Nucleoskeleton-Cytoskeleton Connections and Cell Polarity in Aging

衰老过程中的核骨架-细胞骨架连接和细胞极性

• 批准号: 10394870
• 负责人: Gregg G Gundersen
• 金额: $ 41.65万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10394870
• 关键词: Actins; Actomyosin; Address; Adhesions; Age; Aging; Cell Aging; Cell Nucleus; Cell Polarity; Cell physiology; Cells; Cellular Morphology; Centrosome; Child; Complex; Cytoplasm; Cytoskeletal Proteins; Cytoskeleton; Data; Defect; Dermis; Disease; Dynein ATPase; Fibroblasts; Filament; Functional disorder; Genes; Human; Impaired wound healing; Individual; Lead; Mass Spectrum Analysis; Mediating; Mendelian disorder; Methods; Microfilaments; Microtubules; Molecular; Movement; Mus; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Lamina; Nuclear Outer Membrane; Physiological; Play; Process; Progeria; Protein Isoforms; Proteins; RNA Splicing; Role; Skin; Skin wound healing; Splice-Site Mutation; Syndrome; System; Testing; Transgenic Mice; Variant; age related; aged; autocrine; cell age; healing; human subject; in vivo; insight; lamin C; migration; normal aging; novel; overexpression; paracrine; prelamin A; protein purification; reconstitution; release factor; skin wound; tissue culture; wound; wound healing

Project Summary The cytoskeleton and its connections to the nucleus play fundamental roles in establishing cellular morphology, polarity, migration and adhesion. Because of their essential roles in these cellular functions, it is critically important to understand the changes that take place in cytoskeletal systems and their nuclear connections during the normal process of cellular aging. Despite this importance, remarkably little is known about the role of the cytoskeleton in physiologically aging. Our preliminary studies have led to the discovery of a fundamental cell polarity defect that occurs in fibroblasts from children with the accelerated aging disorder Hutchinson- Gilford progeria syndrome and also in fibroblasts from physiologically-aged individuals. This defect results from unbalanced connections between the nuclear lamina on the inner aspect of the inner nuclear membrane and two major cytoskeletal protein systems: actin microfilaments and microtubules. These connections are mediated by the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex composed of inner nuclear membrane SUN and outer nuclear membrane KASH/nesprin proteins. In aging, there is a preferential increased interaction of nesprin-2G with microtubules (supported by SUN1) versus actin microfilaments (supported by SUN2). This has led to our overall hypothesis that altered nucleocytoskeletal connections mediated by the LINC complex causes an intrinsic cell polarity defect in physiological aging as well as an accelerated aging disorder. We further hypothesize that this defect is at least in part mediated by a protein factor secreted by cells from aged individuals. We propose to test these hypotheses in three specific aims. In Aim 1, we will decipher the mechanism underlying the cell polarity defect in physiological aging. We will determine how increased SUN1 levels are established in the nuclear envelope during aging, explore how SUN1 interaction with nesprin-2G biases its interaction toward microtubules and examine how cytoplasmic microtubules are impacted by their excessive interaction with the nuclear envelope. In Aim 2, we will examine the role of the age-dependent cell polarity defect in would healing in vivo. We will examine cutaneous wounds from young and old mice to determine if the same mechanisms that generate polarity defects in tissue culture are operative in vivo as well as examine wound healing and fibroblast polarity in transgenic mice overexpressing SUN1 in fibroblasts. In Aim 3, we will purify and characterize a soluble factor secreted from aged cells that leads to defective cell polarity. We will use complementary methods of mass spectrometry and protein purification to identify this factor we call FRAC, for Factor Released from Aged Cells. Completion of these aims will lead to novel insights into how alterations in nucleocytoskeltal connections at the LINC complex lead to a fundamental cell polarity defect in aging.

项目摘要 细胞骨架及其与细胞核的连接在建立细胞形态中起着基本作用， 极性，迁移和粘附。由于它们在这些细胞功能中的重要作用，因此很重要 了解细胞骨架系统及其核连接中发生的变化很重要 在细胞衰老的正常过程中。尽管很重要，但对 生理衰老的细胞骨架。我们的初步研究导致发现了基本 细胞极性缺陷发生在患有加速衰老疾病的儿童的成纤维细胞中 吉尔福德（Gilford）富裕综合征，以及生理年龄个体的成纤维细胞。这个缺陷来自 核层之间的连接不平衡，在内部核膜的内部和 两个主要的细胞骨架蛋白系统：肌动蛋白微丝和微管。这些连接是 由核骨骼和细胞骨架（LINC）复合物的接头介导，由内部核组成 膜太阳和外核膜Kash/Nesprin蛋白。在衰老中，有一个优先 Nesprin-2g与微管（由SUN1支撑）与肌动蛋白微丝的相互作用增加 （由Sun2支持）。这导致了我们的总体假设，即改变了核细胞骨骼连接 linc络合物介导的生理衰老中的固有细胞极性缺陷以及 加速衰老障碍。我们进一步假设该缺陷至少部分由蛋白质介导 来自老年人的细胞分泌的因素。我们建议以三个特定目标来检验这些假设。在 AIM 1，我们将破译生理衰老中细胞极性缺陷的机制。我们将 确定如何在衰老期间在核包膜中建立升高的SUN1水平，探索如何 Sun1与Nesprin-2g的相互作用偏向于其与微管的相互作用，并检查细胞质如何 微管受到与核包膜过度相互作用的影响。在AIM 2中，我们将检查 年龄依赖性细胞极性缺陷在体内愈合的作用。我们将检查皮肤伤口 从小鼠和老鼠开始，以确定组织培养中产生极性缺陷的相同机制是否存在 在体内手术，并检查转基因小鼠的伤口愈合和成纤维细胞极性 成纤维细胞中过表达的太阳。在AIM 3中，我们将净化并表征从中分泌的可溶因子 导致细胞极性缺陷的老化细胞。我们将使用质谱法的互补方法和 蛋白质纯化以识别我们称为FRAC的因素，以从老化细胞释放的因子。完成 这些目标将导致对林克复合物核细胞连接的改变的新见解 导致衰老中的基本细胞极性缺陷。