Drosophila Cappuccino alleles provide insight into formin mechanism and role in oogenesis.
Drosophila Cappuccino alleles provide insight into formin mechanism and role in oogenesis.
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DOI：
10.1091/mbc.e14-11-1558
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发表时间：
2015-05-15
影响因子：
3.3
通讯作者：
Quinlan ME
中科院分区：
文献类型：
--
作者：
Yoo H;Roth-Johnson EA;Bor B;Quinlan ME
对卡布奇诺基因的七个等位基因的分析表明，这种福明在果蝇卵子发生过程中必须既成核又不断伸长肌动蛋白丝。这些数据还提供了对 formin 总体功能的机制洞察。在果蝇发育过程中，福尔明肌动蛋白成核剂卡布奇诺 (Capu) 有助于在整个卵母细胞中构建细胞质肌动蛋白网。 Capu 的缺失会导致雌性不育，大概是因为极性决定因素在没有网格的情况下无法正确定位。为了更深入地了解 Capu 如何构建这种肌动蛋白网格，我们系统地表征了 7 个 Capu 等位基因，这些等位基因在 Capu 的福尔明同源 2 (FH2) 域中具有错义突变。我们报告说，所有七个等位基因在体内对果蝇繁殖力和肌动蛋白网都有有害影响，但在体外对 Capu 的生化活性有显着不同的影响。通过结合大量和单丝肌动蛋白组装测定，我们发现等位基因对 Capu 成核和持续伸长肌动蛋白丝的能力有不同的影响。我们还在 Capu 的 FH2 结构域的套索区域中发现了一个独特的“循环”。去除该环可增强 Capu 的体外成核、伸长和 F-肌动蛋白捆绑活性。我们对环和七种错义突变的研究结果共同提供了对福尔明功能的一般机制以及卡普在果蝇卵母细胞中的具体作用的机制见解。
Analysis of seven alleles of the cappuccino gene demonstrates that this formin must both nucleate and processively elongate actin filaments during Drosophila oogenesis. The data also provide mechanistic insight into formin function in general. During Drosophila development, the formin actin nucleator Cappuccino (Capu) helps build a cytoplasmic actin mesh throughout the oocyte. Loss of Capu leads to female sterility, presumably because polarity determinants fail to localize properly in the absence of the mesh. To gain deeper insight into how Capu builds this actin mesh, we systematically characterized seven capu alleles, which have missense mutations in Capu's formin homology 2 (FH2) domain. We report that all seven alleles have deleterious effects on fly fertility and the actin mesh in vivo but have strikingly different effects on Capu's biochemical activity in vitro. Using a combination of bulk and single- filament actin-assembly assays, we find that the alleles differentially affect Capu's ability to nucleate and processively elongate actin filaments. We also identify a unique “loop” in the lasso region of Capu's FH2 domain. Removing this loop enhances Capu's nucleation, elongation, and F-actin–bundling activities in vitro. Together our results on the loop and the seven missense mutations provides mechanistic insight into formin function in general and Capu's role in the Drosophila oocyte in particular.