Molecular, Genetic and Biochemical Analysis of the C. elegans Muscle Gene UNC-60

[9728762] Benian Actin是真核细胞中最丰富的蛋白质之一，它可以聚合成微丝，为真核细胞提供结构，既可以作为单独的分散丝，也可以通过肌动蛋白结合蛋白捆绑在一起形成电缆。肌动蛋白微丝与其相关的运动蛋白（肌球蛋白）一起构成了大量细胞内运动（包括肌肉收缩）的基础。虽然肌动蛋白是研究最深入的真核蛋白之一，但对其功能和调控仍知之甚少。该项目承诺对某些蛋白质如何调节肌动蛋白细胞骨架组装的理解有重要的见解。要使用的模型系统是线虫，秀丽隐杆线虫，一种模式生物，其中许多影响肌肉蛋白的突变已被编目。unc-60（“不协调”）基因编码两种蛋白质，这两种蛋白质都是已知的肌动蛋白结合蛋白家族（ADF/cofilin家族）的成员，迄今为止已在所有真核细胞中发现。对这些蛋白质在脊椎动物体内的生化研究表明，它们主要参与肌动蛋白细丝的解聚。Unc-60突变体提供了一个独特的机会，因为它们是任何多细胞生物中这类肌肉蛋白中唯一可用的突变体。Unc-60突变的蠕虫会瘫痪，它们的肌肉会异常地积聚大量的肌动蛋白丝。本研究的工作假设是，在正常蠕虫中，unc-60的解聚作用是调节聚合形式的肌动蛋白的数量，缺乏这种调节会导致细丝的异常积累。Benian实验室已经成功地在细菌中表达了这两种UNC-60亚型，并纯化并初步表征了蛋白质。将合成RNA微注射到秀丽隐杆线虫的种系中，通常会导致相应基因的表型突变。利用这种方法，贝尼亚实验室已经证明，注射unc-60A RNA而不是unc-60B RNA会导致胚胎死亡。这表明UNC-60A的正常功能与UNC-60B不同，UNC-60A在早期发育中起着至关重要的作用。该项目的目标是：1，确定12个unc-60等位基因的序列变化，并根据表型严重程度和突变在unc-60蛋白结构上的位置解释这些变化；2、确定unc-60编码的两种蛋白的时空表达模式，评估其中一种是肌肉特异性的，另一种是胚胎或非肌肉性的假设；3、研究细菌表达的unc-60A和B对体外肌动蛋白聚合的影响，寻找磷酸化肌苷和丝氨酸磷酸化可能的调控作用；3a，尝试使蛋白质结晶以进行结构分析；4、分析UNC-60特异性抑制基因sup12，看看sup12是否与UNC-60或肌动蛋白相互作用或修饰。SUP-12蛋白的性质很可能为细丝数量的调节提供见解，目前尚不容易通过生化手段获得。这些研究将为细胞如何调节肌动蛋白微丝的聚合提供重要的见解。

9728762 Benian Actin is one of the most abundant proteins in eukaryotic cells It can polymerize into microfilaments that provide structure to eukaryotic cells, either as individual dispersed filaments or bundled together by actin binding proteins into cables. Together with its associated motor proteins, the myosins, actin microfilaments form the basis for a significant amount of intracellular movement, including muscle contraction. Although actin is one of the most intensively studied eukaryotic proteins, there is much about its function and regulation that remains poorly understood. This project promises significant insight into the understanding of how certain proteins regulate the assembly of the actin cytoskeleton. The model system to be used is the nematode worm, C. elegans, a model organism in which many mutations affecting muscle proteins have been catalogued. The unc-60 ("uncoordinated") gene encodes two proteins, both of which are members of a known family of actin-binding proteins (the ADF/cofilin family) which have been found in all eukaryotic cells examined to date. Biochemical studies of these proteins in vertebrates have shown that they are involved primarily in depolymerizing actin filaments. Unc-60 mutants provide a unique opportunity in that they are the only available mutants in this type of muscle protein in any multicellular organism. Unc-60 mutant worms are paralyzed, and their muscles have abnormally large accumulations of actin filaments. The working hypothesis for this study is that in normal worms, depolymerization by unc-60 serves to regulate the amount of actin in polymerized form, and lack of such regulation results in the abnormal accumulation of filaments. The Benian laboratory has successfully expressed both UNC-60 isoforms in bacteria and has purified and initially characterized the proteins. Microinjection of synthetic RNA into the germline of C. elegans often results in a mutant phenotype of the corresponding gene. Using this method, the Be nian lab has shown that injection of unc-60A RNA but not unc-60B RNA results in embryonic lethality. This suggests that the normal function of UNC-60A is different from UNC-60B and that UNC-60A has an essential role in early development. The goals of the project are: 1, to determine the sequence alterations of 12 unc-60 alleles and interpret these with respect to phenotypic severity and position of the mutation on the UNC-60 protein structure; 2, to determine the spatial and temporal patterns of expression of the two proteins encoded by unc-60, to evaluate the hypothesis that one of them is muscle-specific and the other is embryonic or non-muscle; 3, to study the effect of bacterially expressed unc-60A and B on actin polymerization in vitro and look for possible regulation by phosphoinositides and serine phosphorylation; 3a, to attempt to crystallize the proteins for structural analysis; 4, to analyze an unc-60-specific suppressor gene, sup-12, to see if SUP-12 interacts with, or modifies either UNC-60 or actin. The nature of the SUP-12 proteins is likely to provide insight into the regulation of thin filament number, presently not easily obtainable through biochemical means. These studies will provide important insights into how cells regulate the polymerization of actin microfilaments.