The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1

The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein

Regulation of N-WASP and the Arp2/3 Complex by Abp1 Controls Neuronal Morphology

Polymerization and organization of actin filaments into complex superstructures is indispensable for structure and function of neuronal networks. We here report that knock down of the F-actin-binding protein Abp1, which is important for endocytosis and synaptic organization, results in changes in axon development virtually identical to Arp2/3 complex inhibition, i.e., a selective increase of axon length. Our in vitro and in vivo experiments demonstrate that Abp1 interacts directly with N-WASP, an activator of the Arp2/3 complex, and releases the autoinhibition of N-WASP in cooperation with Cdc42 and thereby promotes N-WASP-triggered Arp2/3 complex-mediated actin polymerization. In line with our mechanistical studies and the colocalization of Abp1, N-WASP and Arp2/3 at sites of actin polymerization in neurons, we reveal an essential role of Abp1 and its cooperativity with Cdc42 in N-WASP-induced rearrangements of the neuronal cytoskeleton. We furthermore show that introduction of N-WASP mutants lacking the ability to bind Abp1 or Cdc42, Arp2/3 complex inhibition, Abp1 knock down, N-WASP knock down and Arp3 knock down, all cause identical neuromorphological phenotypes. Our data thus strongly suggest that these proteins and their complex formation are important for cytoskeletal processes underlying neuronal network formation

Role of β-Catenin in Post-Meiotic Male Germ Cell Differentiation

Though roles of β-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that β-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of β-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of β-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that β-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell β-catenin complex to β-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation

Golgi-localized GAP for Cdc42 functions downstream of ARF1 to control Arp2/3 complex and F-actin dynamics

The small GTP-binding ADP-ribosylation factor 1 (ARF1) acts as a master regulator of Golgi structure and function through the recruitment and activation of various downstream effectors. It has been proposed that members of the Rho family of small GTPases also control Golgi function in coordination with ARF1, possibly through the regulation of Arp2/3 complex and actin polymerization on Golgi membranes. Here, we identify ARHGAP10--a novel Rho GTPase-activating protein (Rho-GAP) that is recruited to Golgi membranes through binding to GTP-ARF1. We show that ARHGAP10 functions preferentially as a GAP for Cdc42 and regulates the Arp2/3 complex and F-actin dynamics at the Golgi through the control of Cdc42 activity. Our results establish a role for ARHGAP10 in Golgi structure and function at the crossroads between ARF1 and Cdc42 signalling pathways

Actin acting at the Golgi

The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in traf- ficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers

Splice variant–specific cellular function of the formin INF2 in maintenance of Golgi architecture

INF2 is a unique formin that can both polymerize and depolymerize actin. One INF2 splice variant localizes in an actin-dependent, web-like network in cytoplasm, whereas a second isoform is ER bound. Suppression of the first isoform causes Golgi dispersion. These findings denote isoform-specific cellular functions for INF2