ErbB2 receptor controls microtubule capture by recruiting ACF7 to the plasma membrane of migrating cells.
ABSTRACT: Microtubules (MTs) contribute to key processes during cell motility, including the regulation of focal adhesion turnover and the establishment and maintenance of cell orientation. It was previously demonstrated that the ErbB2 receptor tyrosine kinase regulated MT outgrowth to the cell cortex via a complex including Memo, the GTPase RhoA, and the formin mDia1. But the mechanism that linked this signaling module to MTs remained undefined. We report that ErbB2-induced repression of glycogen synthase kinase-3 (GSK3) activity, mediated by Memo and mDia1, is required for MT capture and stabilization. Memo-dependent inhibition of GSK3 allows the relocalization of APC (adenomatous polyposis coli) and cytoplasmic linker-associated protein 2 (CLASP2), known MT-associated proteins, to the plasma membrane and ruffles. Peripheral microtubule extension also requires expression of the plus-end binding protein EB1 and its recently described interactor, the spectraplakin ACF7. In fact, in migrating cells, ACF7 localizes to the plasma membrane and ruffles, in a Memo-, GSK3-, and APC-dependent manner. Finally, we demonstrate that ACF7 targeting to the plasma membrane is both required and sufficient for MT capture downstream of ErbB2. This function of ACF7 does not require its recently described ATPase activity. By defining the signaling pathway by which ErbB2 allows MT capture and stabilization at the cell leading edge, we provide insights into the mechanism underlying cell motility and steering.
Project description:Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLC?) also contributed to cell guidance. We further showed that PLC? signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.
Project description:The mammalian diaphanous-related formin (mDia1), a Rho-regulated cytoskeletal modulator, has been shown to promote T lymphocyte chemotaxis and interaction with antigen presenting cells, but the mechanisms underpinning mDia1 roles in these processes have not been defined. Here we show that mDia1(-/-) T cells exhibit impaired lymphocyte function-associated antigen 1 (LFA-1)-mediated T cell adhesion, migration and in vivo trafficking. These defects are associated with impaired microtubule (MT) polarization and stabilization, altered MT dynamics and reduced peripheral clustering of the MT plus-end-protein, adenomatous polyposis coli (APC) in migrating T cells following LFA-1-engagement. Loss of mDia1 also leads to impaired inducible inactivation of the glycogen synthase kinase (GSK) 3? as well as hyperphosphorylation and reduced levels of APC in migrating T cells. These findings identify essential roles for the mDia1 formin in modulating GSK3?-dependent MT contributions to induction of T-cell polarity, adhesion and motility.
Project description:Coordinated interactions between microtubule (MT) and actin cytoskeletons are involved in many polarized cellular processes. Spectraplakins are enormous (>500 kDa) proteins able to bind both MTs and actin filaments (F-actin) directly. To elucidate the physiological significance and functions of mammalian spectraplakin ACF7, we've conditionally targeted it in skin epidermis. Intriguingly, ACF7 deficiency compromises the targeting of microtubules along F-actin to focal adhesions (FAs), stabilizes FA-actin networks, and impairs epidermal migration. Exploring underlying mechanisms, we show that ACF7's binding domains for F-actin, MTs, and MT plus-end proteins are not sufficient to rescue the defects in FA-cytoskeletal dynamics and migration functions of ACF7 null keratinocytes. We've uncovered an intrinsic actin-regulated ATPase domain in ACF7 and demonstrate that it is both functional and essential for these roles. Our findings provide insight into the functions of this important cytoskeletal crosslinking protein in regulating dynamic interactions between MTs and F-actin to sustain directional cell movement.
Project description:In migrating fibroblasts, RhoA and its effector mDia1 regulate the selective stabilization of microtubules (MTs) polarized in the direction of migration. The conserved formin homology 2 domain of mDia1 is involved both in actin polymerization and MT stabilization, and the relationship between these two activities is unknown. We found that latrunculin A (LatA) and jasplakinolide, actin drugs that release mDia1 from actin filament barbed ends, stimulated stable MT formation in serum-starved fibroblasts and caused a redistribution of mDia1 onto MTs. Knockdown of mDia1 by small interfering RNA (siRNA) prevented stable MT induction by LatA, whereas blocking upstream Rho or integrin signaling had no effect. In search of physiological regulators of mDia1, we found that actin-capping protein induced stable MTs in an mDia1-dependent manner and inhibited the translocation of mDia on the ends of growing actin filaments. Knockdown of capping protein by siRNA reduced stable MT levels in proliferating cells and in starved cells stimulated with lysophosphatidic acid. These results show that actin-capping protein is a novel regulator of MT stability that functions by antagonizing mDia1 activity toward actin filaments and suggest a novel form of actin-MT cross-talk in which a single factor acts sequentially on actin and MTs.
Project description:Multiple formins regulate microtubule (MT) arrays, but whether they function individually or in a common pathway is unknown. Lysophosphatidic acid (LPA) stimulates the formation of stabilized detyrosinated MTs (Glu MTs) in NIH3T3 fibroblasts through RhoA and the formin mDia1. Here we show that another formin, INF2, is necessary for mDia1-mediated induction of Glu MTs and regulation of MT dynamics and that mDia1 can be bypassed by activating INF2. INF2 localized to MTs after LPA treatment in an mDia1-dependent manner, suggesting that mDia1 regulates INF2. Mutants of either formin that disrupt their interaction failed to rescue MT stability in cells depleted of the respective formin, and the mDia1-interacting protein IQGAP1 regulated INF2's localization to MTs and the induction of Glu MTs by either formin. The N-terminus of IQGAP1 associated with the C-terminus of INF2 directly, suggesting the possibility of a tripartite complex stimulated by LPA. Supporting this, the interaction of mDia1 and INF2 was induced by LPA and dependent on IQGAP1. Our data highlight a unique mechanism of formin action in which mDia1 and INF2 function in series to stabilize MTs and point to IQGAP1 as a scaffold that facilitates the activation of one formin by another.
