Lysyl hydroxylase 2 deficiency promotes filopodia formation and fibroblast migration.

Lysyl hydroxylase 2 (LH2) regulates intermolecular cross-linking of collagen molecules. Accumulation of LH2-modified collagen, which is highly stable and resistant to collagenase cleavage, is one cause of fibrosis. We previously demonstrated that conventional LH2 knockout mice showed embryonic lethality. Here we established LH2 conditional knockout mice using a tamoxifen-inducible Cre system. Morphological analysis of LH2-deficient fibroblasts by microscopy showed a dramatic increase in the number of filopodia, the finger-like cell surface projections that enable cell movement. The tips and leading edges of these filopodia exhibited up-regulated expression of Myosin-X (Myo10), a regulator of filopodial integrity. Wound healing assays demonstrated that migration of LH2-deficient cells was significantly faster than that of control cells. Gene expression profiling data also supported this phenotype. Together these findings indicate that LH2 deficiency may prevent fibrosis through decreased accumulation of LH2-cross-linked collagen, and that fibroblasts with faster migration contribute to enhanced wound healing activity. In conclusion, our cellular models provide evidence that LH2 deficiency plays a critical role in cell migration mediated through filopodia formation. Understanding the precise role of this phenotype in LH2-deficient cells may be helpful to define the pathogenesis of fibrosis. As such, detailed analyses of fibrosis and wound healing using LH2-deficient mouse models are needed.

VASP-mediated actin dynamics activate and recruit a filopodia myosin.

Filopodia are thin, actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a MyTH-FERM (MF) myosin, DdMyo7 in amoeba, are essential for filopodia initiation. DdMyo7 is localized to dynamic regions of the actin-rich cortex. Analysis of VASP mutants and treatment of cells with anti-actin drugs shows that myosin recruitment and activation in Dictyostelium requires localized VASP-dependent actin polymerization. Targeting of DdMyo7 to the cortex alone is not sufficient for filopodia initiation; VASP activity is also required. The actin regulator locally produces a cortical actin network that activates myosin and together they shape the actin network to promote extension of parallel bundles of actin during filopodia formation. This work reveals how filopodia initiation requires close collaboration between an actin-binding protein, the state of the actin cytoskeleton and MF myosin activity.

Lamellipodia-like protrusions and focal adhesions contribute to collective cell migration in zebrafish.

Collective cell migration is a process where cohorts of cells exhibit coordinated migratory behavior. During individual and collective cellular migration, cells must extend protrusions to interact with the extracellular environment, sense chemotactic cues, and act as points of attachment. The mechanisms and regulators of protrusive behavior have been widely studied in individually migrating cells; however, how this behavior is regulated throughout collectives is not well understood. To address this, we used the zebrafish posterior lateral line primordium (pLLP) as a model. The pLLP is a cluster of ~150 â€‹cells that migrates along the zebrafish trunk, depositing groups of cells that will become sensory organs. To define protrusive behavior, we performed mosaic analysis to sparsely label pLLP cells with a transgene marking filamentous actin. This approach revealed an abundance of brush-like protrusions throughout the pLLP that orient in the direction of migration. Formation of these protrusions depends on the Arp2/3 complex, a regulator of dendritic actin. This argues that these brush-like protrusions are an in vivo example of lamellipodia. Mosaic analysis demonstrated that these lamellipodia-like protrusions are located in a close proximity to the overlying skin. Immunostaining revealed an abundance of focal adhesion complexes surrounding the pLLP. Disruption of these complexes specifically in pLLP cells led to impaired pLLP migration. Finally, we show that Erk signaling, a known regulator of focal adhesions, is required for proper formation of lamellipodia-like protrusions and pLLP migration. Altogether, our results suggest a model where the coordinated dynamics of lamellipodia-like protrusions, making contact with either the overlying skin or the extracellular matrix through focal adhesions, promotes migration of pLLP cells.

Beta secretase 1-dependent amyloid precursor protein processing promotes excessive vascular sprouting through NOTCH3 signalling.

