Microtubule dynamics during cell polarity and migration

细胞极性和迁移过程中的微管动力学

基本信息

批准号：
8989112
负责人：
Torsten Wittmann
金额：
$ 30.91万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-05-01 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8989112
关键词：
Abnormal Cell Adenomatous Polyposis Coli Protein Binding Binding Proteins Biochemical Biological Cell Adhesion Cell Communication Cell Polarity Cell Shape Cell-Matrix Junction Cells Cellular biology Characteristics Charge Chromosome Segregation Complex Crosslinker Cytoskeleton Data Development Disease Environment Enzymes Epithelial Epithelial Cells Eukaryotic Cell Exocytosis Extracellular Matrix Extracellular Matrix Degradation F-Actin Filament Focal Adhesions Funding Goals Health Intracellular Transport Light Link Malignant Neoplasms Mechanics Mediating Membrane Membrane Proteins Metalloproteases Microtubule Stabilization Microtubules Modeling Molecular Neoplasm Metastasis Normal Cell Pathway interactions Peptides Peripheral Physiological Plus End of the Microtubule Polymers Process Protein Region Proteins Proteomics Recruitment Activity Regulation Research Role Signal Transduction Site Sorting - Cell Movement System Testing Tissues Tyrosine Phosphorylation Vesicle Transport Pathway base cancer cell cell behavior cell motility design filamin human disease in vitro Assay innovation live cell imaging macromolecular assembly novel overexpression particle receptor research study theories

项目摘要

DESCRIPTION (provided by applicant): Microtubules are dynamic polymers central to many processes in eukaryotic cells, including chromosome segregation, intracellular transport, and cell shape remodeling. Many of these microtubule functions are mediated by protein interactions at growing microtubule plus ends. End-binding proteins (EBs) directly recognize a structural feature of growing microtubule ends. A diverse group of proteins, called +TIPs, bind to EBs through short, conserved SxIP peptide motifs in intrinsically disordered, positively charged protein regions. These characteristics led to the identification of over thirty structurally highly heterogeneous +TIPs, but intracellular +TIP functions remain incompletely understood. A long-term goal of this project is to answer fundamental, unresolved questions of +TIP cell biology: What are molecular functions of specific +TIPs? How is microtubule plus-end-association important for these functions? How are +TIPs spatially and temporally controlled such that different +TIP complexes mediate specific microtubule activities? Based on the unexpected diversity of SxIP-motif-containing +TIPs and findings in the previous funding period, a central hypothesis of this project is that many +TIPs act as adaptors that promote spatially and temporally controlled capture of EB-positive growing microtubule ends to polarize microtubule-dependent activities, which extends the classic search-and-capture theory by providing intracellular receptors to facilitate specific interactions with EB-covered growing microtubule ends. The current application focuses on the function of CLASPs and other +TIPs that are associated with focal adhesions (FAs), multi-layered macromolecular assemblies that mediate dynamic cell interactions with the extracellular matrix (ECM). Based on preliminary data that CLASPs cluster around FAs, tether microtubules to FAs, and facilitate FA turnover, a novel mechanism is proposed in which FA-associated +TIPs establish vesicle transport tracks toward FAs to promote localized ECM remodeling and facilitate outside-in FA disassembly. Experiments in this application will define how +TIPs and microtubules control cell-matrix adhesion remodeling by employing biochemical, cell biological and advanced live cell imaging approaches: Aim 1 asks how growing and mature FAs generate a signal that recruits specific +TIPs to a zone adjacent to FAs. Aim 2 defines molecular mechanisms by which CLASPs capture and link MTs to FAs, and investigates a novel mechanism of EB regulation. Aim 3 asks how localized exocytosis promotes FA turnover, tests whether cell-matrix release is sufficient to trigger FA disassembly by developing a highly innovative light-controlled cell adhesion substrate, and analyzes the consequences of +TIP-mediated local ECM remodeling during epithelial remodeling in a physiological 3D environment. Because abnormal cell-matrix interactions contribute to cancer metastasis, and EBs are overexpressed in cancer cells indicating increased +TIP activity, in addition to establishing new paradigms relating to +TIP function, our studies are highly relevant to understanding pathological cell behavior.

描述（由申请人提供）：微管是真核细胞中许多过程的中心动态聚合物，包括染色体分离，细胞内转运和细胞形状重塑。这些微管功能中的许多都是由生长的微管加末端处的蛋白质相互作用介导的。末端结合蛋白（EB）直接识别生长的微管末端的结构特征。一种称为 +尖端的多种蛋白质，通过简短的保守的SXIP肽基序与本质上无序的，带正电荷的蛋白质区域结合。这些特征导致了三十多个结构上高度识别异质 +尖端，但细胞内 +尖端功能仍未完全理解。该项目的一个长期目标是回答 +TIP细胞生物学的基本，未解决的问题：特定 +尖端的分子功能是什么？微管加末端关联对这些功能至关重要？ +尖端如何在空间和时间控制上，使不同的 +尖端配合物介导了特定的微管活动？ Based on the unexpected diversity of SxIP-motif-containing +TIPs and findings in the previous funding period, a central hypothesis of this project is that many +TIPs act as adaptors that promote spatially and temporally controlled capture of EB-positive growing microtubule ends to polarize microtubule-dependent activities, which extends the classic search-and-capture theory by providing intracellular receptors to facilitate specific interactions with EB-covered growing microtubule结束。当前的应用集中于与焦点粘附（FAS），多层大分子组件相关的扣子和其他 +尖端的功能，这些大分子分子组件介导了与细胞外矩阵（ECM）的动态细胞相互作用。基于将围绕FAS群簇的初步数据，链球微管群，直到FAS并促进FA的转换，提出了一种新型机制，其中FA相关 +TIPS +TIPS向FAS建立了囊泡运输轨道，以促进局部ECM重塑，并促进外部FA In-In-In-In-In In-In-In-In-In In-In In-In In-In-In-In In-In In-In Insabsbly。该应用程序中的实验将定义 +TIPS和微管如何通过采用生化，细胞生物学和先进的活细胞成像方法来控制细胞矩阵粘附重塑：AIM 1询问生长和成熟的FAS如何产生一个信号，从而将特定的 +尖端募集到与FAS相邻的区域相邻的特定 +尖端。 AIM 2定义了clasps捕获并将MT与FAS联系起来的分子机制，并研究了EB调节的新机制。 AIM 3询问局部胞吐作用如何促进FA的周转，测试细胞矩阵释放是否足以通过开发高度创新的光控制的细胞粘附底物来触发FA拆卸，并分析 +TIP TIP介导的局部ECM重塑在上皮重塑过程中的 +TIP介导的局部ECM重塑的后果。由于异常细胞 - 矩阵相互作用有助于癌症转移，并且EB在表明 +尖端活性增加的癌细胞中过表达，除了建立与 +尖端功能有关的新范式外，我们的研究与理解病理细胞行为高度相关。