Signaling Pathways and Microtubule Function

描述(申请人提供)：该项目的长期目标是了解调节微管和微管依赖过程的信号通路。指导这个项目的一般假设是，皮质事件激活了影响微管稳定性的信号通路。这一假说源于PI实验室关于渗透支持对微管及其相关蛋白质的影响的研究，并与PI更好地了解微管细胞骨架是如何调节的长期目标非常吻合。初步研究的主要发现是，CinSp是一种在有丝分裂中发挥作用的酵母微管相关马达蛋白，其突变损害了细胞壁的完整性，渗透压支持稳定了微管，并抑制了与这些突变相关的温度敏感性表型。这一假说的另一个基础是发现了两个多拷贝抑制基因GIC1和KRE6，它们抑制cin8突变，并与调节渗透条件和细胞完整性的MAPK途径相关。渗透条件如何通过与信号通路的相互作用影响微管稳定性尚不清楚，这是这个新项目的重点。渗透支持对cin8突变的明显抑制为鉴定渗透条件诱导的信号转导通路的组成提供了有效的工具。该方案的具体目的是通过研究渗透压稳定剂对微管细胞骨架的影响以及阐明影响微管稳定的信号通路来检验这一假说。三种实验方法将被用来检验这一假设。在目标1中，将利用微生物、生化、分子和细胞生物学技术来分析渗透调节物质对微管动力学和功能的影响。在目标2中，突变分析将识别参与渗透诱导的稳定微管的信号通路的基因。在目标3中，这些基因的上位性分析将用于构建影响微管稳定性的一连串事件的模型。该项目的成果将增强我们目前对细胞如何调节其微管的知识，并将对细胞骨架和信号转导领域产生重大影响。因为一些癌症治疗药物是基于控制微管稳定性的，所以关于这种机制的新信息可以用于寻找针对调控癌细胞微管稳定性的通路组件的新药。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the signaling pathways that regulate microtubules and microtubule-dependent processes. The general hypothesis guiding this project is that cortical events activate signaling pathways that affect microtubule stability. This hypothesis stems from studies performed in the PI's laboratory on the effect of osmotic support on microtubules and their associated proteins, and fits well with the PI's long-term goals to better understand how the microtubule cytoskeleton is regulated. The main finding of the preliminary studies is that mutations in CinSp, a yeast microtubule-associated motor protein that functions in mitosis, compromise cell wall integrity and that osmotic support stabilizes microtubules and suppresses the temperature sensitivity phenotype associated with these mutations. An additional basis for the hypothesis is the identification of two multicopy suppressors, GIC1 and KRE6, which suppress the cin8 mutations and are associated with MAP kinase pathways that regulate osmotic conditions and cell integrity. How osmotic conditions affect microtubule stability through interaction with signaling pathways is not known and is the focus of this new project. The clear suppression of the cin8 mutations by osmotic support provides an efficient tool for identification of components of the pathways that are induced by osmotic conditions. The specific aims in this proposal will test the hypothesis by studying the effect of osmotic stabilizers on the microtubule cytoskeleton and elucidating the signaling pathways that affect microtubule stabilization. Three experimental approaches will be used to test the hypothesis. In Aim 1, microbial, biochemical, molecular and cell biology techniques will be employed to assay the effect of osmolytes on microtubule dynamics and function. In Aim 2, mutational analysis will identify genes that participate in osmotically-induced signaling pathways that stabilize microtubules. In Aim 3, epistasis analysis of these genes will be used to construct models for the cascade of events that affect microtubule stability. Results obtained under this project will enhance our current knowledge on how cells regulate their microtubules and will significantly impact the fields of cytoskeleton and signal transduction. Because some cancer therapeutic drugs are based on controlling microtubule stability, new information on such mechanisms could be applied to the search of new drugs that target components of pathways that regulate microtubule stability in cancerous cells.