Amyloid beta peptides (Aß) proteins play a key role in vascular pathology in Alzheimer's Disease (AD) including impairment of the blood-brain barrier and aberrant angiogenesis. Although previous work has demonstrated a pro-angiogenic role of Aß, the exact mechanisms by which amyloid precursor protein (APP) processing and endothelial angiogenic signalling cascades interact in AD remain a largely unsolved problem. Here, we report that increased endothelial sprouting in human-APP transgenic mouse (TgCRND8) tissue is dependent on ß-secretase (BACE1) processing of APP. Higher levels of Aß processing in TgCRND8 tissue coincides with decreased NOTCH3/JAG1 signalling, overproduction of endothelial filopodia and increased numbers of vascular pericytes. Using a novel in vitro approach to study sprouting angiogenesis in TgCRND8 organotypic brain slice cultures (OBSCs), we find that BACE1 inhibition normalises excessive endothelial filopodia formation and restores NOTCH3 signalling. These data present the first evidence for the potential of BACE1 inhibition as an effective therapeutic target for aberrant angiogenesis in AD.

Micromolar concentrations of citalopram or escitalopram inhibit glycoprotein VI-mediated and integrin αIIbß3-mediated signaling in human platelets.

Collagen and convulxin induce platelet aggregation through glycoprotein VI (GPVI)-FcRγ-Syk signaling pathway. In addition, fibrinogen induces platelet activation through integrin αIIbß3-FcγRIIa-Syk signaling pathway. We previously reported that high concentrations of selective serotonin reuptake inhibitors (SSRI) reduce platelet aggregation induced by collagen. We further investigated the effects of SSRI on GPVI- and αIIbß3-mediated signaling pathway. Citalopram and escitalopram, two relatively pure SSRI, were used in this study. Both citalopram and escitalopram concentration-dependently inhibited convulxin-induced platelet aggregation, serotonin (5-HT) release and the activation of αIIbß3. 5-HT concentration in washed platelets was unchanged after short-term treatment with citalopram. The additional 5-HT failed to fully rescue the inhibitory effect of citalopram on convulxin-induced aggregation. Convulxin-induced phosphorylation of Syk, LAT, and Akt was inhibited by citalopram and escitalopram. Citalopram inhibited the interaction between FcRγ and Syk, whereas the phosphorylation of FcRγ in response to convulxin remained unaltered. Further, citalopram inhibited the increase of the interaction between serotonin transporter and Syk induced by convulxin. In the presence of Mn2+, escitalopram inhibited the formation of lamellipodia on immobilized fibrinogen. Escitalopram did not influence the binding of fibrinogen to platelets. It inhibited the phosphorylation of Syk and PAK triggered by the adhesion on fibrinogen. Our data demonstrate that micromolar concentrations of citalopram and escitalopram inhibit GPVI- and αIIbß3-mediated platelet functions. The mechanism of the inhibitory effect of citalopram or escitalopram is not the influence on the activation of GPVI or the interaction between fibrinogen and αIIbß3, but the interaction between Syk and its upstream molecules.

ARL4C stabilized by AKT/mTOR pathway promotes the invasion of PTEN-deficient primary human glioblastoma.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) deficiency in primary human glioblastoma (GBM) is associated with increased invasiveness and poor prognosis with unknown mechanisms. Therefore, how loss of PTEN promotes GBM progression remains to be elucidated. Herein, we identified that ADP-ribosylation factor like-4C (ARL4C) was highly expressed in PTEN-deficient human GBM cells and tissues. Mechanistically, loss of PTEN stabilized ARL4C protein due to AKT/mTOR pathway-mediated inhibition of ARL4C ubiquitination. Functionally, ARL4C enhanced the progression of GBM cells in vitro and in vivo. Moreover, microarray profiling and GST pull-down assay identified that ARL4C accelerated tumor progression via RAC1-mediated filopodium formation. Importantly, targeting PTEN potently inhibited GBM tumor progression in vitro and in vivo, whereas overexpression of ARL4C reversed the tumor progression impaired by PTEN overexpression. Clinically, analyses with patients' specimens validated a negative correlation between PTEN and ARL4C expression. Elevated ARL4C expression but PTEN deficiency in tumor was associated with poorer disease-free survival and overall survival of GBM patients. Taken together, ARL4C is critical for PTEN-deficient GBM progression and acts as a novel prognostic biomarker and a potential therapeutic candidate. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

NAA80 is actin's N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility.

