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.     

Mammalian Fat1 cadherin regulates actin dynamics and cell-cell contact

Fat cadherins form a distinct subfamily of the cadherin gene superfamily, and are featured by their unusually large extracellular domain. In this work, we investigated the function of a mammalian Fat cadherin. Fat1 was localized at filopodial tips, lamellipodial edges, and cell-cell boundaries, overlapping with dynamic actin structures. RNA interference-mediated knockdown of Fat1 resulted in disorganization of cell junction-associated F-actin and other actin fibers/cables, disturbance of cell-cell contacts, and also inhibition of cell polarity formation at wound margins. Furthermore, we identified Ena/vasodilator-stimulated phosphoproteins as a potential downstream effector of Fat1. These results suggest that Fat1 regulates actin cytoskeletal organization at cell peripheries, thereby modulating cell contacts and polarity.     

PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho-guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4-green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.     

Rapid and dynamic arginylation of the leading edge β‐actin is required for cell migration

β‐actin plays key roles in cell migration. Our previous work demonstrated that β‐actin in migratory non‐muscle cells is N‐terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here, we examined the function of β‐actin arginylation in cell migration. We found that arginylated β‐actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact‐inhibited cells in confluent cultures, suggesting that arginylated β‐actin at the cell leading edge is coupled to active migration. Arginylated actin levels exhibit dynamic changes in response to cell stimuli, lowered after serum starvation and dramatically elevating within minutes after cell stimulation by readdition of serum or lysophosphatidic acid. These dynamic changes require active translation and are not seen in confluent contact‐inhibited cell cultures. Microinjection of arginylated actin antibodies into cells severely and specifically inhibits their migration rates. Together, these data strongly suggest that arginylation of β‐actin is a tightly regulated dynamic process that occurs at the leading edge of locomoting cells in response to stimuli and is integral to the signaling network that regulates cell migration.      

Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton

T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76-associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3-coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton.     

Profilin1 is required for glial cell adhesion and radial migration of cerebellar granule neurons

Cerebellar granule neurons (CGNs) exploit Bergmann glia (BG) fibres for radial migration, and cell-cell contacts have a pivotal role in this process. Nevertheless, little is known about the mechanisms that control CGN-BG interaction. Here we demonstrate that the actin-binding protein profilin1 is essential for CGN-glial cell adhesion and radial migration. Profilin1 ablation from mouse brains leads to a cerebellar hypoplasia, aberrant organization of cerebellar cortex layers and ectopic CGNs. Conversely, neuronal progenitor proliferation, tangential migration of neurons and BG morphology appear to be independent of profilin1. Our mouse data and the mapping of developmental neuropathies to the chromosomal region of PFN1 suggest a similar function for profilin1 in humans.     

NudC regulates actin dynamics and ciliogenesis by stabilizing cofilin 1

Emerging data indicate that actin dynamics is associated with ciliogenesis. However, the underlying mechanism remains unclear. Here we find that nuclear distribution gene C (NudC), an Hsp90 co-chaperone, is required for actin organization and dynamics. Depletion of NudC promotes cilia elongation and increases the percentage of ciliated cells. Further results show that NudC binds to and stabilizes cofilin 1, a key regulator of actin dynamics. Knockdown of cofilin 1 also facilitates ciliogenesis. Moreover, depletion of either NudC or cofilin 1 causes similar ciliary defects in zebrafish, including curved body, pericardial edema and defective left-right asymmetry. Ectopic expression of cofilin 1 significantly reverses the phenotypes induced by NudC depletion in both cultured cells and zebrafish. Thus, our data suggest that NudC regulates actin cytoskeleton and ciliogenesis by stabilizing cofilin 1.     

Integrins and dystroglycan regulate astrocyte wound healing: the integrin beta1 subunit is necessary for process extension and orienting the microtubular network

Monolayers of astrocytes in culture respond to a scrape wound by orienting towards the wound and extending processes that will repair it. We show here that they also upregulate the expression of extracellular matrix (ECM) proteins, laminin, and chondroitin sulfated proteoglycan, that are deposited in astrocytic scars in vivo. We have previously shown that the major functional ECM receptors on astrocytes are dystroglycan (DG) plus integrins alpha1beta1, alpha5beta1, alpha6beta1, and alphavbeta3. Consistent with this, laminin fragments that activate alpha1beta1 integrin, alpha6beta1 integrin, and DG all contribute to attachment. During astrocyte attachment, or process extension, integrins and DG are found at the leading edge of the lammelipodium, though they change in distribution with the extent of attachment and the alpha and beta subunits of DG can be spatially uncoupled. Functionally, inhibitory antibodies to DG and integrin alpha1beta1 or the RGD peptide all inhibit process extension, showing that ligand engagement of integrins and DG contribute to process extension. Astrocytes differentiated from DG or beta1 null ES cells respond very differently to wounding. The former fail to extend process and cell polarization is disrupted partially. However, beta1 null astrocytes not only fail to extend processes perpendicular to the wound, but cell polarization is completely disrupted and cells migrate randomly into the wound. We conclude that integrins are essential for astrocyte polarity.     

ARHGAP30 is a Wrch-1-interacting protein involved in actin dynamics and cell adhesion

The atypical Rho GTPase Wrch-1 has been proposed roles in cell migration, focal adhesion dissolution, stress fibre break down and tight junction heterogeneity. A screen for Wrch-1 binding-partners identified the novel RhoGAP protein, ARHGAP30, as a Wrch-1 interactor. ARHGAP30 is related to the Cdc42- and Rac1-specific RhoGAP CdGAP, which was likewise found to bind Wrch-1. In contrast to CdGAP, ARHGAP30 serves as a Rac1- and RhoA-specific RhoGAP. Ectopic expression of ARHGAP30 results in membrane blebbing and dissolution of stress-fibres and focal adhesions. Our data suggest roles for ARHGAP30 and CdGAP in regulation of cell adhesion downstream of Wrch-1.     