Project description:A widespread belief in phagocyte biology is that FcgammaR-mediated phagocytosis utilizes membrane pseudopods, whereas Mac-1-mediated phagocytosis does not involve elaborate plasma membrane extensions. Here we report that dynamic membrane ruffles in activated macrophages promote binding of C3bi-opsonized particles. We identify these ruffles as components of the macropinocytosis machinery in both PMA- and LPS-stimulated macrophages. C3bi-particle capture is facilitated by enrichment of high-affinity Mac-1 and the integrin-regulating protein talin in membrane ruffles. Membrane ruffle formation and C3bi-particle binding are cytoskeleton dependent events, having a strong requirement for F-actin and microtubules (MTs). MT disruption blunts ruffle formation and PMA- and LPS-induced up-regulation of surface Mac-1 expression. Furthermore, the MT motor, kinesin participates in ruffle formation implicating a requirement for intracellular membrane delivery to active membrane regions during Mac-1-mediated phagocytosis. We observed colocalization of Rab11-positive vesicles with CLIP-170, a MT plus-end binding protein, at sites of particle adherence using TIRF imaging. Rab11 has been implicated in recycling endosome dynamics and mutant Rab11 expression inhibits both membrane ruffle formation and C3bi-sRBC adherence to macrophages. Collectively these findings represent a novel membrane ruffle "capture" mechanism for C3bi-particle binding during Mac-1-mediated phagocytosis. Importantly, this work also demonstrates a strong functional link between integrin activation, macropinocytosis and phagocytosis in macrophages.
Project description:In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with non-APC-decorated MTs. Endogenous APC associated briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, we monitored EB1-green fluorescent protein and endogenous APC concomitantly in living cells. Only a small fraction of EB1 colocalized with APC at any one time. APC-deficient cells and EB1 small interfering RNA showed that EB1 and APC localized at MT ends independently. Depletion of EB1 did not change the growth-stabilizing effects of APC on MT plus ends. In addition, APC remained bound to MTs stabilized with low nocodazole, whereas EB1 did not. Thus, we demonstrate that the association of endogenous APC with MT ends correlates directly with their increased growth stability, that this can occur independently of its association with EB1, and that APC and EB1 can associate with MT plus ends by distinct mechanisms.
Project description:Formins constitute a large family of proteins that regulate the dynamics and organization of both the actin and microtubule cytoskeletons. Previously we showed that the formin mDia1 helps tether microtubules at the cell cortex, acting downstream of the ErbB2 receptor tyrosine kinase. Here we further study the contributions of mDia1 and its two most closely related formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2-dependent breast carcinoma cell migration. We find that depletion of each of these three formins strongly disrupts chemotaxis without significantly affecting actin-based structures. Further, all three formins are required for formation of cortical microtubules in a nonredundant manner, and formin proteins defective in actin polymerization remain active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identify differential binding partners of the formin-homology domain 2 (FH2) of mDia1, mDia2, and mDia3, which may explain their nonredundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacts with Rab6-interacting protein 2 (Rab6IP2). Further, mDia1 is required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupts cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge.
Project description:Microtubules (MTs) govern actin network remodeling in a wide range of biological processes, yet the mechanisms underlying this cytoskeletal cross-talk have remained obscure. We used single-molecule fluorescence microscopy to show that the MT plus-end-associated protein CLIP-170 binds tightly to formins to accelerate actin filament elongation. Furthermore, we observed mDia1 dimers and CLIP-170 dimers cotracking growing filament ends for several minutes. CLIP-170-mDia1 complexes promoted actin polymerization ~18 times faster than free-barbed-end growth while simultaneously enhancing protection from capping proteins. We used a MT-actin dynamics co-reconstitution system to observe CLIP-170-mDia1 complexes being recruited to growing MT ends by EB1. The complexes triggered rapid growth of actin filaments that remained attached to the MT surface. These activities of CLIP-170 were required in primary neurons for normal dendritic morphology. Thus, our results reveal a cellular mechanism whereby growing MT plus ends direct rapid actin assembly.
Project description:Adenomatous polyposis coli (APC) tumor suppressor protein has been shown to be localized near the distal ends of microtubules (MTs) at the edges of migrating cells. We expressed green fluorescent protein (GFP)-fusion proteins with full-length and deletion mutants of Xenopus APC in Xenopus epithelial cells, and observed their dynamic behavior in live cells. During cell spreading and wound healing, GFP-tagged full-length APC was concentrated as granules at the tip regions of cellular extensions. At higher magnification, APC appeared to move along MTs and concentrate as granules at the growing plus ends. When MTs began to shorten, the APC granules dropped off from the MT ends. Immunoelectron microscopy revealed that fuzzy structures surrounding MTs were the ultrastructural counterparts for these GFP signals. The COOH-terminal region of APC was targeted to the growing MT ends without forming granular aggregates, and abruptly disappeared when MTs began to shorten. The APC lacking the COOH-terminal region formed granular aggregates that moved along MTs toward their plus ends in an ATP-dependent manner. These findings indicated that APC is a unique MT-associated protein that moves along selected MTs and concentrates at their growing plus ends through their multiple functional domains.