FIRCA: Probing the Nucleus- Cytoskeleton Connection Using Magnetic Tweezers

描述（由申请人提供）：这项研究将主要在智利天主教大学与阿尔弗雷多·塞莱登合作，作为NIH批准号R 01 GM 08420401，09/30/08 - 08/31/12的延伸。核被膜有一个称为核层的内壳，这是一个由A型和B型核层组成的中间细丝组成的薄网。编码A型核纤层蛋白的Lmna的沿着分布的突变与广泛的人类疾病相关，统称为核纤层蛋白病。在哺乳动物细胞中，LINC复合物，一种与哺乳动物细胞的核纤层和细胞骨架相互作用的进化保守的蛋白质复合物的最新表征表明，细胞核和细胞骨架是密切相连的。我们的主要假设是，核被膜是机械连接到肌动蛋白丝网络和MTOC直接通过特定的接头蛋白，包括emerin和LINC复合物，这些连接被破坏的核纤层蛋白病。 PI实验室中获得的最新结果表明，核纤层蛋白A/C的耗尽以及LINC复合物的特异性破裂对细胞运动性和细胞内力学都产生了负面影响。此外，结合免疫荧光的剪切流测定的结果表明，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缺陷型细胞和核纤层蛋白A/C缺陷型细胞在流动方向上不能粘附相关。然而，缺乏肌动蛋白细胞骨架和MTOC实际上都连接到核膜，并且这些物理连接分别由LINC复合物和emerin介导的直接证明。在这里，我们建议使用磁性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MTOC之间的联系的机械强度。我们还首次利用磁镊测量了活细胞中细胞核的微力学性质。

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.