The nucleus-cytoskeleton connection in health and disease

描述(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型板层赋予，A型和B型板层是核膜下的蛋白质网络，形成核板层。散布在编码A-型层蛋白的Lmna上的突变与一系列人类疾病有关，统称为椎板病。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复合体的描述表明，细胞核与细胞骨架紧密相连。LINC复合体是一种进化保守的蛋白质复合体，跨越核膜，物理上连接核层和哺乳动物细胞的细胞骨架。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突变的有害影响在于严重破坏了介导基本生理细胞过程的物理连接，如核动力学、细胞机械刚性和极化。1)利用活细胞显微镜和基于流动的分析方法，比较缺乏A-型层粘连蛋白的小鼠胚胎成纤维细胞(来源于人类肌营养不良和心肌病的小鼠模型)和它们的野生型成纤维细胞的核和微管组织中心(MTOC)的移位率以及MTOC/核的实时和3D距离。通过定量成像，我们还将确定A型层粘连蛋白缺乏是否影响基于细胞骨架的关键细胞功能，包括单细胞运动性和MTOC极化。2)基于LINC复合体参与A-型层粘连蛋白控制的几个生理过程的假设，还将研究整个LINC复合体及其各个组成部分对细胞机械硬度和基于细胞骨架的细胞功能的贡献。3)疾病相关的Lmna突变对小鼠成纤维细胞和成肌细胞LINC复合体完整性和细胞骨架功能的影响将被检测。我们预计，该项目将阐明控制细胞核动力学和核-细胞骨架连接的生物物理原理，并将识别调节这种相互连接的关键分子连接子，并在细胞机械硬度、极化和运动性方面发挥关键作用。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椎板病相关的各种疾病表型的生物物理学基础。公共卫生相关性：散布在编码A型板层的Lmna基因上的突变与广泛的人类疾病有关，统称为椎板病。这项来自生物工程和细胞生物学的拟议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.