Actin, one of the most abundant proteins in nature, participates in countless cellular functions ranging from organelle trafficking and pathogen motility to cell migration and regulation of gene transcription. Actin's cellular activities depend on the dynamic transition between its monomeric and filamentous forms, a process exquisitely regulated in cells by a large number of actin-binding and signaling proteins. Additionally, several posttranslational modifications control the cellular functions of actin, including most notably N-terminal (Nt)-acetylation, a prevalent modification throughout the animal kingdom. However, the biological role and mechanism of actin Nt-acetylation are poorly understood, and the identity of actin's N-terminal acetyltransferase (NAT) has remained a mystery. Here, we reveal that NAA80, a suggested NAT enzyme whose substrate specificity had not been characterized, is Nt-acetylating actin. We further show that actin Nt-acetylation plays crucial roles in cytoskeletal assembly in vitro and in cells. The absence of Nt-acetylation leads to significant differences in the rates of actin filament depolymerization and elongation, including elongation driven by formins, whereas filament nucleation by the Arp2/3 complex is mostly unaffected. NAA80-knockout cells display severely altered cytoskeletal organization, including an increase in the ratio of filamentous to globular actin, increased filopodia and lamellipodia formation, and accelerated cell motility. Together, the results demonstrate NAA80's role as actin's NAT and reveal a crucial role for actin Nt-acetylation in the control of cytoskeleton structure and dynamics.

Afadin Facilitates Vascular Endothelial Growth Factor-Induced Network Formation and Migration of Vascular Endothelial Cells by Inactivating Rho-Associated Kinase Through ArhGAP29.

OBJECTIVE: We previously reported that afadin, an actin filament-binding protein, regulated vascular endothelial growth factor-induced angiogenesis. However, the underlying molecular mechanisms are poorly understood. Here, we investigated the mechanisms of how Rho-associated kinase is activated in afadin-knockdown human umbilical vein endothelial cells (HUVECs) and how its activation is involved in defects of vascular endothelial growth factor-induced network formation and migration of the cells. APPROACH AND RESULTS: Knockdown of afadin or ArhGAP29, a GTPase-activating protein for RhoA, increased Rho-associated kinase activity and reduced the vascular endothelial growth factor-induced network formation and migration of cultured HUVECs, accompanied by the defective formation of membrane protrusions, such as lamellipodia and peripheral ruffles. Treatment of the afadin- or ArhGAP29-knockdown HUVECs with Rho-associated kinase inhibitors, Y-27632 or fasudil, partially restored the reduced network formation and migration as well as the defective formation of membrane protrusions. ArhGAP29 bound to afadin and was colocalized with afadin at the leading edge of migrating HUVECs. The defective formation of membrane protrusions in ArhGAP29-knockdown HUVECs was restored by expression of mutant ArhGAP29 that bound to afadin and contained a RhoGAP domain but not mutant ArhGAP29 that could bind to afadin and lacked the RhoGAP domain or mutant ArhGAP29 that could not bind to afadin and contained the RhoGAP domain. This suggested the requirement of both the interaction of afadin with ArhGAP29 and RhoGAP activity of ArhGAP29 for migration of HUVECs. CONCLUSIONS: Our results highlight a critical role of the afadin-ArhGAP29 axis for the regulation of Rho-associated kinase activity during vascular endothelial growth factor-induced network formation and migration of HUVECs.

FGD5 Regulates VEGF Receptor-2 Coupling to PI3 Kinase and Receptor Recycling.

