Robert H. Insall

Scar1 and the related Wiskott–Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex

BACKGROUND The actin-related proteins Arp2 and Arp3 are part of a seven-protein complex which is localized in the lamellipodia of a variety of cell types, and in actin-rich spots of unknown function. The Arp2/3 complex enhances actin nucleation and causes branching and crosslinking of actin filaments in vitro; in vivo it is thought to drive the formation of lamellipodia and to be a control center for actin-based motility. The Wiskott-Aldrich syndrome protein, WASP, is an adaptor protein implicated in the transmission of signals from tyrosine kinase receptors and small GTPases to the actin cytoskeleton. Scar1 is a member of a new family of proteins related to WASP, and it may also have a role in regulating the actin cytoskeleton. Scar1 is the human homologue of Dictyostelium Scar1, which is thought to connect G-protein-coupled receptors to the actin cytoskeleton. The mammalian Scar family contains at least four members. We have examined the relationships between WASP, Scar1, and the Arp2/3 complex. RESULTS We have identified WASP and its relative Scar1 as proteins that interact with the Arp2/3 complex. We have used deletion analysis to show that both WASP and Scar1 interact with the p21 subunit of the Arp2/3 complex through their carboxyl termini. Overexpression of carboxy-terminal fragments of Scar1 or WASP in cells caused a disruption in the localization of the Arp2/3 complex and, concomitantly, induced a complete loss of lamellipodia and actin spots. The induction of lamellipodia by platelet-derived growth factor was also suppressed by overexpression of the fragment of Scar1 that binds to the Arp2/3 complex. CONCLUSIONS We have identified a conserved sequence domain in proteins of the WASP family that binds to the Arp2/3 complex. Overexpression of this domain in cells disrupts the localization of the Arp2/3 complex and inhibits lamellipodia formation. Our data suggest that WASP-related proteins may regulate the actin cytoskeleton through the Arp2/3 complex.

Actin Dynamics at the Leading Edge: From Simple Machinery to Complex Networks

Cell migration is an essential feature of eukaryotic life, required for processes ranging from feeding and phagoctyosis to development, healing, and immunity. Migration requires the actin cytoskeleton, specifically the localized polymerization of actin filaments underneath the plasma membrane. Here we summarize recent developments in actin biology that particularly affect structures at the leading edge of the cell, including the structure of actin branches, the multiple pathways that lead to cytoskeleton assembly and disassembly, and the role of blebs. Future progress depends on connecting these processes and components to the dynamic behavior of the whole cell in three dimensions.

Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased choices between random protrusions

Current models of eukaryotic chemotaxis propose that directional sensing causes localized generation of new pseudopods. However, quantitative analysis of pseudopod generation suggests a fundamentally different mechanism for chemotaxis in shallow gradients: first, pseudopods in multiple cell types are usually generated when existing ones bifurcate and are rarely made de novo; second, in Dictyostelium cells in shallow chemoattractant gradients, pseudopods are made at the same rate whether cells are moving up or down gradients. The location and direction of new pseudopods are random within the range allowed by bifurcation and are not oriented by chemoattractants. Thus, pseudopod generation is controlled independently of chemotactic signalling. Third, directional sensing is mediated by maintaining the most accurate existing pseudopod, rather than through the generation of new ones. Finally, the phosphatidylinositol 3-kinase (PI(3)K) inhibitor LY294002 affects the frequency of pseudopod generation, but not the accuracy of selection, suggesting that PI(3)K regulates the underlying mechanism of cell movement, rather than control of direction.

PIP3, PIP2, and cell movement--similar messages, different meanings?

The inositol lipids PI(4,5)P(2) and PI(3,4,5)P(3) are important regulators of actin polymerization, but their different temporal and spatial dynamics suggest that they perform separate roles. PI(3,4,5)P(3) seems to act as an instructive second messenger, inducing local actin polymerization. PI(4,5)P(2) appears to be present at too high a concentration and homogeneous a distribution to fulfil a similar role. Instead, we suggest that PI(4,5)P(2) acts permissively, restricting new actin polymerization to the region of the plasma membrane.

