HSPC300 and its role in neuronal connectivity

<p>Abstract</p> <p>Background</p> <p>The WAVE/SCAR complex, consisting of CYFIP (PIR121 or Sra1), Kette (Nap1), Abi, SCAR (WAVE) and HSPC300, is known to regulate the actin nucleating Arp2/3 complex in a Rac1-dependent manner. While <it>in vitro </it>and <it>in vivo </it>studies have demonstrated that CYFIP, Kette, Abi and SCAR work as subunits of the complex, the role of the small protein HSPC300 remains unclear.</p> <p>Results</p> <p>In the present study, we identify the <it>HSPC300 </it>gene and characterize its interaction with the WAVE/SCAR complex in the <it>Drosophila </it>animal model. On the basis of several lines of evidence, we demonstrate that HSPC300 is an indispensable component of the complex controlling axonal and neuromuscular junction (NMJ) growth. First, the <it>Drosophila HSPC300 </it>expression profile resembles that of other members of the WAVE/SCAR complex. Second, <it>HSPC300 </it>mutation, as well as mutations in the other complex subunits, results in identical axonal and NMJ growth defects. Third, like with other complex subunits, defects in NMJ architecture are rescued by presynaptic expression of the respective wild-type gene. Fourth, HSPC300 genetically interacts with another subunit of the WAVE/SCAR complex. Fifth, HSPC300 physically associates with CYFIP and SCAR.</p> <p>Conclusion</p> <p>Present data provide the first evidence for HSPC300 playing a role in nervous system development and demonstrate <it>in vivo </it>that this small protein works in the context of the WAVE/SCAR complex.</p

Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin

Ezrin, a membrane–actin cytoskeleton linker, which participates in epithelial cell morphogenesis, is held inactive in the cytoplasm through an intramolecular interaction. Phosphatidylinositol 4,5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (T567) are involved in the activation process that unmasks both membrane and actin binding sites. Here, we demonstrate that ezrin binding to PIP2, through its NH2-terminal domain, is required for T567 phosphorylation and thus for the conformational activation of ezrin in vivo. Furthermore, we found that the T567D mutation mimicking T567 phosphorylation bypasses the need for PIP2 binding for unmasking both membrane and actin binding sites. However, PIP2 binding and T567 phosphorylation are both necessary for the correct apical localization of ezrin and for its role in epithelial cell morphogenesis. These results establish that PIP2 binding and T567 phosphorylation act sequentially to allow ezrin to exert its cellular functions

Cortical branched actin determines cell cycle progression

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Specific GFP-binding artificial proteins ( Rep): a new tool for in vitro to live cell applications

International audienceA family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes

A bacterial protein targets the BAHD1 chromatin complex to stimulate type III interferon response

International audienceIntracellular 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-$\lambda$ 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-$\lambda$-mediated immune response to control bacterial colonization of the host

AMOTL1 Promotes Breast Cancer Progression and Is Antagonized by Merlin

AbstractThe Hippo signaling network is a key regulator of cell fate. In the recent years, it was shown that its implication in cancer goes well beyond the sole role of YAP transcriptional activity and its regulation by the canonical MST/LATS kinase cascade. Here we show that the motin family member AMOTL1 is an important effector of Hippo signaling in breast cancer. AMOTL1 connects Hippo signaling to tumor cell aggressiveness. We show that both canonical and noncanonical Hippo signaling modulates AMOTL1 levels. The tumor suppressor Merlin triggers AMOTL1 proteasomal degradation mediated by the NEDD family of ubiquitin ligases through direct interaction. In parallel, YAP stimulates AMOTL1 expression. The loss of Merlin expression and the induction of Yap activity that are frequently observed in breast cancers thus result in elevated AMOTL1 levels. AMOTL1 expression is sufficient to trigger tumor cell migration and stimulates proliferation by activating c-Src. In a large cohort of human breast tumors, we show that AMOTL1 protein levels are upregulated during cancer progression and that, importantly, the expression of AMOTL1 in lymph node metastasis appears predictive of the risk of relapse. Hence we uncover an important mechanism by which Hippo signaling promotes breast cancer progression by modulating the expression of AMOTL1

Free Brick1 Is a Trimeric Precursor in the Assembly of a Functional Wave Complex

Background: The Wave complex activates the Arp2/3 complex, inducing actin polymerization in lamellipodia and membrane ruffles. The Wave complex is composed of five subunits, the smallest of which, Brick1/Hspc300 (Brk1), is the least characterized. We previously reported that, unlike the other subunits, Brk1 also exists as a free form. Principal Findings: Here we report that this free form of Brk1 is composed of homotrimers. Using a novel assay in which purified free Brk1 is electroporated into HeLa cells, we were able to follow its biochemical fate in cells and to show that free Brk1 becomes incorporated into the Wave complex. Importantly, incorporation of free Brk1 into the Wave complex was blocked upon inhibition of protein synthesis and incorporated Brk1 was found to associate preferentially with neosynthesized subunits. Brk1 depleted HeLa cells were found to bleb, as were Nap1, Wave2 or ARPC2 depleted cells, suggesting that this blebbing phenotype of Brk1 depleted cells is due to an impairment of the Wave complex function rather than a specific function of free Brk1. Blebs of Brk1 depleted cells were emitted at sites where lamellipodia and membrane ruffles were normally emitted. In Brk1 depleted cells, the electroporation of free Brk1 was sufficient to restore Wave complex assembly and to rescue the blebbing phenotype. Conclusion: Together these results establish that the free form of Brk1 is an essential precursor in the assembly of

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