OBJECTIVE: VEGF (vascular endothelial growth factor-A) signaling to the endothelial cell (EC) through VEGFR2 (VEGF receptor-2) is the principal cue driving new blood vessel formation. FGD5 (faciogenital dysplasia-5)-a Rho-family guanine nucleotide exchange factor-is selectively expressed in EC. Deficiency of FGD5 is embryonically lethal in mice and perturbs angiogenesis and VEGF signal transduction. However, the mechanism of FGD5 regulation of VEGF signaling is poorly understood. APPROACH AND RESULTS: Angiogenic sprouting and EC cytoskeletal remodeling were evaluated in a 3-dimensional in vitro model. We examined the subcellular localization of FGD5 and VEGFR2 in EC by immunofluorescent staining and studied the association by immunoprecipitation. FGD5 deficiency reduced the number of angiogenic sprouts and tip cell filopodia by ≈80% and ≈70%, respectively. These defects were accompanied by downregulation of the expression of tip cell-specific markers. FGD5 inactivation led to a decrease in EC migration and early protrusion (lamellipodia) formation. In resting and VEGF-stimulated EC, FGD5 forms a complex with VEGFR2 and was enriched at the leading edge of the cell and among endosomes. FGD5 loss reduced mTORC2 (mammalian target of rapamycin complex-2)/Akt-dependent cortactin activation downstream of VEGFR2 but did not alter VEGFR2 plasma membrane expression, Y1175 phosphorylation, or endocytosis. However, FGD5 loss decreased endosomal VEGFR2 coupling to phosphoinositide-3 kinase and diverted VEGFR2 to lysosomal degradation. CONCLUSIONS: FGD5 regulates VEGFR2 retention in recycling endosomes and coupling to PI3 (phosphoinositide-3) kinase/mTORC2-dependent cytoskeletal remodeling.

The helicase, DDX3X, interacts with poly(A)-binding protein 1 (PABP1) and caprin-1 at the leading edge of migrating fibroblasts and is required for efficient cell spreading.

DDX3X, a helicase, can interact directly with mRNA and translation initiation factors, regulating the selective translation of mRNAs that contain a structured 5' untranslated region. This activity modulates the expression of mRNAs controlling cell cycle progression and mRNAs regulating actin dynamics, contributing to cell adhesion and motility. Previously, we have shown that ribosomes and translation initiation factors localise to the leading edge of migrating fibroblasts in loci enriched with actively translating ribosomes, thereby promoting steady-state levels of ArpC2 and Rac1 proteins at the leading edge of cells during spreading. As DDX3X can regulate Rac1 levels, cell motility and metastasis, we have examined DDX3X protein interactions and localisation using many complementary approaches. We now show that DDX3X can physically interact and co-localise with poly(A)-binding protein 1 and caprin-1 at the leading edge of spreading cells. Furthermore, as depletion of DDX3X leads to decreased cell motility, this provides a functional link between DDX3X, caprin-1 and initiation factors at the leading edge of migrating cells to promote cell migration and spreading.

Myristoylated Alanine-Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation.

BACKGROUND: Transcription of the myristoylated alanine-rich C kinase substrate (MARCKS) is upregulated in animal models of intimal hyperplasia. MARCKS knockdown inhibits vascular smooth muscle cell (VSMC) migration in vitro; however, the mechanism is as yet unknown. We sought to elucidate the mechanism of MARCKS-mediated motility and determine whether MARCKS knockdown reduces intimal hyperplasia formation in vivo. METHODS AND RESULTS: MARCKS knockdown blocked platelet-derived growth factor (PDGF)-induced translocation of cortactin to the cell cortex, impaired both lamellipodia and filopodia formation, and attenuated motility of human coronary artery smooth muscle cells (CASMCs). Activation of the small GTPases, Rac1 and Cdc42, was prevented by MARCKS knockdown. Phosphorylation of MARCKS resulted in a transient shift of MARCKS from the plasma membrane to the cytosol. MARCKS knockdown significantly decreased membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Cotransfection with an intact, unphosphorylated MARCKS, which has a high binding affinity for PIP2, restored membrane-associated PIP2 levels and was indispensable for activation of Rac1 and Cdc42 and, ultimately, VSMC migration. Overexpression of MARCKS in differentiated VSMCs increased membrane PIP2 abundance, Rac1 and Cdc42 activity, and cell motility. MARCKS protein was upregulated early in the development of intimal hyperplasia in the murine carotid ligation model. Decreased MARKCS expression, but not total knockdown, attenuated intimal hyperplasia formation. CONCLUSIONS: MARCKS upregulation increases VSMC motility by activation of Rac1 and Cdc42. These effects are mediated by MARCKS sequestering PIP2 at the plasma membrane. This study delineates a novel mechanism for MARCKS-mediated VSMC migration and supports the rational for MARCKS knockdown to prevent intimal hyperplasia.

Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers.

The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity.

Loss of oligophrenin1 leads to uncontrolled Rho activation and increased thrombus formation in mice.

