Cytoskeleton, Nucleus and Integrin Recycling in Cell Migration

摘要 本行政补充申请书要求使用荧光、FRET和TIRF显微镜 父代R35的目标是理解我们了解有限的细胞迁移的两个方面：核 定位和整合素回收。这两个过程对细胞迁移都是必不可少的，但它们如何在 缺乏机械性水平的细胞迁移。母公司R35赠款寻求在之前的两年中取得的成果的基础上再接再厉 甘德森实验室以前的项目得到了NIGMS的支持。对于核定位，我们将研究如何 核骨架和细胞骨架(LINC)复合体的连接物是机械加固的，以抵抗必要的大作用力 移动原子核，并将使用和开发新的FRET张力传感器来直接测量原子核上的力。我们 将决定LINC复合体如何选择肌动蛋白细丝或微管进行核运动和功能 这些相互作用对不同的细胞迁移模式的影响。我们将测试新的假设，即原子核 作为肌动蛋白细丝的“张力电阻”和微管运输的极性因子。对于整合素 回收，我们将检验回收的整合素以活跃的构象运行的总体假设，以及这一种子 在前缘附近以极化方式形成新的粘连。我们将使用新的整合素探针和新的整合素 循环系统结合TIRF显微镜鉴定整合素的细胞内循环途径，其位置 与新形成的粘连相关的整合素胞吐。我们还将使用TIRF显微镜来确定是否回收 整合素在迁移过程中来自细胞后部，并有助于在前缘附近形成新生黏附。 我们将确定我们假设的负责粘连极化改造的微管机制。 从回收的整合素中提取整合素，并测试回收的整合素在整合素信号中发挥作用的可能性。我们将探索 通过确定张力传感器的FRET成像是否可以将细胞迁移的这两个方面联系起来 原子核机械地与基片粘合。我们对这两个过程的拟议研究将会取得进展 了解细胞迁移的基本机制和潜在的确定新的干预靶点 细胞迁移出了问题。拟议的研究也将为博士后研究员和研究生提供肥沃的土壤。 促进学生的培训，发展他们自己的项目。

SUMMARY This application for an Administrative Supplement requests an epifluorescence, FRET and TIRF microscope to pursue the goals of the parent R35 grant to understand two aspects of cell migration for which we have limited understanding: nuclear positioning and integrin recycling. Both these processes are essential for cell migration, yet how they contribute at a mechanistic level to cell migration is lacking. The parent R35 grant seeks to build on results previously obtained in two previous projects in the Gundersen laboratory supported by NIGMS. For nuclear positioning, we will examine how the linker of nucleoskeleton and cytoskeleton (LINC) complex is mechanically reinforced to resist the large forces necessary to move the nucleus and will use and develop new FRET tension sensors to directly measure forces on the nucleus. We will determine how the LINC complex selects actin filament or microtubules for nuclear movement and the functional consequences of these interactions for different modes of cell migration. We will test new hypotheses that the nucleus functions as a “tension resistor” for actin filaments and as a polarity factor for microtubule trafficking. For integrin recycling, we will test the overall hypothesis that recycled integrins travel in an active conformation and that this seeds new adhesion formation in a polarized manner near the leading edge. We will use new integrin probes and a new integrin recycling system coupled with TIRF microscopy to identify the intracellular pathway of recycling integrins, their sites of integrin exocytosis relative to newly formed adhesions. We will also use TIRF microscopy to determine whether recycled integrin derives from the cell rear during migration and contributes to nascent adhesion formation near the leading edge. We will identify the microtubule machinery that we hypothesize is responsible for the polarized reformation of adhesions from recycled integrins and test the possibility that the recycled integrin plays a role in integrin signaling. We will explore whether these two aspects of cell migration can be linked by determining with FRET imaging of tension sensors whether the nucleus mechanically engages substrate adhesions. Our proposed studies on these two processes will advance understanding of the basic mechanisms of cell migration and potential identify new targets for intervening in cases when cell migration goes awry. The proposed studies will also provide fertile ground for postdoctoral fellows and graduate students to advance their training and develop their own projects.