Role of Nucleo-cytoskeleton Interactions in Cell Migration

 描述(申请人提供)：几乎所有的细胞都将它们的细胞核定位在特定的位置，以反映细胞和组织的功能。参与核定位通路的蛋白质的破坏会导致细胞生理改变和人类疾病，包括肌肉营养不良、心肌病、无脑畸形、小脑性共济失调和肌张力障碍。核定位如何影响细胞功能尚不清楚。我们的目标是探索迁移细胞中核位置的机制和功能，其中核始终位于细胞的区域。我们已经建立了一个模型损伤单层系统，在该系统中，核运动建立了这种向后的位置，可以受到外部因素的刺激并进行定量测量。我们还将开发一种新的方法来人工置换粘附物中的细胞核 并将我们的研究扩展到3D迁移系统，在这种系统中，核运动可能是迁移的速率限制。我们早期的研究证实，后向核的位置是肌动蛋白和肌球蛋白依赖的，需要在肌动蛋白缆线和核之间建立连接。这种联系是通过LINC复合体成分Nesprin-2G在外核膜和SUN2在内核膜中的聚集而介导的。这种聚集导致类似于“核粘连”的结构的形成，我们称之为跨膜肌动蛋白相关核线的TAN线。我们将通过令人兴奋的初步研究进一步探索TAN系列的组装和功能，这些研究表明，除了机械偶联细胞核和肌动蛋白细胞骨架外，TAN系列还展示了影响核运动的机械力化学信号。通过我们人工移位细胞核的新方法，我们发现了令人惊讶的证据，即细胞的细胞核位置与肌动蛋白或微管机制有关，这取决于细胞核是否移位到细胞后部的前部。我们将使用这个系统来定义一个新的LINC复合体是如何与微管和微管马达相联系的，并测试细胞在极化和细胞迁移的活跃阶段是否在LINC复合体之间切换。为了了解核位置如何影响迁移，我们将检验核与焦点粘连耦合的假设，并通过基于内部张力的机制来调节它们的动力学。最后，我们将探索不同的LINC复合体对3D基质中细胞迁移的贡献，并检验当核变形时可能需要不同的LINC复合体来挤压基质中的小孔。这些研究将揭示LINC复合体定位细胞核的新机制，并为核定位如何影响细胞迁移提供新的见解。这些信息将有助于理解与核定位通路相关的疾病是如何产生的，以便能够设计有效的治疗方法。

 DESCRIPTION (provided by applicant): Virtually all cells position their nucleus in specific locations that reflect cell and tissue function. Disruption of proteins involved in nuclear positioning pathways leads to altered cell physiology and human diseases including muscular dystrophies, cardiomyopathy, lissencephaly, cerebellar ataxia and dystonia. How nuclear positioning contributes to cell function is unclear. Our goal is to explore the mechanisms and function of nuclear position in migrating cells where nuclei are consistently positioned in the rea of cells. We have developed a model wounded monolayer system where nuclear movement establish this rearward position and can stimulated by external factors and measured quantitatively. We will also developed a novel method of artificially displacing nuclei in adherent cells by centrifugal force and will extend our studies to 3D migration systems where nuclear movements may be rate limiting for migration. Our earlier studies established that rearward nuclear position is actin- and myosin-dependent and requires the establishment of a connection between actin cables and the nucleus. This connection is mediated by the clustering of LINC complex components nesprin-2G in the outer nuclear membrane and SUN2 in the inner nuclear membrane. This clustering results in the formation of structures that resemble "nuclear adhesions" that we termed TAN lines for transmembrane actin-associated nuclear lines. We will further explore the assemble and function of TAN lines by pursuing exciting preliminary studies that suggest that in addition to mechanically coupling nuclei to the actin cytoskeleton, TAN lines exhibit mechanochemical signaling that affects nuclear movement. With our new method for artificially displacing nuclei, we have found surprising evidence that cells position their nuclei y actin or microtubule mechanisms depending on whether nuclei are displaced to the front of rear of the cells. We will use this system to define how a novel LINC complex associate with microtubules and microtubule motors and test whether cells switch between LINC complexes during polarization and active phases of cell migration. To understand how nuclear position influences migration, we will test the hypothesis that nuclei are coupled to focal adhesions and modulate their dynamics by an internal tension based mechanism. Lastly, we will explore the contribution of different LINC complexes to cell migration in 3D matrices and test the specific hypothesis that different LINC complexes may be required when nuclei deform to squeeze through small pores in the matrix. These studies will uncover novel mechanisms of LINC complex positioning of nuclei and provide new insights into how nuclear positioning influences cell migration. This information will contribute to understanding of how diseases associated with nuclear positioning pathways originate so that effective treatments can be designed.