Genetic Analysis of the Regulation of Cytoplasmic Dynein

细胞质动力蛋白是一种多亚单位复合体，其功能是作为微管相关的马达，参与核的运动和定位、纺锤体的组装、染色体分离、高尔基体的组织、内质网到高尔基体的运输以及轴突中细胞器的逆行运输。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单位复合体，称为dynactin。动力蛋白重链是细胞中最大的蛋白质之一(4000个残基)，C-末端三分之二形成运动域，N-末端形成延伸的尾巴，在货物相互作用中发挥作用。在后生动物(如老鼠和果蝇)中，细胞质动力蛋白功能是必不可少的；然而，在丝状真菌中，动力蛋白对生存并不是必需的，这极大地简化了对这一巨大复合体的遗传分析。利用丝状真菌粗面脉孢霉(Neuropora Crassa)(最近公布了其全基因组序列)，已分离出1000株具有胞质动力蛋白或动力蛋白功能缺陷的突变株，其中包括300株独立的动力蛋白重链突变株。对克隆的dynein/dynactin基因的序列分析以及对粗毛裸藻全基因组序列的测定表明，该复合体相对于后生动物来说是高度保守的，支持将粗劣新藻作为分析动力蛋白结构、功能和调控的模型系统。在这一奖项下要做的工作中，大量的粗毛拟青霉动力蛋白突变体将被用于探索动力蛋白运动活动的调节。以往的工作表明，在动力蛋白功能(即货物相互作用)或Lis1复合体功能缺陷的突变体中，粗面新月球藻细胞质动力蛋白马达ATPase活性被磷酸化灭活。这种失活不是通过动力蛋白重链的磷酸化发生的，而是通过与重链相关的小蛋白(8-和20-kDa)的磷酸化而发生的。为了确定这些蛋白质的特性，将对动力蛋白重链进行标记、亲和纯化，并通过2D凝胶对相关蛋白质进行解析。与重链相关的多肽将从2D凝胶中分离出来，并通过结合粗劣新月球藻完整基因组序列的质谱分析来确定它们的身份。编码这些动力蛋白相关亚单位的基因零突变的影响将通过检测动力蛋白ATPase活性来确定。其他工作将集中在确定特定重链突变的影响上。对于那些产生全长多肽的突变体，将确定至少100个独立的动力蛋白重链突变。这些突变体将被检查动力蛋白ATPase活性、微管结合以及与其他动力蛋白亚基结合的缺陷。来自这一分析的信息将补充上述工作，并将提供对动力蛋白马达沿微管转移的机制的洞察。细胞质动力蛋白是细胞质中最复杂的运动，而粗毛拟青霉代表了唯一有可能快速分离数百个动力蛋白功能缺陷突变株的模式生物。这项工作的完成将有助于了解动力蛋白运动活动的调节，并将提供第一次大规模的动力蛋白重链的基因解剖。各少数民族本科生在当前和过去的研究活动中发挥了重要作用，将鼓励更多的学生参与这些研究努力。

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.