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的智利天主教大学与Alfredo Celedon合作进行，作为NIH第号补助金的延伸。R01GM08420401，09/30/08-08/31/12。核膜有一个叫做核膜的内壳，这是一个由A型和B型板层组成的中间丝组成的薄网。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。在哺乳动物细胞中，最近对LINC复合体的描述表明，核和细胞骨架是紧密相连的。LINC复合体是一种进化保守的蛋白质复合体，与哺乳动物细胞的核膜和细胞骨架相互作用。我们的主要假设是，核膜通过特定的连接蛋白，包括Emerin和LINC复合体，直接机械地连接到肌动蛋白细丝网络和MTOC，并且这些连接在椎板病变中被破坏。PI实验室的最新结果表明，层蛋白A/C的耗尽以及LINC复合体的特异性断裂对细胞运动和细胞内机制都产生了负面影响。此外，剪切流分析结合免疫荧光的结果表明，在Emerin耗尽后，MTOC与核膜之间的距离大大增加，并且这种MTOC的松动与Emerin缺陷细胞和Lamin A/C缺陷细胞不能在流动方向极化有关。然而，缺乏直接的证据表明肌动蛋白细胞骨架和MTOC实际上都连接到核膜上，并且这些物理连接分别由LINC复合体和Emerin介导。在这里，我们建议使用磁钳和磁性纳米棒来证明这些分子连接的存在，破译Emerin和LINC复合体在这些连接中的作用，并确定细胞核与肌动蛋白细丝网络和MTOC之间的连接的机械强度。我们还首次使用磁镊子测量活细胞中细胞核的微观力学性质。

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.