SNARE‐mediated membrane traffic modulates RhoA‐regulated focal adhesion formation

Gonon, Eva M.; Skalski, Michael; Kean, Michelle J.; Coppolino, Marc G.

doi:10.1016/j.febslet.2005.09.090

Cited by 12 publications

(11 citation statements)

References 49 publications

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“…Stability of the largest FCs, called focal adhesions, may depend on the fact that opportunities for subunit accretion or turnover at the FC are limited to FC ends by the overlying actin structure (Gov, 2006). Thus, RhoA N19 could limit the stress fibers (Harrington et al, 2003;Gonon et al, 2005), which are thought to constrain cell shape. Stress fibers that anchor on focal adhesions were previously reported parallel to the cell membrane and crossing the center of 1,000 W cells (Li et al, 2006).…”

Section: N19mentioning

confidence: 98%

Relationship of p21‐activated kinase (PAK) and filopodia to persistence and oncogenic transformation

Heckman

Demuth

Deters

et al. 2009

Journal Cellular Physiology

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Previously, we found that oncogenically transformed cells had fewer filopodia and more large, p21-activated kinase (PAK)-dependent features than normal cells. These large protrusions (LPs) were increased in cells expressing RhoA(N19) with Cdc42-associated kinase (ACK). Here, we determine how GTPase-mediated mechanisms of focal contact (FC) regulation affect these protrusions. Constructs encoding various proteins were introduced into cells which were then studied by microscopy and computerized image processing and analysis. Constructs that prevented PAK recruitment by PAK-interacting exchange factor (PIX) or restricted PAK residence time on FCs decreased both protrusions. Thus, filopodia were also PAK-dependent. A comparison of FC distribution in cells expressing PAK in the presence or absence of PAK kinase inhibitor domain (KID) suggested that PAK enlarged FCs without affecting the prevalence of either protrusion. KID or Nck expression increased LPs but not filopodia. Nck failed to synergize with KID or ACK and RhoA(N19) in enhancing LPs. Nck and KID synergistically enhanced filopodia, possibly because Nck recruited PAK to FCs while KID prevented their dissociation by PAK-mediated autophosphorylation. Coexpression of Nck, ACK, and RhoA(N19) abrogated filopodia and replicated the transformed phenotype. Since Nck recruitment of PAK is implicated in persistence of directional movement, we studied the PAK-Nck interface. Filopodia were eliminated by the Nck PAK-binding domain and LPs by the PAK Nck-binding domain. The results suggested that filopodia formation has more stringent requirements than LP formation, and Nck and PAK are used differently in the protrusions. Loss of filopodia in transformed cells may reflect defective regulation of GTPase mechanisms.

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Section: N19mentioning

confidence: 98%

Relationship of p21‐activated kinase (PAK) and filopodia to persistence and oncogenic transformation

Heckman

Demuth

Deters

et al. 2009

Journal Cellular Physiology

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“…To determine whether force transduction signals altered focal adhesions, we quantified the number and area of focal adhesions (Gonon et al, 2005;Zamir et al, 1999) based on analyses of immunofluorescence images of vinculin at the basal plane. Focal adhesions were distributed mainly at the cell perimeter (Fig.…”

Section: E-cadherin-specific Mechanotransduction Triggers Focal Adhesmentioning

confidence: 99%

“…Confocal images of immunostained vinculin were analyzed with a custom MATLAB code (from Brenton Hoffman, Duke University), based on the watershed algorithm (Zamir et al, 1999). Focal adhesion size and number were quantified using the 'Analyze Particles' function in ImageJ (NIH) with a minimum threshold area of 0.3 μm 2 (Gonon et al, 2005).…”

Section: Confocal Immunofluorescencementioning

confidence: 99%

E-cadherin-mediated force transduction signals regulate global cell mechanics

Muhamed

Sehgal

et al. 2016

Journal of Cell Science

104

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This report elucidates an E-cadherin-based force-transduction pathway that triggers changes in cell mechanics through a mechanism requiring epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), and the downstream formation of new integrin adhesions. This mechanism operates in addition to local cytoskeletal remodeling triggered by conformational changes in the E-cadherin-associated protein α-catenin, at sites of mechanical perturbation. Studies using magnetic twisting cytometry (MTC), together with traction force microscopy (TFM) and confocal imaging identified force-activated E-cadherin-specific signals that integrate cadherin force transduction, integrin activation and cell contractility. EGFR is required for the downstream activation of PI3K and myosin-II-dependent cell stiffening. Our findings also demonstrated that α-catenin-dependent cytoskeletal remodeling at perturbed E-cadherin adhesions does not require cell stiffening. These results broaden the repertoire of E-cadherin-based force transduction mechanisms, and define the force-sensitive signaling network underlying the mechano-chemical integration of spatially segregated adhesion receptors.

