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J Victor Small

Publications and source records attributed to J Victor Small.

At least 19 recordsLinked to original sources
PubMed2006

PREL1 provides a link from Ras signalling to the actin cytoskeleton via Ena/VASP proteins.

Ena/VASP family proteins are important modulators of cell migration and localize to focal adhesions, stress fibres and the very tips of lamellipodia and filopodia. Proline-rich proteins like vinculin and zyxin are well established interaction partners, which mediate Ena/VASP-recruitment via their EVH1-domains to focal adhesions and stress fibres. However, it is still unclear, which binding partners Ena/VASP proteins may have at lamellipodia tips and how their recruitment to these cellular protrusions is regulated. Here, we report the identification of a novel protein with high similarity to the C. elegans MIG-10 protein, which we termed PREL1 (Proline Rich EVH1 Ligand). PREL1 is a 74 kDa protein and shares homology with the Grb7-family of signalling adaptors. We show that PREL1 directly binds to Ena/VASP proteins and co-localizes with them at lamellipodia tips and at focal adhesions in response to Ras activation. Moreover, PREL1 directly binds to activated Ras in a phosphoinositide-dependent manner. Thus, our data pinpoint PREL1 as the first direct link between Ras signalling and cytoskeletal remodelling via Ena/VASP proteins during cell migration and spreading.

Actin Cytoskeleton↗
PubMed2006

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes.

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.

Actin-Related Protein 2-3 Complex↗
PubMed2005

E-cadherin endocytosis regulates the activity of Rap1: a traffic light GTPase at the crossroads between cadherin and integrin function.

The coordinate modulation of cadherin and integrin functions plays an essential role in fundamental physiological and pathological processes, including morphogenesis and cancer. However, the molecular mechanisms underlying the functional crosstalk between cadherins and integrins are still elusive. Here, we demonstrate that the small GTPase Rap1, a crucial regulator of the inside-out activation of integrins, is a target for E-cadherin-mediated outside-in signaling. In particular, we show that a strong activation of Rap1 occurs upon adherens junction disassembly that is triggered by E-cadherin internalization and trafficking along the endocytic pathway. By contrast, Rap1 activity is not influenced by integrin outside-in signaling. Furthermore, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and controlled by an increased Src kinase activity, and is paralleled by the colocalization of Rap1 and E-cadherin at the perinuclear Rab11-positive recycling endosome compartment, and the association of Rap1 with a subset of E-cadherin-catenin complexes that does not contain p120ctn. Conversely, Rap1 activity is suppressed by the formation of E-cadherin-dependent cell-cell junctions as well as by agents that inhibit either Src activity or E-cadherin internalization and intracellular trafficking. Finally, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and is required for the formation of integrin-based focal adhesions. Our findings provide the first evidence of an E-cadherin-modulated endosomal signaling pathway involving Rap1, and suggest that cadherins may have a novel modulatory role in integrin adhesive functions by fine-tuning Rap1 activation.

Actins↗
PubMed2005

Raf-1 regulates Rho signaling and cell migration.

Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

Animals↗
PubMed2005

PREL1 provides a link from Ras signalling to the actin cytoskeleton via Ena/VASP proteins.

Ena/VASP family proteins are important modulators of cell migration and localize to focal adhesions, stress fibres and the very tips of lamellipodia and filopodia. Proline-rich proteins like vinculin and zyxin are well established interaction partners, which mediate Ena/VASP-recruitment via their EVH1-domains to focal adhesions and stress fibres. However, it is still unclear, which binding partners Ena/VASP proteins may have at lamellipodia tips and how their recruitment to these cellular protrusions is regulated. Here, we report the identification of a novel protein with high similarity to the C. elegans MIG-10 protein, which we termed PREL1 (Proline Rich EVH1 Ligand). PREL1 is a 74 kDa protein and shares homology with the Grb7-family of signalling adaptors. We show that PREL1 directly binds to Ena/VASP proteins and co-localizes with them at lamellipodia tips and at focal adhesions in response to Ras activation. Moreover, PREL1 directly binds to activated Ras in a phosphoinositide-dependent manner. Thus, our data pinpoint PREL1 as the first direct link between Ras signalling and cytoskeletal remodelling via Ena/VASP proteins during cell migration and spreading.

Actin Cytoskeleton↗
PubMed2005

The last but not the least: the origin and significance of trailing adhesions in fibroblastic cells.

Mature adhesions in a motile fibroblast can be classified as stationary "towing" adhesions in the front and sliding trailing adhesions that resist the traction force. Adhesions formed at the front of motile fibroblasts rarely reach the trailing zone, due to disassembly promoted by intensive microtubule targeting. Here, we show that the majority of adhesions found at the trailing edge originate within small short-lived protrusions that extend laterally and backwards from the cell edge. These adhesions enlarge by sliding and by fusion with neighboring adhesions. A further subset of trailing adhesions is initiated at a novel site proximal to trailing stress fibre termini. Following tail retraction, trailing adhesions are actively regenerated and the stress fibre system is remodeled accordingly; the tensile forces elaborated by the contractile actin system are consequently redirected according to trailing adhesion location. We conclude that persistent and dynamic anchorage of the cell rear is needed for the maintenance of continuous unidirectional movement of fibroblasts.

