Alexis Gautreau

Generation of branched actin networks: assembly and regulation of the N-WASP and WAVE molecular machines

The Arp2/3 complex is a molecular machine that generates branched actin networks responsible for membrane remodeling during cell migration, endocytosis, and other morphogenetic events. This machine requires activators, which themselves are multiprotein complexes. This review focuses on recent advances concerning the assembly of stable complexes containing the most‐studied activators, N‐WASP and WAVE proteins, and the level of regulation that is provided by these complexes. N‐WASP is the paradigmatic auto‐inhibited protein, which is activated by a conformational opening. Even though this regulation has been successfully reconstituted in vitro with isolated N‐WASP, the native dimeric complex with a WIP family protein has unique additional properties. WAVE proteins are part of a pentameric complex, whose basal state and activated state when bound to the Rac GTPase were recently clarified. Moreover, this review attempts to put together diverse observations concerning the WAVE complex in the conceptual frame of an in vivo assembly pathway that has gained support from the recent identification of a precursor.

Retromer-mediated endosomal protein sorting: All WASHed up!

Endosomal protein sorting governs the fate of many physiologically important proteins involved in a panoply of cellular functions. Recent discoveries have revealed a vital role for endosomally localised branched actin patches in facilitating protein sorting. The formation of the actin patches has been shown to require the function of the WASH complex - the major endosomal actin polymerisation-promoting complex - which stimulates the activity of the ubiquitously expressed Arp2/3 complex. Another key component of the endosomal protein-sorting machinery is the retromer complex. Studies now show that retromer mediates the recruitment of the WASH complex and its regulators to endosomes. In this review, recent progress in understanding the role of the WASH complex along with retromer in endosomal protein sorting is discussed.

A Bacterial Protein Targets the BAHD1 Chromatin Complex to Stimulate Type III Interferon Response

A virulence factor secreted by Listeria monocytogenes induces expression of interferon-stimulated genes Intracellular pathogens such as Listeria monocytogenes subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate interferon (IFN)–stimulated genes (ISGs). IFN-λ expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with lntA-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1+/– mice or when lntA was constitutively expressed. Thus, the LntA-BAHD1 interplay may modulate IFN-λ−mediated immune response to control bacterial colonization of the host.

Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia.

WASH and exocyst promote pericellular matrix degradation and tumor cell invasion by enabling localized exocytosis of MT1-MMP from late endosomes.

Synergistic BAR-NPF interactions in actin-driven membrane remodeling.

Cell and organelle shape can profoundly influence proper cellular function. In recent years, two machineries have emerged as major regulators of membrane shape: Bin-Amphiphysin-Rvs161/167 (BAR) domain-containing proteins, which induce membrane invaginations or protrusions, and nucleation promoting factors (NPFs), which activate the Arp2/3 complex and are thus responsible for the generation of branched actin networks that push on membranes. Several BAR-NPF interactions have been shown to induce various types of protrusions, such as lamellipodia or filopodia, or invaginations, including trafficking organelles such as caveolae, endosomes and clathrin-coated pits (CCPs). This review focuses on how collaboration between these two interacting machineries, which emerges as a unified mechanism of membrane remodeling, accounts for such a variety of membrane shapes.

Endosomal recruitment of the WASH complex: Active sequences and mutations impairing interaction with the retromer

The Wiskott‐Aldrich syndrome protein and scar homolog (WASH) complex is the major Arp2/3 activator at the surface of endosomes. The branched actin network, that the WASH complex induces, contributes to cargo sorting and scission of transport intermediates destined for most endosomal routes. A major challenge is to understand how the WASH molecular machine is recruited to the surface of endosomes. The retromer endosomal machinery has been proposed by us and others to play a role in this process.