BACKGROUND: Platelet cytoskeletal reorganization is essential for platelet adhesion and thrombus formation in hemostasis and thrombosis. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of platelets and induce filopodia and lamellipodia formation and actin polymerization to strongly increase the platelet surface upon activation. Moreover, they are important for platelet secretion, integrin activation and arterial thrombus formation. OBJECTIVES: Rho GTPases are regulated by GTPase-activating proteins (GAPs) that stimulate their GTPase activity to terminate Rho signaling. The regulation of Rho GTPase activity in platelets is not well defined. Recently, we identified oligophrenin1 (OPHN1), a RhoGAP in platelets that exhibits strong GTPase-stimulating activity towards RhoA, Cdc42 and Rac1. RESULTS: In the present study we show for the first time, that deficiency of OPHN1 led to abnormal Rho activation and increased platelet cytoskeletal reorganization, including cell adhesion and lamellipodia formation on fibrinogen. Furthermore, platelets from ophn1(-/-) mice showed enhanced susceptibility to platelet activation with alterations in actin distribution and early release of granules. Platelet activation was enhanced following GPVI and PAR4 stimulation. This translated into elevated platelet thrombus formation and promoted arterial thrombosis under low shear conditions with altered hemostasis, as detected by tail bleeding time. CONCLUSIONS: The results of the present study identified OPHN1 as an important regulator of platelet cytoskeletal reorganization and demonstrate that abnormal regulation of Rho proteins leads to increased platelet adhesion and thrombus formation under low shear conditions in vitro and in vivo, suggesting a prothrombotic phenotype of mice critical for acute thrombotic occlusions.

Mouse macrophages completely lacking Rho subfamily GTPases (RhoA, RhoB, and RhoC) have severe lamellipodial retraction defects, but robust chemotactic navigation and altered motility.

RhoA is thought to be essential for coordination of the membrane protrusions and retractions required for immune cell motility and directed migration. Whether the subfamily of Rho (Ras homolog) GTPases (RhoA, RhoB, and RhoC) is actually required for the directed migration of primary cells is difficult to predict. Macrophages isolated from myeloid-restricted RhoA/RhoB (conditional) double knock-out (dKO) mice did not express RhoC and were essentially "pan-Rho"-deficient. Using real-time chemotaxis assays, we found that retraction of the trailing edge was dissociated from the advance of the cell body in dKO cells, which developed extremely elongated tails. Surprisingly, velocity (of the cell body) was increased, whereas chemotactic efficiency was preserved, when compared with WT macrophages. Randomly migrating RhoA/RhoB dKO macrophages exhibited multiple small protrusions and developed large "branches" due to impaired lamellipodial retraction. A mouse model of peritonitis indicated that monocyte/macrophage recruitment was, surprisingly, more rapid in RhoA/RhoB dKO mice than in WT mice. In comparison with dKO cells, the phenotypes of single RhoA- or RhoB-deficient macrophages were mild due to mutual compensation. Furthermore, genetic deletion of RhoB partially reversed the motility defect of macrophages lacking the RhoGAP (Rho GTPase-activating protein) myosin IXb (Myo9b). In conclusion, the Rho subfamily is not required for "front end" functions (motility and chemotaxis), although both RhoA and RhoB are involved in pulling up the "back end" and resorbing lamellipodial membrane protrusions. Macrophages lacking Rho proteins migrate faster in vitro, which, in the case of the peritoneum, translates to more rapid in vivo monocyte/macrophage recruitment.

Molecular cloning, sequence analysis, prokaryotic expression, and function prediction of foot-specific peroxidase in Hydra magnipapillata Chinese strain.

The cDNA sequence of foot-specific peroxidase PPOD1 from the Chinese strain of Hydra magnipapillata was cloned by reverse transcription-polymerase chain reaction. The cDNA sequence contained a coding region with an 873-bp open reading frame, a 31-bp 5'-untranslated region, and a 36-bp 3'-untranslated region. The structure prediction results showed that PPOD1 contains 10.34% of α-helix, 38.62% of extended strand, 12.41% of ß-turn, and 38.62% of random coil. The structural core was α-helix at the N terminus. The GenBank protein blast server showed that PPOD1 contains 2 fascin-like domains. In addition, high-level PPOD1 activity was only present in the ectodermal epithelial cells located on the edge of the adhesive face of the basal disc, and that these cells extended lamellipodia and filopodia when the basal disc was tightly attached to a glass slide. The fascin-like domains of Hydra PPOD1 might contribute to the bundling of the actin filament of these cells, and hence, the formation of filopodia. In conclusion, these cells might play an important role in strengthening the adsorbability of the basal disc to substrates.