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“…SNAREs are operationally divided into two groups: those found primarily on the transport vesicle, known as v-SNAREs (or VAMPs), and those found primarily on the target membrane, called t-SNAREs (syntaxins and SNAPs) (9,10). The demonstration that VAMPs and SNAPs are involved in ␣5␤1 integrin trafficking suggested that SNARE proteins more generally play roles in the regulation of integrin trafficking (6,(13)(14)(15)(16). Recently, we showed that the functional t-SNARE syntaxin 6 is required for the post-Golgi trafficking of VEGFR2/KDR/Flk-1 in vitro, and for VEGF-induced angiogenesis both in vitro and in vivo (17).…”

mentioning

confidence: 99%

“…Internalization of the ␣5␤1 integrin complexes occurs via both clathrin-dependent and clathrin-independent endocytic pathways (4). Subsequently, these complexes traffic to early endosomes (EEs) and then to the perinuclear recycling compartment, from which they are recycled to the PM (5)(6)(7). This transport requires Rab11 and the activity of PKB/GSK3b (5).…”

mentioning

confidence: 99%

Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated α5β1 Integrin Recycling

Tiwari

Jung

Inamdar

et al. 2011

Journal of Biological Chemistry

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The ␣5␤1 integrin heterodimer regulates many processes that contribute to embryonic development and angiogenesis, in both physiological and pathological contexts. As one of the major adhesion complexes on endothelial cells, it plays a vital role in adhesion and migration along the extracellular matrix. We recently showed that angiogenesis is modulated by syntaxin 6, a Golgi-and endosome-localized t-SNARE, and that it does so by regulating the post-Golgi trafficking of VEGFR2. Here we show that syntaxin 6 is also required for ␣5␤1 integrin-mediated adhesion of endothelial cells to, and migration along, fibronectin. We demonstrate that syntaxin 6 and ␣5␤1 integrin colocalize in EEA1-containing early endosomes, and that functional inhibition of syntaxin 6 leads to misrouting of ␤1 integrin to the degradation pathway (late endosomes and lysosomes) rather transport along recycling pathway from early endosomes; an increase in the pool of ubiquitinylated ␣5 integrin and its lysosome-dependent degradation; reduced cell spreading on fibronectin; decreased Rac1 activation; and altered Rac1 localization. Collectively, our data show that functional syntaxin 6 is required for the regulation of ␣5␤1-mediated endothelial cell movement on fibronectin. These syntaxin 6-regulated membrane trafficking events control outside-in signaling via haptotactic and chemotactic mechanisms.Adhesion molecules present on the endothelial cell (EC) 2 surface are vital regulators of vascular homeostasis and angiogenesis. Integrins are heterodimeric cell-adhesion receptors for extracellular matrix proteins, and are composed of noncovalently bound ␣ and ␤ subunits (1). During embryonic vascular development, as well as during tumor angiogenesis, the extracellular matrix protein fibronectin serves as an adhesive support and signals through ␣5␤1 integrin to regulate the spreading, migration, and contractility of ECs (1-3). Integrins do not have intrinsic enzymatic activity; their ability to transduce signals depends on recruitment of cytoplasmic linker and signaling proteins, and on the assembly of focal adhesions. Hence the processes that regulate integrin turnover at the cell surface may have implications for the regulation of angiogenesis in the context of therapies.The levels of ␣5␤1 integrin on the cell surface are maintained by recycling of endocytosed complexes back to the plasma membrane (PM). Internalization of the ␣5␤1 integrin complexes occurs via both clathrin-dependent and clathrin-independent endocytic pathways (4). Subsequently, these complexes traffic to early endosomes (EEs) and then to the perinuclear recycling compartment, from which they are recycled to the PM (5-7). This transport requires Rab11 and the activity of PKB/GSK3b (5). Binding of fibronectin to the ␣5␤1 integrin triggers endocytosis that does not involve integrin recycling; in this case the integrin complexes move from the EEs into the multivesicular endosomes for degradation (8). This phenomenon highlights the importance of ␣5␤1 integrin trafficking at the EE compartme...

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SNARE‐mediated membrane traffic modulates RhoA‐regulated focal adhesion formation

Cited by 12 publications

References 49 publications

Relationship of p21‐activated kinase (PAK) and filopodia to persistence and oncogenic transformation

Relationship of p21‐activated kinase (PAK) and filopodia to persistence and oncogenic transformation

E-cadherin-mediated force transduction signals regulate global cell mechanics

Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated α5β1 Integrin Recycling

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