Identification of a novel candidate gene for non-syndromic autosomal recessive intellectual disability: the WASH complex member SWIP

High-throughput sequencing has greatly facilitated the elucidation of genetic disorders, but compared with X-linked and autosomal dominant diseases, the search for genetic defects underlying autosomal recessive diseases still lags behind. In a large consanguineous family with autosomal recessive intellectual disability (ARID), we have combined homozygosity mapping, targeted exon enrichment and high-throughput sequencing to identify the underlying gene defect. After appropriate single-nucleotide polymorphism filtering, only two molecular changes remained, including a non-synonymous sequence change in the SWIP [Strumpellin and WASH (Wiskott-Aldrich syndrome protein and scar homolog)-interacting protein] gene, a member of the recently discovered WASH complex, which is involved in actin polymerization and multiple endosomal transport processes. Based on high pathogenicity and evolutionary conservation scores as well as functional considerations, this gene defect was considered as causative for ID in this family. In line with this assumption, we could show that this mutation leads to significantly reduced SWIP levels and to destabilization of the entire WASH complex. Thus, our findings suggest that SWIP is a novel gene for ARID.

Molecular dissection of Salmonella-induced membrane ruffling versus invasion

Type III secretion system‐mediated injection of a cocktail of bacterial proteins drives actin rearrangements, frequently adopting the shape of prominent protuberances of ruffling membrane, and culminating in host cell invasion of Gram‐negative pathogens like Salmonella typhimurium. Different Salmonella effectors are able to bind actin and activate Rho‐family GTPases, which have previously been implicated in mediating actin‐dependent Salmonella entry by interacting with N‐WASP or WAVE‐complex, well‐established activators of the actin nucleation machine Arp2/3‐complex. Using genetic deletion and RNA interference studies, we show here that neither individual nor collective removal of these Arp2/3‐ complex activators affected host cell invasion as efficiently as Arp2/3‐complex knock‐down, although the latter was also not essential. However, interference with WAVE‐complex function abrogated Salmonella‐induced membrane ruffling without significantly affecting entry efficiency, actin or Arp2/3‐complex accumulation. In addition, scanning electron microscopy images captured entry events in the absence of prominent membrane ruffles. Finally, localization and RNA interference studies indicated a relevant function in Salmonella entry for the novel Arp2/3‐complex regulator WASH. These data establish for the first time that Salmonella invasion is separable from bacteria‐induced membrane ruffling, and uncover an additional Arp2/3‐complex activator as well as an Arp2/3‐complex‐independent actin assembly activity that contribute to Salmonella invasion.

Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes

Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel colocalization method that is insensitive to the heterogeneity of size and shape of endosomes, we further show that preventing the generation of branched actin networks induces endosomal accumulation of the WASH complex. Moreover, we found that actin depolymerization induces a dramatic decrease in the recovery of endosomal WASH after photobleaching. This result suggests a built-in turnover, where the actin network, i.e. the product of the WASH complex, contributes to the dynamic exchange of the WASH complex by promoting its detachment from endosomes. Our experiments also provide evidence for a role of actin polymerization in the lateral compartmentalization of endosomes: several WASH domains exist at the surface of enlarged endosomes, however, the WASH domains coalesce upon actin depolymerization or Arp2/3 depletion. Branched actin networks are thus involved in the regulation of the size of WASH domains. The potential role of this regulation in membrane scission are discussed.

Function and Regulation of the Endosomal Fusion and Fission Machineries

Organelles within the endomembrane system are connected via vesicle flux. Along the endocytic pathway, endosomes are among the most versatile organelles. They sort cargo through tubular protrusions for recycling or through intraluminal vesicles for degradation. Sorting involves numerous machineries, which mediate fission of endosomal transport intermediates and fusion with other endosomes or eventually with lysosomes. Here we review the recent advances in our understanding of these processes with a particular focus on the Rab GTPases, tethering factors, and retromer. The cytoskeleton has also been recently recognized as a central player in membrane dynamics of endosomes, and this review covers the regulation of the machineries that govern the formation of branched actin networks through the WASH and Arp2/3 complexes in relation with cargo recycling and endosomal fission.