Cytoskeletal force generation on the nucleus

描述（由申请人提供）：细胞爬行是生理过程的核心，如伤口愈合、组织维持、分化、发育（形态发生）和癌症转移。爬行细胞在执行其正常任务（伸出、粘附和后缘缩回）时如何定位其核尚不清楚。我们提出两个具体目标：目标1：解释细胞核是如何通过机械力作用于爬行成纤维细胞核表面而定位于中心的。目的2：解释细胞核和肌动蛋白细胞骨架之间的机械连接如何介导成纤维细胞爬行的方向持久性。该提议具有创新性，原因如下：1）大多数关于细胞核运动的体外研究都是在受伤细胞单层边缘的运动性背景下进行的，重点是初始极化事件（其中细胞核被推离前缘）。相比之下，本研究将确定一个孤立的爬行细胞如何将其细胞核平移到细胞爬行的方向。2)我们从新颖的概念观点出发，认为核定位是相互竞争的前后力平衡的结果，前后力可以是推或拉。3)我们建议使用工程和生物分子工具的组合来扰乱这种核力平衡，并推导出驱动核定位的主导细胞骨架力的方向和相对大小。这项工作的医学意义是由于这样一个事实，即异常的核-细胞骨架力转移被认为涉及许多疾病，包括Emery-Dreifuss肌营养不良症，扩张型心肌病，Hutchinson-Gilford早衰综合征和Dunnigan型家族性部分脂肪营养不良。虽然这些疾病的分子机制尚不清楚，但已经假设这些疾病可能（部分）由于从细胞骨架到细胞核的异常力传递而导致。我们在这个建议中的方法依赖于突变与这些疾病相关的核-细胞骨架连接体，并检查力平衡的改变。因此，这项工作具有科学和医学意义。

DESCRIPTION (provided by applicant): Cell crawling is central to physiological processes like wound healing, maintenance of tissue, differentiation, development (morphogenesis) and cancer metastasis. How a crawling cell positions its nucleus as it executes its normal tasks of protrusion, adhesion, and retraction of the trailing edge is poorly understood. We propose two specific aims: Aim 1: Explain how the nucleus is positioned centrally through generation of mechanical forces on the nuclear surface in crawling fibroblasts. Aim 2: Explain how the mechanical linkages between the nucleus and the F-actin cytoskeleton mediate directional persistence of fibroblast crawling. This proposal is innovative for the following reasons: 1) The majority of in vitro studies on nuclear motion have been performed in the context of motility at the edge of a wounded cell monolayer with an emphasis on initial polarization events (where the nucleus is pushed away from the leading edge). In contrast, this study will determine how an isolated crawling cell translates its nucleus in the direction of cell crawling. 2) We work from th novel conceptual view that nuclear positioning is a result of a balance of competing forward and rearward forces which can be pushing or pulling. 3) We propose to use a combination of engineering and biomolecular tools to perturb this nuclear force balance and deduce the direction and relative magnitude of the dominant cytoskeletal forces driving nuclear positioning. The medical significance of this work is due to the fact that abnormal nuclear-cytoskeletal force transfer is thought to be involved in a number of diseases including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, Hutchinson-Gilford progeria syndrome and Dunnigan-type familial partial lipodystrophy. Although the molecular mechanism underlying these diseases remains unclear, it has been hypothesized that these diseases may result (in part) due to abnormal force transmission from the cytoskeleton to the nucleus. Our approach in this proposal relies on mutating nuclear- cytoskeletal linkers that are associated with these diseases and examining alterations in the force balance. This work therefore has both scientific and medical significance.