Genetic Analysis of the Regulation of Cytoplasmic Dynein

细胞质动力蛋白是一种多亚基复合物，其作为微管相关马达发挥功能，所述微管相关马达是核运动和定位、纺锤体组装、染色体分离、高尔基体组织、ER至高尔基体运输以及轴突中细胞器的逆行运输所需的。动力蛋白的功能和与各种货物的相互作用需要一个额外的多亚基复合物，称为动力蛋白。动力蛋白重链是细胞中最大的蛋白质之一（4000个残基），C-末端三分之二形成马达结构域，N-末端区域形成在货物相互作用中起作用的延伸尾部。细胞质动力蛋白功能在后生动物中是必不可少的（例如，小鼠和果蝇）;然而，在丝状真菌中，动力蛋白对于生存力不是必需的，这大大简化了对这种巨大复合体的遗传分析。使用丝状真菌粗糙脉孢菌（最近公布了其完整的基因组序列），已分离出1000个细胞质动力蛋白或动力蛋白功能缺陷的突变体，包括300个独立的动力蛋白重链突变体。克隆的动力蛋白/动力肌动蛋白基因的序列分析以及整个N. crassa基因组序列分析表明，该复合体在N. crassa相对于后生动物支持使用N. crass作为动力蛋白结构、功能和调控分析的模型系统。在这个奖项下要做的工作中，大量的N。crassa动力蛋白突变体将用于探索动力蛋白运动活性的调节。已有的研究表明，N. crassa细胞质动力蛋白马达ATP酶活性在动力蛋白功能缺陷的突变体中通过磷酸化而失活（即，货物相互作用）或LIS 1复合物的功能。这种失活不是通过动力蛋白重链的磷酸化，而是通过与重链相关的小蛋白（8-和20-kDa）的磷酸化发生的。为了确定这些蛋白质的身份，将标记动力蛋白重链，亲和纯化，并通过2D凝胶解析相关蛋白质。将重链相关多肽从2D凝胶上切下，并通过使用质谱分析结合完整的N. crassa基因组序列。编码这些动力蛋白相关亚基的基因中无效突变的影响将通过测定动力蛋白ATP酶活性来确定。额外的工作将集中在定义特定重链突变的影响。对于产生全长多肽的突变体，将鉴定至少100个独立的动力蛋白重链突变。将检查这些突变体的动力蛋白ATP酶活性、微管结合以及与其他动力蛋白亚基的结合缺陷。来自该分析的信息将补充上述工作，并将提供动力蛋白马达沿沿着微管易位的机制的见解。 胞质动力蛋白是胞质中最复杂的运动蛋白，N。crassa代表了唯一的模式生物，其中可以快速分离出数百种动力蛋白功能缺陷的突变体。这项工作的完成将有助于理解动力蛋白运动活性的调节，并将首次对动力蛋白重链进行大规模的遗传解剖。各少数群体的本科生在当前和过去的研究活动中发挥了重要作用，并将鼓励更多的学生参与这些研究工作。

Cytoplasmic dynein is a multisubunit complex that functions as a microtubule-associated motor required for nuclear movement and positioning, assembly of the spindle, chromosome segregation, organization of Golgi, ER to Golgi trafficking, and retrograde transport of organelles in axons. An additional multisubunit complex known as dynactin is required for dynein function and interaction with various cargoes. The dynein heavy chain is one of the largest proteins in the cell (4000 residues) with the C-terminal two-thirds forming the motor domain and the N-terminal region forming an extended tail that functions in cargo interaction. Cytoplasmic dynein function is essential in metazoans (e.g., mouse and Drosophila); however, in filamentous fungi, dynein is not essential for viability and this has greatly simplified genetic analysis of this enormous complex. Using the filamentous fungus Neurospora crassa (for which the complete genome sequence was recently published), 1000 mutants defective for cytoplasmic dynein or dynactin function have been isolated, including 300 independent dynein heavy chain mutants. Sequence analysis of cloned dynein/dynactin genes as well as the determination of the entire N. crassa genome sequence has revealed that this complex is highly conserved in N. crassa relative to metazoans supporting the use of N. crass as a model system for the analysis of dynein structure, function and regulation. In the work to be done under this award, the vast array of N. crassa dynein mutants will be used to explore the regulation of dynein motor activity. Previous work has shown that N. crassa cytoplasmic dynein motor ATPase activity is inactivated by phosphorylation in mutants defective in dynactin function (i.e., cargo interaction) or function of the LIS1 complex. This inactivation occurs not by phosphorylation of the dynein heavy chain, but by phosphorylation of small proteins (8- and 20-kDa) associated with the heavy chain. To determine the identity of these proteins, dynein heavy chain will be tagged, affinity purified and the associated proteins will be resolved by 2D gels. The heavy chain- associated polypeptides will be excised from the 2D gels and their identity determined by using mass spectrometry analysis in conjunction with the complete N. crassa genome sequence. The effects of null mutations in the genes encoding these dynein-associated subunits will be determined by assaying dynein ATPase activity. Additional work will focus on defining the effects of specific heavy chain mutations. A minimum of 100 independent dynein heavy chain mutations will be identified for those mutants that produce full-length polypeptide. These mutants will be examined for defects in dynein ATPase activity, microtubule binding and association with other dynein subunits. The information from this analysis will complement the work described above and will provide insight into the mechanism by which the dynein motor translocates along microtubules. Cytoplasmic dynein is the most complicated motor operating in the cytoplasm, and N. crassa represents the only model organism in which it is possible to rapidly isolate hundreds of mutants defective in dynein function. Completion of this work will be of great benefit in understanding the regulation of the dynein motor activity, and it will provide the first large-scale genetic dissection of the dynein heavy chain. Undergraduate students of various minority groups have played important roles in current and past research activities, and additional students will be encouraged to participate in these research efforts.