Animals↗
PubMed2005

The comings and goings of actin: coupling protrusion and retraction in cell motility.

Cells utilize actin filaments to produce protrusive and contractile arrays that cooperate to drive cell motility. The generation of the two arrays and the coupling between them result from the unique properties of the lamellipodium, a protrusive leaflet of cytoplasm at the cell edge. From the lamellipodium into the lamella behind, there is a transition from a fast retrograde flow of actin polymer driven by polymerization to a slow flow driven by the interaction of anti-parallel arrays of actin with myosin. In addition to driving protrusion, the lamellipodium appears to play a role in supplying filaments to the lamella for the assembly of the contractile network required for traction.

Actins↗
PubMed2003

Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport.

Bicaudal D is an evolutionarily conserved protein, which is involved in dynein-mediated motility both in Drosophila and in mammals. Here we report that the N-terminal portion of human Bicaudal D2 (BICD2) is capable of inducing microtubule minus end-directed movement independently of the molecular context. This characteristic offers a new tool to exploit the relocalization of different cellular components by using appropriate targeting motifs. Here, we use the BICD2 N-terminal domain as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate the rapid dynein-mediated transport of selected organelles. Surprisingly, unlike other cytoplasmic dynein-mediated processes, this transport shows very low sensitivity to overexpression of the dynactin subunit dynamitin. The dynein-recruiting activity of the BICD2 N-terminal domain is reduced within the full-length molecule, indicating that the C-terminal part of the protein might regulate the interaction between BICD2 and the motor complex. Our findings provide a novel model system for dissection of the molecular mechanism of dynein motility.

Drosophila Proteins↗
PubMed2003

Nanometer targeting of microtubules to focal adhesions.

Although cell movement is driven by actin, polarization and directional locomotion require an intact microtubule cytoskeleton that influences polarization by modulating substrate adhesion via specific targeting interactions with adhesion complexes. The fidelity of adhesion site targeting is precise; using total internal reflection fluorescence microscopy (TIRFM), we now show microtubule ends (visualized by incorporation of GFP tubulin) are within 50 nm of the substrate when polymerizing toward the cell periphery, but not when shrinking from it. Multiple microtubules sometimes followed similar tracks, suggesting guidance along a common cytoskeletal element. Use of TIRFM with GFP- or DsRed-zyxin in combination with either GFP-tubulin or GFP-CLIP-170 further revealed that the polymerizing microtubule plus ends that tracked close to the dorsal surface consistently targeted substrate adhesion complexes. This supports a central role for the microtubule tip complex in the guidance of microtubules into adhesion foci, and provides evidence for an intimate cross-talk between microtubule tips and substrate adhesions in the range of molecular dimensions.

Animals↗
PubMed2003

IRSp53 is colocalised with WAVE2 at the tips of protruding lamellipodia and filopodia independently of Mena.

The insulin receptor tyrosine kinase substrate p53 (IRSp53) links Rac and WAVE2 and has been implicated in lamellipodia protrusion. Recently, however, IRSp53 has been reported to bind to both Cdc42 and Mena to induce filopodia. To shed independent light on IRSp53 function we determined the localisations and dynamics of IRSp53 and WAVE2 in B16 melanoma cells. In cells spread well on a laminin substrate, IRSp53 was localised by antibody labelling at the tips of both lamellipodia and filopodia. The same localisation was observed in living cells with IRSp53 tagged with enhanced green florescence protein (EGFP-IRSp53), but only during protrusion. From the transfection of deletion mutants the N-terminal region of IRSp53, which binds active Rac, was shown to be responsible for its localisation. Although IRSp53 has been reported to regulate filopodia formation with Mena, EGFP-IRSp53 showed the same localisation in MVD7 Ena/VASP (vasodilator stimulated phosphoprotein) family deficient cells. WAVE2 tagged with DsRed1 colocalised with EGFP-IRSp53 at the tips of protruding lamellipodia and filopodia and, in double-transfected cells, the IRSp53 signal in filopodia decreased before that of WAVE2 during retraction. These results suggest an alternative modulatory role for IRSp53 in the extension of both filopodia and lamellipodia, through WAVE2.

Animals↗
PubMed2003

SWAP-70 identifies a transitional subset of actin filaments in motile cells.

Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3'-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actin cytoskeleton as an effector or adaptor protein in response to agonist stimulated phosphatidylinositol (3,4)-bisphosphate production and cell protrusion.

Actin Cytoskeleton↗
PubMed2003

Microtubules meet substrate adhesions to arrange cell polarity.

