Kinesin-Like Proteins in Eukaryotic Flagella and Cilia

9808654伯恩斯坦运动蛋白是依赖于细胞质微管(MT)的马达蛋白的一个多样化的超家族，它将化学能转化为机械力，在各种关键的细胞事件中应用，包括有丝分裂、减数分裂和囊泡运输。目前已鉴定出80多种不同的激动素样蛋白(KLP)，它们的已知功能可分为两大类：参与膜性货物运输的KLP和有丝分裂过程中所需的KLP。然而，大多数KLP的确切功能尚不清楚。最近，鞭毛和纤毛中的KLPS被描述，这是真核细胞用来在水环境中游泳或移动水环境通过细胞表面的基于MT的结构。本项目的目的是利用分子遗传学、生物化学和细胞学的方法，结合单细胞真核生物衣藻和四膜虫，了解两种动蛋白在纤毛和鞭毛功能中的作用。Klp1是在鞭毛中央对MTS中发现的一种鞭毛衣藻。有人认为Klp1是鞭毛敲打所必需的。为了验证这一假设，我们将确定Klp1缺失鞭毛的表型。通过定点改变Klp1的ATP结合位点，在体外构建显性阴性的Klp1突变，然后将突变基因转化回衣藻细胞，从而实现这一缺陷。表达突变形式的Klp1的细胞将被检测精细结构的细胞学缺陷，以及体外和体内的鞭毛功能缺陷。免疫印迹分析表明，Klp1也存在于四膜虫的纤毛中。利用聚合酶链式反应克隆了四膜虫Klp1基因，构建了四膜虫Klp1缺失突变体。将对四膜虫KLP1缺失突变体进行纤毛运动缺陷和超微结构细胞学缺陷的检查。如果Klp1基因缺失的纤毛缺少纤毛多肽，则可以使用二维凝胶分析来检测其含量。为了确定中心对MTS的正确定位是否需要Klp1，将检查一系列薄片，以比较突变型纤毛和野生型纤毛中中心对MTS相对于外部双对MTS的位置。鞭毛激动素样蛋白Fla10是鞭毛衣藻动蛋白II/Kif3亚家族中的一员，是鞭毛膜相关运动以及鞭毛生长和维持所必需的。通过聚合酶链式反应从四膜虫中克隆了一个与Fla10相关的新的激动素Kin5。将获得Kin5的完整基因组克隆，并用于转化和基因替换方案，以产生四膜虫Delta-kin5缺失突变体。将分析Delta-kin5突变体的纤毛跳动，并将使用视频增强DIC显微镜检查突变体纤毛的膜相关运动。这些实验的结果将促进我们对细胞骨架的结构和功能的了解，特别是对动蛋白样蛋白和真核纤毛和鞭毛的功能的理解。由于纤毛和鞭毛、纤毛和鞭毛的高度保守的结构和生化组成，以及类动蛋白，通过研究这些容易操纵的原生动物，对动蛋白和纤毛/鞭毛功能的深入研究将适用于高等真核细胞的纤毛/鞭毛。此外，通过研究鞭毛和纤毛运动蛋白获得的概念可能有助于理解其他更复杂和动态的MT阵列，包括有丝分裂纺锤体。这些研究描述了影响科学教育和人力资源开发，因为它们将作为研究生培训的一部分进行。通过开展上述研究，学生将在学术和技术上接受培训，成为细胞生物学领域的独立科学家，预计他们最终将在学术或工业环境中为智力和技术进步做出重大贡献。

9808654 Bernstein The kinesins are a diverse superfamily of cytoplasmic microtubule (MT)-dependent motor proteins that transduce chemical energy into mechanical forces that are applied during a variety of critical cellular events, including mitosis, meiosis, and vesicular transport. Over eighty different kinesin-like proteins (klps) have been identified, whose known roles fall into two broad categories: those involved in transport of membranous cargo and those required during mitosis. The precise function of most klps, however, is unknown. Recently, klps have been described in flagella and cilia, the MT-based structures that eukaryotic cells use to either swim through an aqueous environment or move the aqueous environment past the cell's surface. The objective of this project is to understand the function of two kinesins in ciliary and flagellar function, using a combination of molecular genetic, biochemical and cytological approaches with the unicellular eukaryotes, Chlamydomonas and Tetrahymena. Klp1 is a Chlamydomonas flagellar klp found in flagellar central pair MTs. It has been suggested that Klp1 is required for flagellar beating. To test this hypothesis, the phenotype of Klp1-deficient flagella will be determined. The deficiency will be achieved by construction of dominant-negative Klp1 mutations in vitro by site-directed alteration of the ATP binding site of Klp1, followed by transformation of the mutant genes back into Chlamydomonas cells. Cells expressing mutant forms of Klp1 will be assayed for fine-structural cytological defects and both in vitro and in vivo for defects in flagellar function. Immunoblot analysis has indicated that Klp1 is also present in the cilia of Tetrahymena. The Tetrahymena Klp1 gene will be cloned using PCR and a klp1 null mutant of Tetrahymena will be constructed. The Tetrahymena klp1 null mutants will be examined for defective ciliary motility and for ultrastructural cytological defects. Two-dimensional gel analysis will be used to det ermine if ciliary polypeptides are missing from Klp1-deficient cilia. To determine if Klp1 is required for proper orientation of central pair MTs, serial thin sections will be examined to compare the position of the central pair MTs relative to the outer doublet MTs in mutant versus wild type cilia. The flagellar kinesin-like protein Fla10 is a member of the kinesin II/Kif3 subfamily of kinesins that is present in Chlamydomonas flagella, and which is required for flagellar membrane-associated motilities and for flagellar growth and maintenance. A new Fla10-related kinesin, Kin5, has been cloned from Tetrahymena by PCR. The complete genomic clone for Kin5 will be obtained and used in transformation and gene replacement protocols to generate a Tetrahymena delta-kin5 deletion mutant. The delta-kin5 mutant will be analyzed for ciliary beating, and video enhanced DIC microscopy will be used to examine mutant cilia for the membrane-associated motility. The results of these experiments will advance our understanding of the structure and function of the cytoskeleton, and specifically the functions of kinesin-like proteins and eukaryotic cilia and flagella. Due to the highly conserved structures and biochemical compositions of cilia and flagella, MTs, and the kinesin-like proteins, the insights into kinesin and ciliary/flagellar function that will be gained by studying these easily manipulated protozoans will be applicable to klps and cilia/flagella of higher eukaryotic cells. In addition, the concepts gained by studying flagellar and ciliary kinesins may be useful in understanding other, more complex and dynamic, MT arrays, including the mitotic spindle. The studies described impact science education and human resource development in that they will be carried out as part of the training of graduate students. By carrying out the studies described, the students will be trained academically and technically to become independent scientists in the field of cell biology who are expected to eventually contribute significantly to intellectual and technical advances in either an academic or industrial setting.