The role of PAM-1 in the regulation of the cortical cytoskeleton and polarity establishment in C. elegans

项目摘要 在不同生物体的发育过程中，许多细胞成为极化的一部分， 分化过程。这个事件的一个例子发生在单细胞C中。秀丽线虫胚胎 建立前后（AP）身体轴的过程。在许多细胞类型中， 通过肌动球蛋白和微管之间的相互作用提示极化事件 细胞骨架。尽管有很多关于极性的工作，但这些极性之间的相互作用和调节 事件仍然没有完全了解。PAM-1是嘌呤霉素敏感的氨肽酶， 在C.优雅嘌呤霉素敏感 氨肽酶（PSA）是广泛保守的，并且在哺乳动物中具有重要的发育作用。 减数分裂、细胞周期进程和从植物到小鼠的生物体的繁殖。此外，本发明还 PSA已被认为在神经退行性疾病的发展中起保护作用。 老年痴呆症和亨廷顿舞蹈症等疾病。拟议的工作旨在描述 PAM-1在调节皮质肌动球蛋白细胞骨架中的作用。此外， 将描述PAM-1，以确定PAM-1对肌动球蛋白的调节是否可与肌动球蛋白的表达分离。 中心体定位和极性作用。此外，抑制子的基因特性将 被确定。将分析携带GFP标记蛋白的菌株的延时成像， 遵循肌动球蛋白细胞骨架的组织，中心体的位置， PAM-1突变体中极性的建立。与细胞骨架组分的RNAi组合， 将揭示PAM-1在这些过程中的作用。类似的分析将在 抑制菌株比较细胞骨架组织，中心体运动，和极性 在每一个被抑制的菌株中单核苷酸多态性作图和全基因组 将采用测序来鉴定每个抑制基因突变，并将候选者 通过RNAi、蛋白质定位和遗传分析验证。据推测，克隆和 这些抑制突变的特征将揭示新的蛋白质， 与PAM-1结合，并作为氨肽酶的靶点， 细胞骨架和极性的建立。考虑到PSA在神经变性中的作用， C.目标识别秀丽线虫也可能促进对疾病的理解 机制等此外，中心体定位的新参与者将被识别，这可能是 适用于其他系统。拟议的实验将主要由以下人员进行： 本科生谁将被辅导，为他们提供指导和专业知识所需的， 在科学事业和研究生工作中取得成功。

PROJECT SUMMARY During development of diverse organisms, many cells become polarized as part of the differentiation process. One example of this event occurs in the one-cell C. elegans embryo, a process that establishes the anterior-posterior (AP) body axis. In many cell types, this polarization event is cued through interactions between the actomyosin and microtubule cytoskeletons. Despite much work on polarity, the interactions between and regulation of these events are still not fully understood. PAM-1 is a puromycin-sensitive aminopeptidase required for centrosome positioning during polarity establishment in C. elegans. Puromycin-sensitive aminopeptidases (PSAs) are widely conserved and have important developmental roles in meiosis, cell cycle progression, and reproduction in organisms from plants to mice. Additionally, PSAs have been suggested to play a protective role in the development of neurodegenerative diseases such as Alzheimer’s and Huntington’s. The proposed work seeks to characterize the role of PAM-1 in regulation of the cortical actomyosin cytoskeleton. In addition, suppressors of pam-1 will be described to determine if actomyosin regulation by PAM-1 is separable from the centrosome positioning and polarity roles. In addition, the gene identities of the suppressors will be determined. Time-lapse imaging of strains bearing GFP-tagged proteins will be analyzed to follow the organization of the actomyosin cytoskeleton, the position of the centrosome, and polarity establishment in pam-1 mutants. In combination with RNAi of cytoskeletal components, the role of PAM-1 in these processes will be uncovered. Similar analysis will be done in suppressed strains to compare cytoskeletal organization, centrosome movements, and polarity in each suppressed strain. Single-nucleotide polymorphism mapping and whole genome sequencing will be employed to identify each suppressor mutation and candidates will be verified by RNAi, protein localization, and genetic analysis. It is hypothesized that cloning and characterization of these suppressor mutations will reveal novel proteins that work in conjunction with PAM-1 and as targets of the aminopeptidase during regulation of the cytoskeleton and polarity establishment. Given the implication of PSAs in neurodegeneration, identification of targets in C. elegans may also advance the understanding of disease mechanisms. Additionally, new players in centrosome positioning will be identified which may be applicable to other systems. The proposed experiments will be largely carried out by undergraduates who will be mentored to provide them the guidance and expertise needed for success in science careers and graduate work.