Analysis of Enabled (Ena) and GUK-holder (GUKh) in regulating WAVE-driven actin dynamics in Drosophila

肌动蛋白细胞骨架在形态发生过程中提供驱动细胞形状变化和细胞迁移的机械力。肌动蛋白聚合由WASP/WAVE蛋白家族成员控制，该家族成员激活肌动蛋白成核复合体Arp2/3。WASP和WAVE存在于调节果蝇发育中Arp2/3功能不同方面的多蛋白复合物中。我们最近的结构-功能分析揭示了WAVE复合物成员和不同肌动蛋白辅助蛋白之间复杂的相互作用。我们发现WAVE复合体的中心亚基Abi直接结合到果蝇肌动蛋白延长子Enabled （Ena）的EVH1结构域。这种相互作用对WAVE复合体在体内的功能至关重要。Ena在体内是如何调节WAVE活性的？我们将继续对Ena在果蝇发育过程中调节WAVE功能中的作用进行分子分析。此外，我们开始对含有WAVE同源结构域的保守蛋白GUK-holder （GUKh）进行遗传和生化分析。共免疫沉淀实验证明了GUKh和WAVE复合物成员之间的相互作用。实时成像研究表明，GUKh表现出微管+末端跟踪能力，并将Abi招募到微管中。我们未来研究的目标是了解GUKh如何作用于WAVE功能，以及GUKh如何在体内整合肌动蛋白和微管动力学。

The actin cytoskeleton provides mechanical forces to drive cell shape changes and cell migration during morphogenesis. Actin polymerization is controlled by members of the WASP/WAVE protein family that activate the actin nucleating complex Arp2/3. WASP and WAVE exist in multi-protein complexes regulating distinct aspects of Arp2/3 function in Drosophila development. Our recent structure-function analyses revealed an intricate interplay between members of the WAVE complex and different actin accessory proteins. We have found that the central subunit of the WAVE complex, Abi binds directly to the EVH1 domain of the actin elongator Enabled (Ena) in Drosophila. This interaction is crucial for WAVE complex function in vivo. How does Ena regulate WAVE activity in vivo? We will continue our molecular analysis of the role of Ena in regulating WAVE function during Drosophila development. In addition, we started a genetic and biochemical analysis of GUK-holder (GUKh), a conserved WAVE homology domain containing protein. Co-immunoprecipitation experiments demonstrate an interaction between GUKh and members of the WAVE complex. Live-imaging studies revealed that GUKh exhibit microtubule plus end tracking ability and recruits Abi to microtubules. The goal of our future studies is to understand how GUKh acts on WAVE function and how GUKh might integrate actin and microtubule dynamics in vivo.