PIR121 Regulates Pseudopod Dynamics and SCAR Activity in Dictyostelium

BACKGROUND The WASP/SCAR family of adaptor proteins coordinates actin reorganization by coupling different signaling molecules, including Rho-family GTPases, to the activation of the Arp2/3 complex. WASP binds directly to Cdc42 through its GTPase binding domain (GBD), but SCAR does not contain a GBD, and no direct binding has been found. However, SCAR has recently been found to copurify with four other proteins in a complex. One of these, PIR121, binds directly to Rac. RESULTS We have identified four of the members of this complex in Dictyostelium and disrupted the pirA gene, which encodes PIR121. The resulting mutant cells are unusually large, maintain an excessive proportion of their actin in a polymerized state and display severe defects in movement and chemotaxis. They also continually extend new pseudopods by widening and splitting existing leading edges rather than by initiating new pseudopods. Comparing these cells to scar null mutants shows behavior that is broadly consistent with overactivation of SCAR. Deletion of the pirA gene in a scar(-) mutant resulted in cells resembling their scar(-) parents with no obvious changes, confirming that PIR121 mainly acts through SCAR in vivo. Surprisingly given their hyperactive phenotype, we find that pirA(-) mutants contain very little intact SCAR protein despite normal levels of mRNA, suggesting a posttranscriptional downregulation of activated SCAR. CONCLUSIONS Our results demonstrate a genetic connection between the pirA and scar genes. PIR121 appears to inhibit the activity of SCAR in the absence of activating signals. The location of the newly formed protrusions indicates that unregulated SCAR is acting at the edges of existing pseudopods, not elsewhere in the cell. We suggest that active SCAR protein released from the inhibitory complex is rapidly removed and that this is an important and novel mechanism for controlling actin dynamics.

The aimless RasGEF is required for processing of chemotactic signals through G-protein-coupled receptors in Dictyostelium

BACKGROUND Ras proteins are small GTP-binding proteins that play an essential role in a wide range of processes, particularly in mammalian growth control. They act as molecular switches, being inactive when GDP is bound, and active when associated with GTP. Activation is accomplished by guanine nucleotide exchange factors (RasGEFs); when RasGEFs interact with Ras proteins, GDP is allowed to escape, and is replaced by GTP. Dictyostelium responds to chemoattractants through typical seven transmembrane domain receptors and heterotrimeric G proteins. There are at least five different Dictyostelium Ras genes, whose functions are not yet known. RESULTS We have isolated the aimless gene, which encodes the Dictyostelium homologue of RasGEFs, during a screen for insertional mutants that fail to aggregate. We found that aimless null mutants grew at a normal rate, but were severely impaired in both chemotaxis and activation of adenylyl cyclase, both of which are critical for the early stages of development. Although coupling between receptors and their G proteins is unaffected, and several cyclic AMP (cAMP)-mediated responses appear normal, activation of adenylyl cyclase by receptors and GTP gamma S (a non-hydrolyzable GTP analogue) is reduced by up to 95%. The motility of mutant cells appears normal, suggesting a true defect in gradient sensing. CONCLUSIONS The discovery of the aimless gene adds an interesting new member to the family of RasGEFs. Our data suggest an unforeseen role for a RasGEF, and therefore presumably a complete Ras pathway, in the processing of chemotactic signals through G-protein-coupled receptors.

Chemotaxis: finding the way forward with Dictyostelium

Understanding cell migration is centrally important to modern cell biology. However, despite years of study, progress has been hindered by experimental limitations and the complexity of the process. This has led to the popularity of Dictyostelium discoideum, with its experimentally-friendly lifestyle and small, haploid genome, as a tool to dissect the pathways involved in migration. This humble amoeba is now established at the centre of dramatic changes in our understanding of cell movement. In this review we describe the recent reinterpretation of the role of phosphatidylinositol trisphosphate (PIP(3)) and other intracellular messengers that connect signalling and migration, and the transition to models of chemotaxis driven by multiple, intertwined signalling pathways. In shallow gradients, pseudopods are generated with random directions, and we discuss how chemotaxis can operate by biasing this process. Overall we describe how Dictyostelium has the potential to unlock many fundamental questions in the cell motility field.

Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

WASH coats mature lysosomes and is required for exocytosis of indigestible material.

N-WASP coordinates the delivery and F-actin–mediated capture of MT1-MMP at invasive pseudopods

N-WASP is critical for cancer cell invasion through its promotion of the trafficking and capture of MT1-MMP in invasive pseudopods.

Chemotaxis: A Feedback-Based Computational Model Robustly Predicts Multiple Aspects of Real Cell Behaviour

A simple feedback model of chemotaxis explains how new pseudopods are made and how eukaryotic cells steer toward chemical gradients.