The nucleus-cytoskeleton connection in health and disease

描述（由申请人提供）：在哺乳动物细胞中，细胞核的结构完整性是由A型和b型层蛋白赋予的，这是一种位于核膜下面并形成核层的蛋白质网络。编码a型层粘连蛋白的Lmna上散布的突变与广泛的人类疾病有关，统称为层粘连病。这些疾病的分子病因尚不清楚。在哺乳动物细胞中，最近对LINC复合物的表征表明，细胞核与细胞骨架紧密相连。LINC复合物是一种进化保守的蛋白质复合物，跨越核膜，物理上将核层与哺乳动物细胞骨架连接起来。根据我们的初步结果和从低等生物获得的数据，我们假设Lmna突变的有害影响包括介导基本生理细胞过程（如核动力学、细胞机械刚度和极化）的物理连接的严重破坏。本项目将发展三个具体目标：1)使用活细胞显微镜和基于流量的测定，将比较缺乏a型层蛋白（源自人类肌肉萎缩症和心肌病小鼠模型）的小鼠胚胎成纤维细胞和它们的野生型成纤维细胞的细胞核和微管组织中心（MTOC）的易位率以及实时和3D的MTOC/核距离。通过定量成像，我们还将确定a型纤层蛋白缺乏是否会影响基于细胞骨架的关键细胞功能，包括单细胞运动和MTOC极化。2)基于LINC复合物介导由a型层蛋白控制的多种生理过程的假设，我们还将研究整个LINC复合物及其各自组分对细胞机械刚度和细胞骨架细胞功能的贡献。3)将在小鼠成纤维细胞和成肌细胞中检测疾病相关突变对LINC复合物完整性和细胞骨架功能的影响。我们预计该项目将阐明控制核动力学和核-细胞骨架连接的生物物理原理，并将确定调节这种连接并在细胞机械刚度，极化和运动性中发挥关键作用的关键分子连接物。使用我们的定量分析的实验结果也可能有助于建立与人类层压板病相关的各种疾病表型的生物物理基础。公共卫生相关性：编码a型层粘连蛋白的Lmna基因上分散的突变与广泛的人类疾病有关，统称为层粘连病。从生物工程和细胞生物学中提出的研究可能有助于为与人类椎板病相关的各种疾病表型建立生物物理基础。

DESCRIPTION (provided by applicant): In mammalian cells, the structural integrity of the nucleus is conferred by A- and B-type lamins, a meshwork of proteins that underlies the nuclear envelope and forms the nuclear lamina. Mutations scattered along Lmna, which encodes A-type lamins, are associated to a broad range of human diseases, collectively called laminopathies. The molecular etiology of these diseases remains unknown. In mammalian cells, the recent characterization of the LINC complex, an evolutionary-conserved protein complex that spans the nuclear envelope and physically connects the nuclear lamina to the cytoskeleton of mammalian cells suggest that nucleus is intimately tethered to the cytoskeleton. Based on our preliminary results and from data available from lower organisms, we hypothesize that the deleterious effect of Lmna mutations consist in the severe disruption of physical connections that mediate essential physiological cellular processes such as nuclear dynamics, cellular mechanical stiffness and polarization. Three specific aims will be developed in this project: 1) Using live-cell microscopy and a flow-based assay, the rates of translocation of both the nucleus and the microtubule organizing center (MTOC) as well as the MTOC/nucleus distance in real time and in 3D will be compared in mouse embryonic fibroblasts lacking A-type lamins (derived from a mouse model of human muscular dystrophy and cardiomyopathy) and their wild-type counterparts. Using quantitative imaging, we also will determine whether A-type lamin deficiency affects key cytoskeleton-based cell functions, including single-cell motility and MTOC polarization. 2) Based on the hypothesis that the LINC complex mediates several physiological processes controlled by A-type lamins, the contribution of the whole LINC complex as well as of its respective components to cellular mechanical stiffness and cytoskeleton-based cell functions will also be investigated. 3) The effect of disease-associated mutations of Lmna on the LINC complex integrity and cytoskeleton functions will be examined in mouse fibroblasts and myoblasts. We anticipate that this project will shed light on the biophysical principles that govern nucleus dynamics and nucleus-cytoskeleton connections, and will identify key molecular linkers that regulate this interconnection and play a critical role in cellular mechanical stiffness, polarization and motility. Results from experiments using our quantitative assays may also help establish a biophysical basis for the wide variety of disease phenotypes associated to human laminopathies. Public Health Relevance: Mutations scattered along the Lmna gene, which encodes A-type lamins, are associated to a broad range of human diseases, collectively called laminopathies. The proposed research drawing from bioengineering and cell biology may help establish a biophysical basis for the wide variety of disease phenotypes associated to human laminopathies.