The Role(s) of Forked Proteins in Actin Fiber Bundle Formation

细胞运动、改变和维持细胞形状，甚至细胞分裂都需要调节肌动蛋白纤维的组装。肌动蛋白结合蛋白在调节这些过程中是必不可少的。在黑腹果蝇中，可以利用突变来研究蛹期参与刚毛发育的肌动蛋白纤维束。这些纤维束在形态上与存在于脊椎动物肠刷缘、耳和肾的静纤毛中的肌动蛋白纤维束非常相似，但要大得多。在分叉和烧焦突变体中，肌动蛋白纤维束在发育刚毛时显著减少。因此，在成年苍蝇中，刚毛扭曲并分叉。该蛋白与海胆束蛋白同源，并在体外捆绑肌动蛋白纤维，提示其可能在肠刷状微绒毛中发挥类似于纤蛋白的作用。分叉的基因编码六种蛋白质，这些蛋白质具有重叠的编码区，其主要差异在于它们的氨基末端。叉状蛋白也是肌动蛋白结合蛋白，它与脊椎动物的肌动蛋白结合蛋白——螺旋蛋白是同源的。这些最初是在大鼠睾丸外质特化中发现的，但现在也在肾脏和肠道微绒毛以及耳纤毛中发现。与其他已知的肌动蛋白结合蛋白相比，预测的espin的氨基酸序列更类似于分叉蛋白，并且espin的抗体在Western blots上特异性识别分叉蛋白。这表明forked属于一个新的肌动蛋白结合蛋白家族，它参与了特定类型的紧密排列的平行肌动蛋白束的组装。这些蛋白质之间的差异可能导致束的位置和结构的差异，而相似性则反映了它们的共同功能。Petersen博士将继续研究分叉蛋白在果蝇鬃毛和脊椎动物细胞系中肌动蛋白纤维束形成中的作用。她的实验室已经证明，在肌动蛋白束形成之前，叉状蛋白在刚毛尖端被合成和浓缩，并且它们可以在瞬时转染的脊椎动物细胞中诱导肌动蛋白束的形成。这表明它们在启动束形成中起着关键作用。计划使用脊椎动物细胞的转染来进一步确定蛋白质束形成所需的基本部分，并使用亲和层析和酵母双杂交系统来识别与其他蛋白质相互作用的分叉区域。在这些区域突变的叉形基因的p因子转化将被用来阐明每个结构域在刷毛肌动蛋白束形成中的作用。

Regulation of the assembly of actin fibers is required for cell movement, changing and maintaining cell shape, and even for cell division. Actin binding proteins are essential in regulating these processes. In Drosophila melanogaster it is possible to use mutations to study the actin fiber bundles involved in bristle development during the pupal stage. These fiber bundles are morphologically very similar to actin fiber bundles present in vertebrate intestinal brush border and in stereocilia in the ear and kidney, but much larger. In the forked and singed mutants, actin fiber bundles are dramatically reduced in developing bristles. As a result, in the adult fly, the bristles are twisted and branched. The singed protein is homologous to sea urchin fascin and bundles actin fibers in vitro, indicating that it may play a role similar to fimbrin in intestinal brush boarder microvilli. The forked gene encodes six proteins with overlapping coding regions which differ primarily in their amino-terminal ends. The forked proteins are also actin binding proteins which are homologous to the vertebrate actin binding proteins, the espins. These were originally identified in rat testis ectoplasmic specializations but have now been also found in kidney and intestinal microvilli and in ear stereocilia. The predicted amino acid sequences of the espins are more similar to forked proteins than to other known actin binding proteins, and an antibody to espin specifically recognizes forked proteins on Western blots. This suggests that forked belongs to a new family of actin binding proteins which are involved in the assembly of specific types of tightly packed parallel actin bundles. The differences between these proteins may contribute to the differences in location and architecture of the bundles, while the similarities should reflect their common function(s). Dr. Petersen will continue to study the role(s) of forked proteins in actin fiber bundle formation in Drosophila bristles and in vertebrate cell lines. Her laboratory has shown that forked proteins are synthesized and concentrated in bristle tips just prior to the formation of actin bundles, and that they can induce actin bundle formation in transiently transfected vertebrate cells. This suggests that they play a critical role in initiating bundle formation. It is planned to use the transfection of vertebrate cells to further define the essential parts of the protein required for bundle formation, and to use affinity chromatography and the yeast two-hybrid system to identify the regions of forked which interact with other proteins. P-factor transformation with forked genes mutated in these regions will then be used to clarify the roles of each domain in actin bundle formation in bristles.