POWRE: Myosin Heads and Muscle Assembly in C. elegans

为了了解肌肉的功能，有必要了解肌肉组织的超微（纳米级）结构以及该结构是如何组织和组装的。肌节是肌肉组织的功能单位，是一个重复的结构，由两种主要蛋白质成分（肌动蛋白和肌球蛋白）和其他蛋白质成分部分重叠平行交叉的细丝组成。当一组平行重叠的纤维（粗纤维）沿着另一组移动时，整个肌节缩短。整个肌肉中肌节的协调缩短导致整体肌肉缩短或收缩。首席研究员De Stasio博士的长期目标是了解肌球蛋白在横纹肌肌节组装中的作用。肌凝蛋白存在于相对于静止的含有肌动蛋白的细丝运动的粗丝中，引起肌节缩短，从而引起肌肉收缩。肌凝蛋白是一种运动蛋白，它具有酶活性的头部结构域，利用ATP水解形式的化学能沿着肌动蛋白丝移动，从而运行收缩周期。然而，对于头部结构域及其atp酶活性在粗丝和肌节的组装和维持中可能发挥的作用，我们知之甚少。遗传上易于处理的模式生物秀丽隐杆线虫已被广泛用于研究肌肉功能和发育。遗传方法已经确定了秀丽隐杆线虫的肌球蛋白B头部结构域的两类突变，这些突变破坏了粗丝和肌节结构。一类肌凝蛋白突变体在肌凝蛋白头部高度进化保守的功能区域有单个氨基酸的改变。第二类突变体在头结构域内含有翻译终止密码子；这些突变基因的表达将产生部分头部结构域，其中缺乏杆段，但确实包含一个高度保守的氨基酸序列，但尚未指定功能。这两类突变体都是显性的。这些动物是作为杂合子的松弛瘫痪，在大多数情况下，纯合子在早期胚胎时死亡。这些表型比缺乏肌球蛋白B的动物更严重，表明突变的肌球蛋白B是有毒的。早期胚胎致死性进一步表明，突变体干扰了粗丝和肌节组装的某些步骤。De Stasio的实验室利用无义肌凝蛋白突变体作为工具来了解肌凝蛋白头在肌肉组装中的作用。通过产生缺乏棒状编码序列的突变肌球蛋白基因，她希望能够确定导致这种破坏性表型的蛋白质区域。长期目标是通过研究分离的肌球蛋白片段的蛋白-蛋白相互作用和atp酶活性，利用这些截断的基因来探测肌球蛋白的功能。该项目包括一组实验，旨在直接验证截断的肌球蛋白头部的表达，特别是那些包括进化保守结构域的表达，对肌肉组装具有破坏性的假设。包括一个非常短的表位标签的突变肌球蛋白基因结构已经产生，这些基因被微注射到秀丽隐杆线虫的性腺中，在那里DNA被发育中的卵母细胞吸收。宿主秀丽隐杆线虫有一个条件表达系统，即使是致命的转基因也可以维持在允许的温度下，并且转基因的表达可以被控制。De Stasio将利用POWRE奖在麻省理工学院H. Robert Horvitz博士的实验室休假，学习完成项目所需的技术。她需要学习原位表位检测和Rnase保护的艺术，作为一种更定量的评估mRNA表达的方法，她希望计划一套更大的实验来测试肌凝蛋白头的功能。她计划利用表位标记构建物作为分离秀丽隐杆线虫肌球蛋白主要体壁异构体的工具。最后，De Stasio博士计划使用新技术开发至少两个新的实验室实验，以纳入她在家乡劳伦斯大学的分子生物学本科课程，并研究分子建模在这门课程和其他课程中的应用。

In order to understand how muscle functions, it is necessary to know the ultramicroscopic (nanoscale) structure of muscle tissue and how that structure is organized and assembled. The functional unit of muscle tissue, the sarcomere, is a repeating structure consisting of partially overlapping parallel interdigitating filaments of two major protein components, actin and myosin, plus additional protein components. When one set of the parallel overlapping filaments (the thick filaments) move along the other set, the entire sarcomere shortens. Coordinated shortening of sarcomeres throughout the muscle results in overall muscle shortening, or contraction.A long-term goal of the Principal Investigator, Dr. De Stasio, is to understand the role of myosin in the assembly of sarcomeres of striated muscle. Myosin is found in the thick filaments which move relative to the stationary actin-containing thin filaments to cause shortening of the sarcomere and, thereby, contraction of muscle. Myosin is the motor protein, which has an enzymatically active head domain that uses chemical energy in the form of ATP hydrolysis to move itself along the actin filaments and thereby run the contraction cycle. Little is known, however, about the role that the head domain and its ATPase activity might have in the assembly and maintenance of thick filaments and sarcomeres.The genetically tractable model organism, the nematode C. elegans, has been used extensively to study muscle function and development. Genetic approaches have identified two classes of mutations in the myosin B head domain of C. elegans that disrupt thick filament and sarcomere structure. One class of myosin mutants have single amino acid changes in highly evolutionarily conserved functional domains of the myosin head. The second class of mutants contain translational stop codons within the head domain; expression of these mutant genes would produce partial head domains which lack rod segments, but which do contain a highly conserved amino acid sequence for which no function is yet assigned. Both classes of mutants are dominant. These animals are flaccidly paralyzed as heterozygotes and, in most cases, homozygotes die as early embryos. These phenotypes are more severe than that of animals lacking myosin B, indicating that the mutant myosin B is toxic. The early embryonic lethality further indicates that the mutants interfere with some step of thick filament and sarcomere assembly.Dr. De Stasio's lab has used the dominant nonsense myosin mutants as a tool to understand the role of myosin heads in muscle assembly. By producing mutant myosin genes lacking rod-coding sequences, she hopes to identify regions of the protein responsible for the disruptive phenotype. The long-term goal is to then use these truncated genes to probe myosin function by studying protein-protein interactions and ATPase activity of the isolated myosin fragments.This project includes a set of experiments designed to directly test the hypothesis that expression of truncated myosin heads, particularly those including the evolutionarily conserved domain, are disruptive to muscle assembly. Mutant myosin gene constructs including a very short epitope tag have been produced and these are being microinjected into C. elegans gonads where the DNA is taken up by developing oocytes. The host C. elegans have a conditional expression system whereby even lethal transgenes can be maintained at the permissive temperature and transgene expression can be controlled.Dr. De Stasio will use this POWRE award to support a sabbatical leave in the laboratory of Dr. H. Robert Horvitz at MIT to learn techniques necessary to finish the project. She needs to learn the art of in situ epitope detection and Rnase protection as a more quantitative method of assessing mRNA expression, and she wishes to plan a larger set of experiments designed to test the function of myosin heads. She plans to take advantage of the epitope-tagged constructs as a tool for isolating the major body wall isoform of myosin from C. elegans. Lastly, Dr. De Stasio plans to develop at least two new laboratory experiments using new techniques for inclusion in her undergraduate course in molecular biology at her home institution, Lawrence University, and to investigate the applications of molecular modeling for this course and others as well.