsequent cell cycle events. A mutant PDGFR chimera that was not able to detectably associate with or activate Src was compromised in its ability to mediate tyrosine phosphorylation of receptor-associated signaling molecules and initiated a submaximal activation of Erk. In contrast to these early cell cycle events, later responses such as entry of cells into S phase and cell proliferation proceeded normally when Src activity did not increase following acute stimulation with PDGF. We conclude that the initial burst of Src activity is required for efficient tyrosine phosphorylation of receptor-associated proteins such as PLCγ, RasGAP, Shc, and SHP-2 and for maximal activation of Erk. Surprisingly, these events are not required for PDGF-dependent cell proliferation. Finally, later cell cycle events do not require that Src be activated at the G0/G1 transition and leave open the possibility that events such as the G1/S transition require the basal Src activity and/or activation of Src at later times in G1.