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由A型和B型核纤层蛋白赋予，A型和B型核纤层蛋白是位于核膜下方并形成核纤层的蛋白质网络。Lmna编码A型核纤层蛋白，其沿着Lmna散布的突变与广泛的人类疾病相关，统称为核纤层蛋白病。这些疾病的分子病因学仍然未知。在哺乳动物细胞中，LINC复合物的最新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，跨越核膜并将核纤层与哺乳动物细胞的细胞骨架物理连接。基于我们的初步结果和从低等生物获得的数据，我们假设Lmna突变的有害影响在于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。该项目将制定三个具体目标：1）使用活细胞显微镜和基于流动的测定，将在缺乏A型核纤层蛋白的小鼠胚胎成纤维细胞中比较细胞核和微管组织中心（MTOC）的移位率以及真实的时间和3D中的MTOC/细胞核距离（来源于人肌营养不良症和心肌病的小鼠模型）和它们的野生型对应物。使用定量成像，我们还将确定A型核纤层蛋白缺乏是否会影响基于细胞因子的关键细胞功能，包括单细胞运动和MTOC极化。2)基于LINC复合物介导由A型核纤层蛋白控制的几个生理过程的假设，还将研究整个LINC复合物以及其各自组分对细胞机械刚度和基于细胞骨架的细胞功能的贡献。3)将在小鼠成纤维细胞和成肌细胞中检查Lmna的疾病相关突变对LINC复合体完整性和细胞骨架功能的影响。我们预计，该项目将揭示控制细胞核动力学和细胞核-细胞骨架连接的生物物理学原理，并将确定调节这种相互连接的关键分子接头，并在细胞机械刚度，极化和运动中发挥关键作用。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核纤层蛋白病相关的各种疾病表型的生物物理基础。公共卫生相关性：沿着编码A型核纤层蛋白的Lmna基因散布的突变与广泛的人类疾病相关，统称为核纤层蛋白病。从生物工程和细胞生物学中提取的拟议研究可能有助于为与人类层粘连蛋白病相关的各种疾病表型建立生物物理学基础。

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.