HVEM TOMOGRAPHY OF BASAL BODIES IN MUTANT AND WILD TYPE CHLAMYDOMONAS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人们对基本体和中心粒的组装知之甚少。我们正在使用衣藻作为一个模型系统来识别和表征对基本身体结构和功能重要的分子。在过去的一年里，我们利用改进的标本制备方法和双轴电子断层扫描技术研究了单细胞藻莱茵衣藻基体的结构和组织。这项工作在野生型和突变影响特定微管蛋白亚型的菌株中发现了新的结构。对增量微管蛋白缺失突变体uni3-1的基底体进行的断层扫描重建证实，基底体主要含有双线微管。此外，仅存在于野生型细胞过渡区的星状纤维在uni3-1基本体的核心内重复。在这个突变体中，远端的条纹纤维不完整，根微管可能错位，并观察到多鞭毛细胞。Uni3-1的抑制子，命名为tua2-6，在α-微管蛋白中含有突变。随着增量微管蛋白的缺失，tua2-6；uni3-1细胞构建了两个鞭毛，但它们保留了基本体结构和小根微管定位的缺陷。这些数据表明，特定微管蛋白亚型的存在直接影响衣藻基本体和相关结构的组装和功能。这项工作已经出版(O‘Toole等人，Mol.比奥尔。牢房14：2999-3012,2003)。我们现在正在对BALD2基因和一种新基因TNS1发生突变的菌株进行分析，这种基因在基底组装中很重要。达彻实验室最近发现，BALD2基因编码epsilon-微管蛋白。为了进一步了解BALD2在基底体形态发生中的作用，我们正在分析具有新的bald2等位基因的菌株以及部分恢复基底体结构和功能的基因外抑制基因。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Little is known about the assembly of basal bodies and centrioles. We are using Chlamydomonas as a model system to identify and characterize the molecules important for basal body structure and function. Over the past year, we have used improved methods of specimen preparation and dual-axis electron tomography to study the structure and organization of basal bodies in the unicellular alga, Chlamydomonas reinhardtii. This work has revealed novel structures in wild-type and in strains with mutations affecting specific tubulin isoforms. Tomographic reconstructions of basal bodies from the delta-tubulin deletion mutant, uni3-1, have confirmed that basal bodies contain mostly doublet microtubules. Moreover, the stellate fibers that are present only in the transition zone of wild-type cells repeat within the core of uni3-1 basal bodies. The distal striated fiber is incomplete in this mutant, rootlet microtubules can be misplaced, and multiflagellate cells have been observed. A suppressor of uni3-1, designated tua2-6, contains a mutation in alpha-tubulin. In combination with the delta-tubulin deletion tua2-6; uni3-1 cells build both flagella, yet they retain defects in basal body structure and rootlet microtubule positioning. These data suggest that the presence of specific tubulin isoforms directly affects the assembly and function of basal bodies and associated structures in Chlamydomonas. This work has been published (O'Toole et al., Mol. Biol. Cell 14: 2999-3012, 2003). We are now pursuing analysis of strains that have mutations in the BALD2 gene and a new gene, TNS1 that is important in basal body assembly. The Dutcher lab has recently found that the BALD2 gene encodes epsilon-tubulin. To further understand the role of BALD2 in basal body morphogenesis, we are analyzing strains with new bald2 alleles as well as extragenic suppressors that partially restore basal body structure and function.