NONMUSCLE TROPOMYOSIN ISOFORMS IN DROSOPHILA: IDENTIFICATION AND MODIFICATIONS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 肌动蛋白在整个细胞中形成不同的结构，包括运动细胞前缘的分支肌动蛋白网络，丝状伪足中的平行肌动蛋白束，以及胞质分裂期间的肌动球蛋白收缩装置。 原肌球蛋白通过控制进入肌动蛋白丝参与许多这些结构。 人们对原肌球蛋白在肌肉收缩中的作用了解很多，但对其在非肌肉细胞中的作用了解甚少。 在非肌肉细胞中研究原肌球蛋白是困难的，因为高等真核生物可以表达来自四种不同基因的多达40种不同的剪接变体，在任何一种非肌肉细胞类型中表达多达7种异构体。 这种异构体的多样性使得很难梳理出每种原肌球蛋白类型的贡献。 解决非肌肉原肌球蛋白之谜的关键是找到一个简单的模式生物，具有最少数量的非肌肉原肌球蛋白异构体，可以单独研究。 黑腹果蝇似乎是完美的，因为只有一个非肌肉原肌球蛋白异构体是已知的，以确定其他非肌肉异构体（S）一直没有定论。 我们相信UCSF质谱设备可以帮助我们识别未知的非肌肉原肌球蛋白亚型。 我们计划从果蝇非肌肉细胞（S2细胞）中部分分离出原生原肌球蛋白，利用质谱法鉴定蛋白质序列，然后在体外和活体S2细胞中表征其异构体。 这些研究将有助于阐明原肌球蛋白异构体多样性在非肌肉细胞中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Actin makes different structures throughout cells including a branched actin network at the leading edge of motile cells, parallel actin bundles in filopodia, and actomyosin contractile apparatus during cytokinesis. Tropomyosin participates in many of these structures by controlling access to the actin filament. Much is known about tropomyosin's role in muscle contraction, but much less is understood about its role in nonmuscle cells. It is difficult to study tropomyosin in nonmuscle cells because higher eukaryotes can express up to forty different splice variants from four different genes with up to seven isoforms expressed in any one nonmuscle cell type. This isoform diversity makes it difficult to tease out the contributions of each tropomyosin type. The key to solving the mystery of nonmuscle tropomyosin is finding a simple model organism with a minimal number of nonmuscle tropomyosin isoforms that can be individually studied. Drosophila melanogaster seems to be perfect for this task because only one nonmuscle tropomyosin isoform is known to identify the additional nonmuscle isoform(s) have been inconclusive. We believe the UCSF Mass Spectrometry Facility can help us identify the unknown nonmuscle tropomyosin isoforms. We plan to partially isolate native tropomyosin from Drosophila nonmuscle cells (S2 cells), identify the protein sequence using mass spectrometry, and then characterize the isoforms in vitro an in living S2 cells. These studies will help elucidate the role of tropomyosin isoform diversity in nonmuscle cells.