PDK1-mediated activation of MRCKα regulates directional cell migration and lamellipodia retraction.

Directional cell migration is of paramount importance in both physiological and pathological processes, such as development, wound healing, immune response, and cancer invasion. Here, we report that 3-phosphoinositide-dependent kinase 1 (PDK1) regulates epithelial directional migration and invasion by binding and activating myotonic dystrophy kinase-related CDC42-binding kinase α (MRCKα). We show that the effect of PDK1 on cell migration does not involve its kinase activity but instead relies on its ability to bind membrane phosphatidylinositol (3,4,5)-trisphosphate. Upon epidermal growth factor (EGF) stimulation, PDK1 and MRCKα colocalize at the cell membrane in lamellipodia. We demonstrate that PDK1 positively modulates MRCKα activity and drives its localization within lamellipodia. Likewise, the retraction phase of lamellipodia is controlled by PDK1 through an MRCKα-dependent mechanism. In summary, we discovered a functional pathway involving PDK1-mediated activation of MRCKα, which links EGF signaling to myosin contraction and directional migration.

SRF phosphorylation by glycogen synthase kinase-3 promotes axon growth in hippocampal neurons.

The growth of axons is an intricately regulated process involving intracellular signaling cascades and gene transcription. We had previously shown that the stimulus-dependent transcription factor, serum response factor (SRF), plays a critical role in regulating axon growth in the mammalian brain. However, the molecular mechanisms underlying SRF-dependent axon growth remains unknown. Here we report that SRF is phosphorylated and activated by GSK-3 to promote axon outgrowth in mouse hippocampal neurons. GSK-3 binds to and directly phosphorylates SRF on a highly conserved serine residue. This serine phosphorylation is necessary for SRF activity and for its interaction with MKL-family cofactors, MKL1 and MKL2, but not with TCF-family cofactor, ELK-1. Axonal growth deficits caused by GSK-3 inhibition could be rescued by expression of a constitutively active SRF. The SRF target gene and actin-binding protein, vinculin, is sufficient to overcome the axonal growth deficits of SRF-deficient and GSK-3-inhibited neurons. Furthermore, short hairpin RNA-mediated knockdown of vinculin also attenuated axonal growth. Thus, our findings reveal a novel phosphorylation and activation of SRF by GSK-3 that is critical for SRF-dependent axon growth in mammalian central neurons.

Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1.

The class III phosphatidylinositol 3-kinase, VPS34, phosphorylates the D3 hydroxyl of inositol generating phosphatidylinositol 3-phosphate (ptdins(3)p). Initial studies suggested that ptdins(3)p solely functioned as a component of vesicular and endosomal membranes and that VPS34 did not function in signal transduction. However, VPS34 has recently been shown to be required for insulin-mediated activation of S6 kinase 1 (S6K1). Whether VPS34 activity is directly regulated by insulin is unclear. It is also not known whether VPS34 activity can be spatially restricted in response to extracellular stimuli. Data presented here demonstrate that in response to insulin, VPS34 is activated and translocated to lamellipodia where it produces ptdins(3)p. The localized production of ptdins(3)p is dependent on Src phosphorylation of VPS34. In cells expressing VPS34 with mutations at Y231 or Y310, which are Src-phosphorylation sites, insulin-stimulated VPS34 translocation to the plasma membrane and lamellipodia formation are blocked. mTOR also colocalizes with VPS34 and ptdins(3)p at lamellipodia following insulin-stimulation. In cells expressing the VPS34-Y231F mutant, which blocks lamellipodia formation, mTOR localization at the plasma membrane and insulin-mediated S6K1 activation are reduced. This suggests that mTOR localization at lamellipodia is important for full activation of S6K1 induced by insulin. These data demonstrate that insulin can spatially regulate VPS34 activity through Src-mediated tyrosine phosphorylation and that this membrane localized activity contributes to lamellipodia formation and activation of mTOR/S6K1signaling.

Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells.

The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.

Myosin 3A kinase activity is regulated by phosphorylation of the kinase domain activation loop.

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.