FIRCA: Probing the Nucleus- Cytoskeleton Connection Using Magnetic Tweezers

FIRCA：使用磁镊探测细胞核-细胞骨架连接

基本信息

批准号：
8080552
负责人：
Denis Wirtz
金额：
$ 5.81万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-06-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8080552
关键词：
Actins Affect Biological Assay Cell Nucleus Cells Chile Collaborations Complement Complex Cytoplasm Cytoskeleton Dilated Cardiomyopathy Disease Down-Regulation Embryo Emery-Dreifuss Muscular Dystrophy Fibroblasts Fluorescence Genes Grant Health Human Immunofluorescence Immunologic Intermediate Filaments Lamin Type A Lamin Type B Lamins Life Link Magnetism Mammalian Cell Measurement Measures Mechanics Mediating Membrane Proteins Microfilaments Microscopy Microtubule-Organizing Center Missense Mutation Molecular Monitor Motion Mus Mutation Myoblasts Nuclear Nuclear Envelope Nuclear Lamina Parents Phase Property Proteins Regulation Research Role Rotation Rupture Shapes Small Interfering RNA Specimen Testing The Sun Time Torque United States National Institutes of Health Universities Wound Healing base cell motility disease phenotype emerin essays human disease migration nanorod novel parent grant protein complex research study

项目摘要

DESCRIPTION (provided by applicant): This research will be done primarily in Chile at the Catholic university of Chile in collaboration with Alfredo Celedon, as an extension of NIH Grant No. R01GM08420401, 09/30/08 - 08/31/12." The nuclear envelope has an internal shell called nuclear lamina, a thin meshwork of intermediate filaments composed of A- and B-type lamins. Mutations scattered along Lmna, which encodes A-type lamins, have been associated with a broad range of human diseases, collectively called laminopathies. In mammalian cells, the recent characterization of the LINC complex, an evolutionary-conserved protein complex that interacts both with the nuclear lamina and the cytoskeleton of mammalian cells suggests that nucleus and cytoskeleton are intimately connected. Our main hypothesis is that the nuclear envelope is mechanically connected to the actin filament network and the MTOC directly through specific linker proteins, including emerin and the LINC complexes, and that these connections are disrupted in laminopathies. Recent results obtained in the PI's lab suggest that the depletion of lamin A/C, as well as the specific rupture of the LINC complexes negatively affects both cell motility and intracellular mechanics. Moreover, results from a shear flow assay combined with immuno-fluorescence show that the distance between MTOC and nuclear envelope is greatly increased following emerin depletion and that this loosening of the MTOC correlates with the inability of emerin- deficient cells and lamin A/C-deficient cells to polarize in the flow direction. However, a direct demonstration that the actin cytoskeleton and the MTOC are actually both connected to the nuclear envelope and that these physical connections are mediated by the LINC complexes and emerin, respectively, is lacking. Here, we propose to use magnetic tweezers and magnetic nanorods to demonstrate the existence of these molecular connections, decipher the role of emerin and the LINC complexes in these connections and determine the mechanical strength of the links between the nucleus and both the actin filament network and the MTOC. We also use magnetic tweezers to measure for the first time the micromechanical properties of nucleus in live cells. 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. Using a new, single cell magnetic tweezers strategy, the proposed research may help establish a biophysical basis for the wide variety of disease phenotypes associated to human laminopathies.

描述（由申请人提供）：这项研究将主要在智利的智利天主教大学与 Alfredo Celedon 合作进行，作为 NIH 拨款号 R01GM08420401（2008 年 9 月 30 日至 2012 年 8 月 31 日）的延伸。”核膜有一个称为核纤层的内壳，是一种由分散在其中的 A 型和 B 型核纤层组成的中间丝的薄网。 Lmna 编码 A 型核纤层蛋白，与多种人类疾病（统称为核纤层蛋白病）有关。在哺乳动物细胞中，LINC 复合物是一种进化保守的蛋白复合物，与核纤层和核纤层相互作用。哺乳动物细胞的细胞骨架表明细胞核和细胞骨架紧密相连，我们的主要假设是核膜通过特定的连接蛋白直接与肌动蛋白丝网络和 MTOC 机械连接。包括 emerin 和 LINC 复合物，并且这些连接在核纤层蛋白病中被破坏。 PI 实验室最近获得的结果表明，核纤层蛋白 A/C 的消耗以及 LINC 复合物的特定破裂会对细胞运动和细胞内力学产生负面影响。此外，剪切流测定结合免疫荧光的结果表明，在 emerin 耗尽后，MTOC 和核膜之间的距离大大增加，并且 MTOC 的这种松动与 emerin 缺陷细胞和核纤层蛋白 A/C- 的无能力相关。缺陷细胞沿流动方向极化。然而，缺乏直接证明肌动蛋白细胞骨架和 MTOC 实际上都与核膜相连，并且这些物理连接分别由 LINC 复合物和 emerin 介导。在这里，我们建议使用磁镊子和磁性纳米棒来证明这些分子连接的存在，破译 emerin 和 LINC 复合物在这些连接中的作用，并确定细胞核与肌动蛋白丝网络和肌动蛋白丝网络之间连接的机械强度。 MTOC。我们还首次使用磁力镊子测量活细胞中细胞核的微观力学特性。公共卫生相关性：分布在编码 A 型核纤层蛋白的 Lmna 基因上的突变与多种人类疾病相关，统称为核纤层蛋白病。使用新的单细胞磁性镊子策略，拟议的研究可能有助于为与人类核纤层蛋白病相关的多种疾病表型建立生物物理学基础。