Mechanisms of mesenchymal chemotaxis

描述（由申请人提供）：细胞在各种环境中向可溶性化学线索迁移，这一过程被称为趋化性。尽管经过近一个世纪的研究，趋化性的机制基础仍然不完全清楚。生长因子的空间梯度指导组织中间充质细胞的运动，以协调和加速生理上重要的过程，如伤口愈合，间充质趋化性与心血管和纤维化疾病等病理状况有关。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变形虫细胞上。间充质趋化性一直难以研究，因为它需要维持稳定的梯度好几个小时。传统的方法，如transwell分析，提供很少或根本没有动态信息，并且很难区分实际定向传感对运动效率的影响。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，同时允许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 > PKC > Myosin II途径控制间质趋化，并需要信号事件和细胞骨架组织的协调。为了阐明间充质细胞趋化的机制，我们建议：1)剖析间充质细胞趋化信号的时空性质；2)理解细胞骨架在趋化过程中的动态组织；3)描述导致复杂趋化行为的信号和细胞骨架事件的协调，如在三维环境中对新线索的重新定向和趋化。这些研究将直接有助于我们了解肿瘤转移、纤维化、心血管疾病等疾病状态的生理基础，以及对伤口愈合等生理过程的认识。

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.