STRUCTURAL STUDIES OF WNT5A CONTROL OF CELL POLARITY AND DIRECTIONAL MOVEMENT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞极性对细胞行为的几乎所有方面都是必不可少的，包括细胞形态、细胞运动和不对称分裂引起的细胞特性。细胞的极性在遗传系统中得到了最广泛的研究，但尽管这导致了许多调控因子的发现，还有更多的调控因子仍有待确定。非规范的Wnt信号(即非依赖于连接素的)控制许多细胞极性的实例，例如在细胞分裂期间或在建立神经元的定向形态时。许多与细胞极性有关的基因已经通过遗传筛选被发现，包括细胞受体、细胞骨架调节因子和Wnt信号分子。然而，目前还不清楚这些基因是如何以机械方式协同工作来调节细胞极性的。我们的目标是将生化方法与EM断层扫描相结合，以建立一个如何控制细胞极性的机制模型。WNT在许多发育系统中调节极化受体的定位，但这是发生在细胞极化的早期还是晚期尚不清楚。我们开发了一种非正统的策略，在使用已知浓度的配体在受控的一段时间内急性刺激Wnt信号后，对细胞进行成像，并发现了一种前所未有的机制来解释细胞极性和定向细胞运动是如何通过Wnt5a响应信号而被控制的。这些被应用于黑色素瘤细胞，其中Wnt5a已被证明促进细胞侵袭[1]。通过用纯化的Wnt5a处理黑色素瘤细胞系，我们发现了Wnt5a(2)诱导的一种新的极化结构。该复合体由一个免疫球蛋白家族细胞黏附分子(黑色素瘤细胞黏附分子，或“MCAM”)、F-肌动蛋白和肌球蛋白IIB组成。这种独特的复合体，我们称之为Wnt-受体-肌动蛋白-肌球蛋白复合体(Wram)“，在接触Wnt5a后30分钟内形成，反映了更广泛的细胞极化。WNT5A诱导WRAM复合体在细胞后部的极化定位，其形成导致膜的快速回缩，导致细胞向前方向移动。因此，WRAM复合体的形成通过调节后膜回缩来控制细胞的定向运动。在趋化梯度的存在下，WRAM复合体相对于高尔基体向远端定向，表明在趋化线索的背景下Wnt5a的细胞极化。这种对急性Wnt5a暴露的新颖反应允许研究引发细胞极化的早期事件，而传统的遗传方法不容易完成这一事件。相关的电子显微镜(EM)和EM断层扫描被用来绘制WRAM复合体形成过程中细胞结构的变化。我们对表达MCAM-绿色荧光蛋白L的活细胞进行成像，并在WNT5a处理后的不同时间快速冷冻，捕捉到WRAM复合体形成的不同步骤。这将使超微结构的变化与wram复合体的变化相关联成为可能。相关的EM将使我们能够观察到MVB在特定步骤上的内化、膜运输以及与细胞骨架和质膜蛋白的相互作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell polarity is essential for virtually all aspects of cell behavior including cell morphology, cell motility, and cell identity arising from asymmetric divisions. Cell polarity has been most extensively studied in genetic systems, but while this has led to the discovery of many regulators, many more remain to be identified. Non-canonical Wnt signaling (i.e. ¿-catenin independent) controls many instances of cell polarity such as during cell division or in establishing directional morphology of neurons. Many genes involved in cell polarity have been identified through genetic screens including cell receptors, cytoskeleton regulators, and Wnt signaling molecules. Yet it is not clear how these genes work together mechanistically to regulate cell polarity. Our goal is to integrate biochemical approaches with EM tomography in order to build a mechanistic model of how cell polarity is controlled. Wnt regulates polarized receptor localization in many developmental systems, but whether this occurs early or late during cell polarization is unclear. We developed an unorthodox strategy of imaging cells after acute stimulation of Wnt signaling using known concentrations of ligand for controlled periods of time, and discovered an unprecedented mechanism to explain how cell polarity and directional cell movement are controlled in response to signaling through Wnt5a. These were applied to melanoma cells, where Wnt5a has been shown to promote cell invasion (1). By treating melanoma cell lines with purified Wnt5a, we discovered a novel polarized structure induced by Wnt5a (2). The complex is composed of an IgG-family cell adhesion molecule (melanoma cell adhesion molecule, or "MCAM"), F-actin, and myosin IIB. This unique complex, which we refer to as the Wnt-Receptor-Actin-Myosin (WRAM) complex", forms within 30 minutes of exposure to Wnt5a and reflects a more widespread polarization of the cell. Wnt5a induces a polarized localization of the WRAM complex at the cell posterior, and its formation leads to rapid retraction of membrane, leading to cell movement towards the anterior direction. Thus, formation of the WRAM complex controls directional cell movement, by regulating posterior membrane retraction. In the presence of a chemotactic gradient, the WRAM complex orients distally with respect to Golgi, indicating cell polarization by Wnt5a in the context of a chemotactic cue. This novel response to acute Wnt5a exposure allows the study of early events which initiate cell polarization, not easily accomplished by classic genetic approaches. Correlative electron microscopy (EM) and EM tomography are being used to map the changing structure of the cell during WRAM complex formation. We are imaging live cells expressing MCAM-GFP l and have frozen by rapid freezing at different times after Wnt5a treatment, capturing different steps in WRAM complex formation. This will make it possible to correlate changes in ultrastructure with changes in the WRAM complex. Correlative EM will allow us to observe MVBs at specific steps distinguished by internalization, membrane trafficking, and interaction with cytoskeletal and plasma membrane proteins.