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

南希·S彼得森9604409（技术摘要）：在黑腹果蝇分叉和烧焦的突变体，肌动蛋白纤维束显着减少发展的刚毛。 因此，在成年苍蝇中，刚毛是扭曲和分支的。 与这两种突变相对应的正常基因最近已被克隆和测序。 该蛋白与海胆肌成束蛋白同源，并在体外将肌动蛋白纤维束成束，表明它可能在肠道刷状微绒毛中发挥类似于fimarin的作用。 果蝇中的分叉基因是正常刚毛发育所必需的，并编码六种具有重叠编码区的蛋白质，这些重叠编码区主要在其氨基末端不同。 初步数据表明，叉状蛋白也是肌动蛋白结合蛋白。 它们的氨基酸序列与一种新发现的肌动蛋白结合蛋白espin的预测序列相似，espin是在大鼠睾丸外质特化中发现的。 此外，espin的抗体在Western印迹上特异性识别叉状蛋白。 这表明，分叉属于一个新的家庭的肌动蛋白结合蛋白，参与肌动蛋白纤维束组装。 本项目的目的是研究分叉蛋白在果蝇刚毛肌动蛋白纤维束形成中的作用。 使用共聚焦显微镜、亲和层析和肌动蛋白沉降测定的初步数据表明，分叉蛋白与肌动蛋白结合，并参与肌动蛋白纤维束形成的启动以及在非常长（100 mm）的纤维束中纵向连接肌动蛋白纤维束。 分叉蛋白中的肌动蛋白结合位点将使用涉及与F-肌动蛋白的体外共沉降的定量测定来鉴定和表征。 将为每种不同的分叉蛋白质制备特异性抗体，这些抗体将用于检查正常脓疱和几种分叉突变（其中某些分叉蛋白质未合成）的刚毛中分叉蛋白质的分布。 肌动蛋白纤维束结构将通过共聚焦显微镜在野生型和突变型puppatients中进行检查。 分叉的同源物将在脊椎动物组织中鉴定，这些组织具有与果蝇刚毛中的肌动蛋白束相似的肌动蛋白束。 (Lay翻译后摘要）：肌动蛋白微丝是一个主要类别的细胞骨架元素无处不在的所有真核生物。 调节肌动蛋白微丝组装成束或纤维是许多细胞功能所必需的，包括细胞运动、改变和维持细胞形状以及细胞分裂。 肌动蛋白结合蛋白在调节这些过程中是必不可少的。 果蝇是遗传学和发育学研究的一个成熟的模型系统，并且正在成为细胞生物学中越来越重要的模型系统。 利用果蝇的适当突变体来研究蛹期刚毛发育过程中涉及的肌动蛋白纤维束是可能的。 这些纤维束在形态上与脊椎动物肠刷状缘和耳及肾中静纤毛中存在的肌动蛋白纤维束非常相似，并且在这些研究中获得的关于肌动蛋白结合蛋白的信息可能不仅与果蝇的生物学有关，而且与一般的真核生物有关。 ***

Nancy S. Petersen 9604409 (Technical Abstract): In Drosophila melanogaster 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 normal genes corresponding to these two mutations have recently been cloned and sequenced. 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 in Drosophila is essential for normal bristle development and encodes six proteins with overlapping coding regions which differ primarily in their amino-terminal ends. Preliminary data indicates that the forked proteins are also actin binding proteins. Their amino acid sequences are similar to the predicted sequence of a newly-identified actin binding protein called espin, has been found in rat testis ectoplasmic specializations. Furthermore, an antibody to espin specifically recognizes forked proteins on Western blots. This suggests that forked belongs to a new family of actin binding proteins that are involved in actin fiber bundle assembly. The aim of this project is to study the role of forked proteins in actin fiber bundle formation in Drosophila bristles. Preliminary data using confocal microscopy, affinity chromatography and actin sedimentation assays indicates that the forked proteins bind to actin and are involved the initiation of actin fiber bundle formation as well as in connecting actin fiber bundles longitudinally in the very long ( 100 mm) bundles. The actin binding sites in the forked proteins will be identified and characterized using quantitative assays involving in vitro co-sedimentation with F-actin. Specific antibodies will be made for each of the different forked proteins, which will be used to examine the distribution of forked proteins in bristles from normal pupae and several forked mutations in which certain of the forked proteins are not synthesized. The actin fiber bundle structure will be examined in wild type and mutant pupae by confocal microscopy. Homologues of forked will be identified in vertebrate tissues which have actin bundles similar to those in Drosophila bristles. (Lay Abstract): Actin microfilaments are a major class of cytoskeletal elements ubiquitous to all eukaryotic organisms. Regulation of the assembly of actin microfilaments into bundles or fibers is required for many cellular functions, including cell movement, changing and maintaining cell shape, and cell division. Actin binding proteins are essential in regulating these processes. The common fruit fly, Drosophila melanogaster, is a well established model system for studies of genetics and development, and is becoming an increasingly important model system for cell biology. It is possible to use appropriate mutants of Drosophila 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, and the information obtained in these studies about actin-binding proteins will likely be relevant not only to the biology of Drosophila but eukaryotic organisms in general. ***