Mechanisms of mesenchymal chemotaxis

描述(由申请人提供)：各种环境中的细胞在一种称为趋化性的过程中向可溶的化学信号迁移。尽管经过了近一个世纪的研究，但趋化性的机制基础仍然不完全清楚。生长因子的空间梯度引导组织中间充质细胞的运动，以协调和加速伤口愈合等重要生理过程，间充质趋化作用与心血管疾病和纤维化疾病等病理疾病有关。然而，绝大多数趋化性研究都集中在白细胞和其他快速移动的阿米巴细胞上。间充质趋化性的研究一直是非常困难的，因为它需要维持稳定的梯度数小时。传统的方法，如井间分析，提供的动态信息很少或根本没有，而且对运动效率和实际定向传感的区分效果很差。为了克服这些技术限制，我们最近建立了一种微流控趋化试验，可以在数小时内以稳定的线性梯度直接观察间充质细胞，从而支持单细胞跟踪和高分辨率活细胞成像方法，如TIRF显微镜。我们的初步数据表明，生长因子受体PDGF-R通过PLC&GT；PKC&GT；肌球蛋白II途径控制间充质趋化，并需要信号事件和细胞骨架组织的协调。我们建议通过1)解剖间充质细胞趋化信号的时空性质来阐明间充质细胞趋化的机制，2)了解这种细胞在趋化过程中细胞骨架的动态组织，以及3)描述导致复杂的趋化行为的信号和细胞骨架事件的协调，例如在3D环境中对新线索的重新定位和趋化。这些研究将直接有助于我们理解肿瘤转移、纤维化和心血管疾病等疾病状态的生理基础，以及我们对伤口愈合等生理过程的理解。

DESCRIPTION (provided by applicant): Cells in a variety of contexts migrate towards soluble chemical cues in a process known as chemotaxis. Despite nearly a century of study, the mechanistic underpinnings of chemotaxis remain incompletely understood. Spatial gradients of growth factors direct the movements of mesenchymal cells in tissues to coordinate and accelerate physiologically important processes such as wound healing, and mesenchymal chemotaxis has been implicated in pathological conditions such as cardiovascular and fibrotic diseases. Yet, the vast majority of chemotaxis studies have focused on leukocytes and other fast-moving, amoeboid cells. Mesenchymal chemotaxis has been prohibitively difficult to study, because it requires maintenance of stable gradients for many hours. Traditional methods such as transwell assays provide little or no dynamic information and poorly discriminate effects on the efficiency of motility from actual directional sensing. To overcome these technical limitations we recently established a microfluidic chemotaxis assay that allows direct observation of mesenchymal cells in stable, linear gradients over many hours, allowing both single-cell tracking and high-resolution live-cell imaging approaches such as TIRF microscopy. Our preliminary data indicate that the growth factor receptor, PDGF-R controls mesenchymal chemotaxis by a PLC > PKC > Myosin II pathway and requires the coordination of signaling events and cytoskeletal organization. We propose to elucidate the mechanisms of mesenchymal chemotaxis by 1) Dissecting the spatio- temporal nature of chemotactic signaling in mesenchymal cells 2) Understanding the dynamic organization of the cytoskeleton during chemotaxis in this cell type and 3) Delineating the coordination of signaling and cytoskeletal events that lead to complex chemotactic behaviors such as re-orientation to new cues and chemotaxis in 3D environments. These studies will directly contribute to our understanding the physiological basis of disease states such tumor metastasis, fibrosis and cardiovascular disease, as well as our understanding of physiological processes such as wound healing.