Liu Jiangshu, Lee Donghoon M, Yu Cao Guo, Angers Stephane, Harris Tony J C
Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON M5S 3G5, Canada Department of Biochemistry, University of Toronto, Toronto, ON M5S 3G5, Canada.
Mol Biol Cell. 2015 Feb 15;26(4):711-25. doi: 10.1091/mbc.E14-11-1554. Epub 2014 Dec 24.
Cytohesin Arf-GEFs are conserved plasma membrane regulators. The sole Drosophila cytohesin, Steppke, restrains Rho1-dependent membrane cytoskeleton activity at the base of plasma membrane furrows of the syncytial embryo. By mass spectrometry, we identified a single major Steppke-interacting protein from syncytial embryos, which we named Stepping stone (Sstn). By sequence, Sstn seems to be a divergent homologue of the mammalian cytohesin adaptor FRMD4A. Our experiments supported this relationship. Specifically, heterophilic coiled-coil interactions linked Sstn and Steppke in vivo and in vitro, whereas a separate C-terminal region was required for Sstn localization to furrows. Sstn mutant and RNAi embryos displayed abnormal, Rho1-dependent membrane cytoskeleton expansion from the base of pseudocleavage and cellularization furrows, closely mimicking Steppke loss-of-function embryos. Elevating Sstn furrow levels had no effect on the steppke phenotype, but elevating Steppke furrow levels reversed the sstn phenotype, suggesting that Steppke acts downstream of Sstn and that additional mechanisms can recruit Steppke to furrows. Finally, the coiled-coil domain of Steppke was required for Sstn binding and in addition homodimerization, and its removal disrupted Steppke furrow localization and activity in vivo. Overall we propose that Sstn acts as a cytohesin adaptor that promotes Steppke activity for localized membrane cytoskeleton restraint in the syncytial Drosophila embryo.
细胞鸟苷酸交换因子（Cytohesin Arf-GEFs）是保守的质膜调节因子。果蝇中唯一的细胞鸟苷酸交换因子Steppke，在合胞体胚胎质膜沟基部抑制Rho1依赖性膜细胞骨架活性。通过质谱分析，我们从合胞体胚胎中鉴定出一种单一的主要与Steppke相互作用的蛋白质，我们将其命名为垫脚石（Stepping stone，Sstn）。从序列来看，Sstn似乎是哺乳动物细胞鸟苷酸交换因子衔接蛋白FRMD4A的一个不同寻常的同源物。我们的实验支持了这种关系。具体而言，在体内和体外，异源卷曲螺旋相互作用将Sstn和Steppke联系在一起，而Sstn定位于沟需要一个单独的C末端区域。Sstn突变体和RNA干扰胚胎表现出异常的、Rho1依赖性的膜细胞骨架从假分裂和细胞化沟基部扩展，这与Steppke功能丧失的胚胎非常相似。提高Sstn在沟处的水平对steppke表型没有影响，但提高Steppke在沟处的水平可逆转sstn表型，这表明Steppke在Sstn下游起作用，并且其他机制可以将Steppke招募到沟处。最后，Steppke的卷曲螺旋结构域对于Sstn结合以及自身同二聚化是必需的，去除该结构域会破坏Steppke在体内的沟定位和活性。总体而言，我们提出Sstn作为一种细胞鸟苷酸交换因子衔接蛋白，促进Steppke的活性，从而在合胞体果蝇胚胎中对局部膜细胞骨架进行限制。
