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: 10619511
• 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 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10619511
• 关键词: Acceleration; Actins; Actomyosin; Address; Adhesions; Age; Aging; Cell Aging; Cell Nucleus; Cell Polarity; Cell physiology; Cell secretion; 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; Protein Secretion; 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.

项目概要 细胞骨架及其与细胞核的连接在建立细胞形态方面发挥着重要作用， 极性、迁移和粘附。由于它们在这些细胞功能中的重要作用，因此至关重要 了解细胞骨架系统及其核连接发生的变化很重要 在细胞衰老的正常过程中。尽管如此重要，但人们对它的作用却知之甚少。 生理衰老中的细胞骨架。我们的初步研究发现了一个基本的 患有加速衰老疾病的儿童的成纤维细胞中发生的细胞极性缺陷 Hutchinson- 吉尔福德早衰综合症以及来自生理年龄个体的成纤维细胞。这个缺陷是由 内核膜内侧的核层和核层之间的连接不平衡 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 由内核组成的核骨架和细胞骨架连接器 (LINC) 复合物介导 膜 SUN 和外核膜 KASH/nesprin 蛋白。老龄化方面，有优惠 与肌动蛋白微丝相比，nesprin-2G 与微管（由 SUN1 支持）的相互作用增强 （由 SUN2 支持）。这导致我们的整体假设改变了核细胞骨架连接 由 LINC 复合物介导的细胞极性缺陷会导致生理衰老过程中的内在细胞极性缺陷以及 加速衰老障碍。我们进一步假设这种缺陷至少部分是由蛋白质介导的 老年人细胞分泌的因子。我们建议在三个具体目标中检验这些假设。在 目标1，我们将破译生理衰老过程中细胞极性缺陷的机制。我们将 确定衰老过程中核膜中 SUN1 水平如何增加，探索如何 SUN1 与 nesprin-2G 的相互作用使其相互作用偏向于微管，并检查细胞质如何 微管受到与核膜过度相互作用的影响。在目标 2 中，我们将检查 年龄依赖性细胞极性缺陷在体内愈合中的作用。我们将检查皮肤伤口 从年轻和年老的小鼠身上确定在组织培养中产生极性缺陷的机制是否相同 在体内有效，并检查转基因小鼠的伤口愈合和成纤维细胞极性 在成纤维细胞中过度表达 SUN1。在目标 3 中，我们将纯化并表征从 老化的细胞会导致细胞极性有缺陷。我们将使用质谱分析的补充方法 蛋白质纯化以鉴定我们称之为 FRAC 的因子，即从老化细胞中释放的因子。完成 这些目标将带来关于 LINC 复合体核细胞骨架连接如何改变的新见解 导致衰老过程中基本的细胞极性缺陷。