Mechanisms of Cell-Matrix Interaction and Signaling in Lens Development

描述：细胞基质粘附位点和细胞骨架之间关键连接的缺失可能是许多疾病的核心。本项目的重点是阐明在发育中的小鼠晶状体中调节这种功能附着的分子机制。我们的中心假设是，焦点粘附激酶（FAK）和相关信号分子将细胞-基质的附着转化为细胞骨架重排和一系列反应，包括1)细胞增殖、分化和迁移，以及2)细胞外基质组织成基底膜。研究这种动态的，双向的相互作用需要检查一个本地的，三维的组织结构。我们的总体策略是在小鼠晶状体中研究这些通路，因为它被体内最厚的基底膜之一包围，经历了一个明确的发展，可以完整地培养，并且是透明的，使其具有独特的成像分析能力。我们将使用条件敲除方法从发育中的小鼠晶状体中删除FAK，其唯一的家族成员Pyk2以及整合素连接激酶（ILK）。Pyk2的缺失将解决功能冗余的问题，ILK的缺失将通过独立于FAK的替代途径破坏细胞-基质-细胞骨架的附着，从而进一步验证我们的假设。我们将使用分子、细胞、生化和结构方法的组合来解决以下问题：目标1)FAK、Pyk2和ILK信号是晶状体发育所必需的吗？这个通路的扰动会导致晶状体病理吗？fak相关信号的破坏是否会导致晶状体细胞迁移、增殖和/或分化中的特定细胞缺陷？涉及哪些信号通路？目的3)fak相关信号的中断如何改变晶状体囊基底膜的组织、合成和重塑？这些拟议的研究将为细胞和细胞外基质之间所需的串扰以及所涉及的信号通路提供机制见解。重要的是，这些结果也将为眼睛发育机制提供独特的见解，并可能有助于更好地理解致盲疾病，如小眼和先天性白内障，我们预测这些疾病可能是由于晶状体细胞发育过程中细胞基质相互作用的缺陷造成的。

DESCRIPTION: Loss of the pivotal connection between cell matrix adhesion sites and the cytoskeleton may lie at the heart of a number of diseases. This project is focused on elucidating the molecular mechanisms regulating this functional attachment in the developing mouse lens. Our central hypothesis is that focal adhesion kinase (FAK) and related signaling molecules translate cell-matrix attachment into cytoskeletal rearrangements and a cascade of responses, including 1) cellular proliferation, differentiation and migration as well as 2) organization of the extracellular matrix into basement membranes. Study of this dynamic, bi-directional interaction requires examination of a native, three-dimensional tissue structure. Our general strategy is to study these pathways in the mouse ocular lens, since it is surrounded by one of the thickest basement membranes in the body, undergoes a defined development, can be cultured intact, and is transparent, rendering it uniquely accessible to imaging analysis. We will use a conditional knockout approach to delete FAK, its only family member Pyk2, as well as integrin-linked kinase (ILK) from the developing mouse lens. Deletion of Pyk2 will address issues of functional redundancy, and deletion of ILK will further test our hypothesis by disrupting cell-matrix-cytoskeletal attachment through an alternative route, independent of FAK. We will use a combination of molecular, cellular, biochemical and structural approaches to address the following questions: Aim 1) Are FAK, Pyk2 and ILK signaling required for lens development? Does perturbation of this pathway result in lens pathology? Aim 2) Does disruption of FAK-related signaling result in specific cellular defects in lens cell migration, proliferation and/or differentiation? What signaling pathways are involved? Aim 3) How does disruption of FAK-related signaling alter how the lens capsule basement membrane is organized, synthesized and re-modeled? These proposed studies will provide mechanistic insight into the crosstalk required between cells and the extracellular matrix, and the signaling pathways involved. Importantly, these results will also provide unique insight into the mechanisms of eye development and potentially contribute to a better understanding of blinding disorders such as microphthalmia and congenital cataracts that we predict may result from faulty cell matrix interactions during lens cell development.