EB1 protein: regulator of actin protrusion and cell motility

描述(申请人提供)：细胞运动是人体内各种生物学过程的基础，包括胚胎发育、伤口愈合、血管发育和癌症转移。两个主要的细胞骨架系统，微管和肌动蛋白，共同协调细胞在运动过程中的突起和回缩。微管的加端进入细胞外周，靶向并调节肌动蛋白细胞骨架的成分。然而，将微管与肌动蛋白突起功能联系起来的信号机制却知之甚少。加端跟踪蛋白选择性地结合生长的微管，使它们成为调节微管和肌动蛋白相互作用的良好候选者。一种特殊的正端跟踪蛋白EB1对于小鼠黑色素瘤细胞中正常的肌动蛋白突起和细胞运动是必需的。EB1蛋白通过调节肌动蛋白信号通路和与细胞外基质的相互作用来控制细胞的运动，这一假设驱动了这一提议的研究目的和实验方法。第一个目的是研究EB1缺失对微管靶向黏附部位和黏附部位转换的影响。这一目标将涉及使用二维反卷积来分析活细胞中黏附位置的动力学。第二个和第三个AIMS将描绘EB1在板脂和丝轴信号通路中的位置。这些目标将使用定量免疫荧光显微镜和生化技术相结合来完成。这项拟议研究的总体目标是了解EB1调节细胞运动的分子机制。 公共卫生相关性：细胞运动过程中突起的空间控制需要微管和肌动蛋白细胞骨架系统之间的协调活动。然而，微管与肌动蛋白突起的功能偶联的分子机制却知之甚少。这项研究的主要目标是了解与癌症转移相关的微管+末端与细胞运动相关的分子细节。

DESCRIPTION (provided by applicant): Cell motility underlies diverse biological processes, both normal and pathological, in the human body including embryonic development, wound healing, development of blood vessels and cancer metastasis. Two major cytoskeletal systems, microtubules and actin, work together to coordinate cell protrusion and retraction during motility. The plus-ends of microtubules enter the cell periphery, target and modulate components of the actin cytoskeleton. However, the signaling mechanisms which functionally link microtubules to actin protrusion are poorly understood. Plus-end tracking proteins selectively bind growing microtubules making them good candidates for mediating the interaction between microtubules and actin. A particular plus- end tracking protein, EB1, is necessary for normal actin protrusion and cell motility in mouse melanoma cells. The research aims and experimental approaches of this proposal are driven by the hypothesis that EB1 protein controls cell motility through regulating actin signaling pathways and interaction with the extracellular matrix. The first aim is to investigate the effect of EB1 depletion on targeting of microtubules to adhesion sites and adhesion site turnover. This aim will involve use of 2-dimensional deconvolution to analyze adhesion site dynamics in live cells. The second and third aims will delineate EB1 position in lamellipodial and filopodial signaling pathways. These aims will be completed using quantitative immunofluorescence microscopy combined with biochemical techniques. The overall goal of the proposed research is to understand the molecular mechanism by which EB1 regulates cell motility. PUBLIC HEALTH RELEVANCE: Spatial control of protrusion during cell motility requires coordinated activities between the microtubule and actin cytoskeleton systems. However, the molecular mechanism that functionally couples microtubules to actin protrusion is poorly understood. The broad goal of this study is to understand the molecular details that link microtubule plus-ends to cell motility relevant to cancer metastasis.