Cell adhesion and cytoskeletal dynamics in skin

描述（由申请人提供）：我们的总体目标是发展上皮干细胞如何在哺乳动物皮肤中进行形态发生，稳态和伤口修复的分子理解，并将这项研究带入临床环境。我们的重点是皮肤干细胞如何利用细胞骨架连接到细胞间和细胞基质连接来生成组织。了解参与协调肌动蛋白丝（AF）细胞骨架动力学与粘附连接（AJs）的蛋白质，并阐明这些连接如何与整合素-AF动力学反向协调，是理解自我更新的皮肤上皮如何维持和修复其表面屏障以及干细胞如何产生毛囊（HF）的关键。阐明微管（MTs）如何与AJs连接对于理解层状表皮如何维持单层分裂细胞，以及当干细胞被激活产生HFs时纺锤体极性如何变化具有重要意义。通过关注四种细胞骨架连接连接蛋白，我们将获得对这些动力学的分子理解，这些蛋白在我们之前的研究中被认为是关键：-catenin，它协调和极化AF-AJ动力学并抑制af -整合素动力学；ACF7，在伤口反应中协调和极化MT-AF和Par3介导的动力学；以及Par3和mInsc，它们的极化依赖于-catenin和1整合素，它们似乎对皮肤形态发生中纺锤体的定向至关重要。通过生物化学和分子方法，我们将定义和表征在皮肤形态发生和伤口修复过程中参与极化和重塑细胞骨架动力学的相关蛋白。通过小鼠遗传学和新的ShRNA策略，我们已经开发出快速敲除成人和胚胎皮肤中的基因，这些蛋白质的功能意义及其与其他蛋白质的各种关联将被确定。最后，随着这些过程中的主要参与者的展开，功能分析将与微阵列数据相结合，以确定aj -细胞骨架基因表达全局变化的潜在意义，这些变化伴随着干细胞接受外部信号来重塑其aj -细胞骨架连接并启动分层和HF形态发生。了解这些细胞骨架联系如何在正常皮肤中被调节是阐明这些过程中的缺陷如何导致包括皮肤癌在内的遗传疾病的先决条件。过去和现在的AR27883研究提供了一个很好的例子，说明分子皮肤生物学如何帮助产生新的和改进的工具，用于诊断和治疗人类皮肤病。

DESCRIPTION (provided by applicant): Our global objective is to develop a molecular understanding of how epithelial stem cells undergo morphogenesis, homeostasis, and wound repair in mammalian skin and bring this research to a clinical setting. Our focus is on how skin stem cells utilize cytoskeletal connections to intercellular and cell substratum junctions to generate tissues. Knowledge of the proteins involved in coordinating actin filament (AF) cytoskeletal dynamics with adherens junctions (AJs) and elucidating how these connections are inversely coordinated with integrin-AF dynamics are key to understanding how self-renewing skin epithelium maintains and repairs its surface barrier and how a stem cell can give rise to a hair follicle (HF). Elucidating how microtubules (MTs) link to AJs is important for understanding how the stratified epidermis maintains a single layer of dividing cells, and how spindle polarity changes when stem cells are activated to produce HFs. A molecular understanding of these dynamics will be obtained by focusing on four cytoskeletal-junction linking proteins that surfaced as being key from our prior studies: -catenin, which coordinates and polarizes AF-AJ dynamics and suppresses AF-integrin dynamics; ACF7, which coordinates and polarizes MT-AF and Par3- mediated dynamics in a wound response; and Par3 and mInsc, whose polarization relies upon -catenin and 1 integrin, and which appear to be critical for orienting spindles in skin morphogenesis. Through biochemical and molecular approaches, we'll define and characterize the associated proteins that are involved in polarizing and remodeling cytoskeletal dynamics during skin morphogenesis and wound repair. Through mouse genetics and novel ShRNA strategies we've developed to rapidly knockdown genes in adult and embryonic skin, the functional significance of these proteins and their varied associations with other proteins will be ascertained. Finally, as the major players in these processes unfold, functional analyses will be combined with microarray data to define the underlying significance of global changes in AJ-cytoskeletal gene expression that occur concomitantly as stem cells receive external cues to remodel their AJ-cytoskeletal connections and initiate stratification and HF morphogenesis. Understanding how these cytoskeletal linkages are regulated in normal skin is a prerequisite to elucidating how defects in these processes lead to genetic disorders, including skin cancers. Past and present AR27883 research provides an excellent illustration of how molecular skin biology can help to generate new and improved tools for the diagnosis and treatment of human skin disease. Public Health Relevance: Stem cells are natural units of tissue repair and homeostasis, and their versatility holds promise for tissue regeneration. This research focuses on deciphering how different tissue structures are derived from multipotent stem cells in the skin. Specifically, we focus on how stem cells within a single layer use cell-cell and cell substratum junctions to remodel their cytoskeleton to generate a stratified, differentiating epidermis or an invaginating hair follicle bud, and how this changes transiently in a wound response. This study is a fundamental prerequisite to understanding how aberrations in these basic properties go awry in skin cancers, including squamous cell carcinomas.