FUNCTIONS OF CYTOPLASMIC DYNEIN

细胞质动力蛋白的功能

基本信息

批准号：
2392005
负责人：
John RICHARD MCINTOSH
金额：
$ 23.28万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-07-01 至 2000-03-31
项目状态：
已结题

项目摘要

We have recently discovered that human cells and tissues synthesize three forms of cytoplasmic dynein. The dynein heavy chains (DHCs) in these enzymes are encoded by three distinct genes. lsotype-specific antibodies have been raised against polypeptides expressed in bacteria from recombinant fragments of each DHC gene. Both these and gene-specific probes reveal different levels of expression in each cell studied. The three dyneins have interestingly different localizations within cells. DHC1, the form already studied in HeLa cells and rodent brain, is in the mitotic spindle, with concentrations on the centrosomes and kinetochores. During interphase, it is associated with cytoplasmic vesicles and perhaps with primary endosomes. DHC2 is concentrated in the Golgi apparatus and on cytoplasmic vesicles. DHC3 is associated with a reticular or stringy cytoplasmic structure during interphase, which may be a subcompartment of the ER or the compartment intermediate between ER and Golgi. We propose to study the functions of these dynein isoforms in vivo and in vitro. Immunological and molecular methods will be used to perturb their actions in living cells, and the resulting phenotypes will be studied by microscopy and a variety of physiological assays that monitor membrane traffic and behavior in vivo. We will purify the holo-enzymes that include each of these isoforms, identify any lower molecular weight polypeptides with which each DHC associates, and characterize any ATPase activities, motor actions on microtubules, and interactions with potential regulatory complexes, like dynactin. We will work with in vitro systems that reconstitute some aspect of.chromosome motion or membrane traffic, so the roles of each dynein isoform can be explored under well controlled experimental circumstances. A laser trap will be used, together with stable or labile microtubules, to control the framework over which different populations of vesicles or chromosomes can move, and the roles of dynein in these movements will be assessed by studying the effects of added enzyme, function blocking antibodies and/or recombinant fragments of the DHCs that might compete for essential binding activities and block the action of the holo-enzymes. Through these experiments we hope to learn the roles played by each isoform of cytoplasmic dynein in cell growth and physiology. Finally, we will improve our knowledge of the gene structures and map positions of each DHC to look for related disease genes or mutations that might help us understand these enzymes, and we will seek additional isoforms of cytoplasmic DHCs.

我们最近发现人体细胞和组织合成三种细胞质动力蛋白的形式。这些中的动力蛋白重链 (DHC) 酶由三个不同的基因编码。同种型特异性抗体已针对细菌中表达的多肽进行了培养每个 DHC 基因的重组片段。这些和基因特异性探针揭示了所研究的每个细胞中不同的表达水平。这有趣的是，三种动力蛋白在细胞内具有不同的定位。 DHC1 的形式已在 HeLa 细胞和啮齿动物大脑中进行了研究，它存在于有丝分裂纺锤体，集中在中心体和动粒上。在间期期间，它与细胞质囊泡相关，也许与初级内体。 DHC2 集中在高尔基体中细胞质囊泡。 DHC3 与网状或丝状结构相关间期的细胞质结构，可能是内质网或内质网和高尔基体之间的中间室。我们建议研究这些动力蛋白亚型在体内和体内的功能体外。将使用免疫学和分子方法来扰乱它们在活细胞中的作用，以及由此产生的表型将被研究显微镜和各种监测膜的生理测定体内的交通和行为。我们将纯化全息酶，包括这些亚型中的每一个，都可以识别任何较低分子量的多肽每个 DHC 都与之相关，并表征任何 ATP 酶活性，微管上的运动动作以及与潜在监管的相互作用复合物，如动力蛋白。我们将与体外系统合作重建染色体运动或膜运输的某些方面，因此可以在良好的控制下探索每种动力蛋白亚型的作用实验情况。将使用激光陷阱以及稳定或不稳定的微管，以控制其框架不同的囊泡或染色体群体可以移动，并且它们的作用将通过研究以下因素的影响来评估动力蛋白在这些运动中的作用：添加酶、功能阻断抗体和/或重组片段 DHC 可能会竞争必要的结合活性并阻止全酶的作用。通过这些实验我们希望了解细胞质动力蛋白的每种同工型在细胞生长和生理。最后，我们将提高对基因结构的了解绘制每个 DHC 的位置以寻找相关的疾病基因或可能有助于我们理解这些酶的突变，我们将寻求细胞质 DHC 的其他亚型。