Cell movement is driven by the regulated and polarised turnover of the actin cytoskeleton and of the adhesion complexes that link it to the extracellular matrix. For most cells, polarisation requires the engagement of microtubules, which exert their effect by mediating changes in the activity of the Rho GTPases. Evidence suggests that these changes are effected in a very localised fashion at sites of substrate adhesion, via specific microtubule-targeting interactions. Targeting serves to bring molecular complexes bound at the tips and along microtubules in close proximity with adhesion complexes, to promote adhesion disassembly and remodelling of the actin cytoskeleton.

Actin Cytoskeleton↗
PubMed2002

Tensile stress stimulates microtubule outgrowth in living cells.

Cell motility is driven by the sum of asymmetric traction forces exerted on the substrate through adhesion foci that interface with the actin cytoskeleton. Establishment of this asymmetry involves microtubules, which exert a destabilising effect on adhesion foci via targeting events. Here, we demonstrate the existence of a mechano-sensing mechanism that signals microtubule polymerisation and guidance of the microtubules towards adhesion sites under increased stress. Stress was applied either by manipulating the body of cells moving on glass with a microneedle or by stretching a flexible substrate that cells were migrating on. We propose a model for this mechano-sensing phenomenon whereby microtubule polymerisation is stimulated and guided through the interaction of a microtubule tip complex with actin filaments under tension.

Actin Cytoskeleton↗
PubMed2002

Visualisation of the actin cytoskeleton by cryo-electron microscopy.

An understanding of the mechanisms driving cell motility requires clarification of the structural organisation of actin filament arrays in the regions of cell protrusion termed lamellipodia. Currently, there is a lack of consensus on lamellipodia organisation stemming from the application of alternative procedures for ultrastructural visualisation of cytoskeleton networks. In this study, we show that cryo-electron microscopy of extracted cytoskeletons embedded in a thin layer of vitreous ice can reveal the organisation of cytoskeletal elements at high resolution. Since this method involves no dehydration, drying and contrasting steps that can potentially introduce subtle distortions of filament order and interactions, its application opens the way to resolving the controversial details of lamellipodia architecture.

Actin Cytoskeleton↗
PubMed2002

Modulation of substrate adhesion dynamics via microtubule targeting requires kinesin-1.

Recent studies have shown that the targeting of substrate adhesions by microtubules promotes adhesion site disassembly (Kaverina, I., O. Krylyshkina, and J.V. Small. 1999. J. Cell Biol. 146:1033-1043). It was accordingly suggested that microtubules serve to convey a signal to adhesion sites to modulate their turnover. Because microtubule motors would be the most likely candidates for effecting signal transmission, we have investigated the consequence of blocking microtubule motor activity on adhesion site dynamics. Using a function-blocking antibody as well as dynamitin overexpression, we found that a block in dynein-cargo interaction induced no change in adhesion site dynamics in Xenopus fibroblasts. In comparison, a block of kinesin-1 activity, either via microinjection of the SUK-4 antibody or of a kinesin-1 heavy chain construct mutated in the motor domain, induced a dramatic increase in the size and reduction in number of substrate adhesions, mimicking the effect observed after microtubule disruption by nocodazole. Blockage of kinesin activity had no influence on either the ability of microtubules to target substrate adhesions or on microtubule polymerisation dynamics. We conclude that conventional kinesin is not required for the guidance of microtubules into substrate adhesions, but is required for the focal delivery of a component(s) that retards their growth or promotes their disassembly.

Animals↗
PubMed2002

The lamellipodium: where motility begins.

Lamellipodia, filopodia and membrane ruffles are essential for cell motility, the organization of membrane domains, phagocytosis and the development of substrate adhesions. Their formation relies on the regulated recruitment of molecular scaffolds to their tips (to harness and localize actin polymerization), coupled to the coordinated organization of actin filaments into lamella networks and bundled arrays. Their turnover requires further molecular complexes for the disassembly and recycling of lamellipodium components. Here, we give a spatial inventory of the many molecular players in this dynamic domain of the actin cytoskeleton in order to highlight the open questions and the challenges ahead.

Actins↗
PubMed2002

Pure F-actin networks are distorted and branched by steps in the critical-point drying method.

Elucidation of the ultrastructural organization of actin networks is crucial for understanding the molecular mechanisms underlying actin-based motility. Results obtained from cytoskeletons and actin comets prepared by the critical-point procedure, followed by rotary shadowing, support recent models incorporating actin filament branching as a main feature of lamellipodia and pathogen propulsion. Since actin branches were not evident in earlier images obtained by negative staining, we explored how these differences arise. Accordingly, we have followed the structural fate of dense networks of pure actin filaments subjected to steps of the critical-point drying protocol. The filament networks have been visualized in parallel by both cryo-electron microscopy and negative staining. Our results demonstrate the selective creation of branches and other artificial structures in pure F-actin networks by the critical-point procedure and challenge the reliability of this method for preserving the detailed organization of actin assemblies that drive motility.

Actins↗