HVEM TOMOGRAPHY OF BASAL BODIES IN MUTANT AND WILD TYPE CHLAMYDOMONAS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。对基体和中心粒的组装知之甚少。我们正在使用衣藻作为模型系统来识别和表征对基础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类莱茵衣藻（Chlamydomonas reinhardtii）的基底结构和组织。这项工作揭示了新的结构在野生型和菌株的突变影响特定的微管蛋白异构体。delta-微管蛋白缺失突变体uni -1对基底体的层析重建证实，基底体主要含有双态微管。此外，仅存在于野生型细胞过渡区的星状纤维在uni -1基体的核心内重复。在这个突变体中，远端条纹纤维不完整，根微管可能错位，并且观察到多鞭毛细胞。uni -1的抑制因子，称为tua2-6，包含α -微管蛋白的突变。结合delta-微管蛋白缺失tua2-6；Uni3-1细胞既有鞭毛，也有鞭毛，但其基底结构和根微管定位存在缺陷。这些数据表明，特定微管蛋白亚型的存在直接影响衣藻基底体和相关结构的组装和功能。这项工作已发表（O'Toole et al., Mol. Biol）。Cell 14: 2999- 3012,2003)。我们现在正在对BALD2基因和一个在基础体组装中很重要的新基因TNS1突变的菌株进行分析。荷兰实验室最近发现，BALD2基因编码epsilon-tubulin。为了进一步了解BALD2在基底体形态发生中的作用，我们正在分析含有新的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因子。

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.