Myosin VI,an actin motor for membrane traffic and cell migration

The actin cytoskeleton and associated myosin motor proteins are essential for the transport and steady-state localization of vesicles and organelles and for the dynamic remodeling of the plasma membrane as well as for the maintenance of differentiated cell-surface structures. Myosin VI may be expected to have unique cellular functions, because it moves, unlike almost all other myosins, towards the minus end of actin filaments. Localization and functional studies indicate that myosin VI plays a role in a variety of different intracellular processes, such as endocytosis and secretion as well as cell migration. These diverse functions of myosin VI are mediated by interaction with a range of different binding partners .     

Ctdnep1 and Eps8L2 regulate dorsal actin cables for nuclear positioning during cell migration

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Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

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Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond

Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.     

The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization

In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)- Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Delta, pea2Delta, bud6Delta, and bni1Delta, required for normal polarization and actin cytoskeleton functions and, of these, bni1Delta affected Kar9p localization most severely. Like kar9Delta, bni1Delta mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Delta, the bni1Delta mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Delta bni1Delta double mutants suggested that Kar9p retained some function in bni1Delta mitotic cells. Unlike the polarization mutants, kar9Delta shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Delta. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks.     

p73 is required for ependymal cell maturation and neurogenic SVZ cytoarchitecture

The adult subventricular zone (SVZ) is a highly organized microenvironment established during the first postnatal days when radial glia cells begin to transform into type B‐cells and ependymal cells, all of which will form regenerative units, pinwheels, along the lateral wall of the lateral ventricle. Here, we identify p73, a p53 homologue, as a critical factor controlling both cell‐type specification and structural organization of the developing mouse SVZ. We describe that p73 deficiency halts the transition of the radial glia into ependymal cells, leading to the emergence of immature cells with abnormal identities in the ventricle and resulting in loss of the ventricular integrity. p73‐deficient ependymal cells have noticeably impaired ciliogenesis and they fail to organize into pinwheels, disrupting SVZ niche structure and function. Therefore, p73 is essential for appropriate ependymal cell maturation and the establishment of the neurogenic niche architecture. Accordingly, lack of p73 results in impaired neurogenesis. Moreover, p73 is required for translational planar cell polarity establishment, since p73 deficiency results in profound defects in cilia organization in individual cells and in intercellular patch orientation. Thus, our data reveal a completely new function of p73, independent of p53, in the neurogenic architecture of the SVZ of rodent brain and in the establishment of ependymal planar cell polarity with important implications in neurogenesis. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 730–747, 2016     

Dapper-1 is essential for Wnt5a induced cardiomyocyte hypertrophy by regulating the Wnt/PCP pathway

The Wnt signaling pathway was identified as crucial mediator of cardiomyocyte hypertrophy. In this study we found that activation of non-canonical Wnt signaling by Wnt5a stimulates protein synthesis and enlargement of cardiomyocyte surface area. These hypertrophic features were inhibited in Dapper-1 (Dpr1) depleted cells. On the molecular level, we observed inhibition of the non-canonical Wnt/planar-cell-polarity (PCP) pathway denoted by reduction of c-jun-n-terminal-kinase (JNK) phosphorylation. Upstream of JNK, increased protein levels of the Wnt/PCP trans-membrane receptor van-Gogh-like-2 (Vangl2) were observed along with an enrichment of Vangl2 in perinuclear located vesicles. The findings suggest that Dpr1 is essential for execution of the Wnt/PCP pathway and regulation of the Vangl2/JNK axis. Depletion of Dpr1 inhibits non-canonical Wnt signaling induced cardiomyocyte hypertrophy by blocking Wnt/PCP signaling.     

Rac1-dependent actin filament organization in growth cones is necessary for β1-integrin–mediated advance but not for growth on poly-D-lysine

The activity of filopodia and lamellipodia determines the advance, motility, adhesion, and sensory capacity of neuronal growth cones. The shape and dynamics of these highly motile structures originate from the continuous reorganization of the actin cytoskeleton in response to extracellular signals. The small GTPases, Rac1, Rho, and CDC42, regulate the organization of actin filament structures in nonneuronal cells; yet, their role in growth cone motility and neurite outgrowth is poorly understood. We investigated in vitro the function of Rac1 in neurite outgrowth and differentiation by introducing purified recombinant mutants of Rac1 into primary chick embryo motor neurons via trituration. Endogenous Rac1 was expressed in growth cone bodies as well as in the tips and shafts of filopodia, where it often colocalized with actin filament structures. The introduction of constitutively active Rac1 resulted in an increase in rhodamine–phalloidin staining, presumably from an accumulation of actin filaments in growth cones, while dominant negative Rac1 caused a decrease in rhodamine–phalloidin staining. Nevertheless, both Rac1 mutants retarded growth cone advance, and hence attenuated neurite outgrowth and inhibited differentiation of neurites into axons and dendrites on laminin and fibronectin. In contrast, on poly-D -lysine, neither Rac1 mutant affected growth cone advance, neurite outgrowth, or neurite differentiation despite inducing similar changes in the amount of rhodamine–phalloidin staining in growth cones. Our data demonstrate that Rac1 regulates actin filament organization in neuronal growth cones and is pivotal for β1 integrin–mediated growth cone advance, but not for growth on poly-D lysine. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 524–540, 1998