STRUCTURAL STUDIES OF WNT5A CONTROL OF CELL POLARITY AND DIRECTIONAL MOVEMENT

WNT5A 控制细胞极性和定向运动的结构研究

• 批准号: 7955059
• 负责人: NATALIE G. AHN
• 金额: $ 1.07万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955059
• 关键词: Actins; Acute; Anterior; Biochemical; CD146 antigen; Cell Adhesion Molecules; Cell Line; Cell Polarity; Cell division; Cell membrane; Cells; Cellular Morphology; Cellular Structures; Complex; Computer Retrieval of Information on Scientific Projects Database; Cues; Cytoskeleton; Development; Electron Microscopy; Event; Exposure to; F-Actin; Family; Freezing; Funding; Genes; Genetic; Genetic Screening; Goals; Golgi Apparatus; Grant; Image; Immunoglobulin G; Institution; Life; Ligands; MCAM gene; Maps; Melanoma Cell; Membrane; Membrane Protein Traffic; Membrane Proteins; Modeling; Morphology; Movement; Myosin ATPase; Neurons; Nonmuscle Myosin Type IIB; Receptor Cell; Research; Research Personnel; Resources; Signal Transduction; Signaling Molecule; Source; Structure; System; Time; United States National Institutes of Health; Work; cell behavior; cell motility; cellular imaging; directional cell; novel; receptor; response; tomography

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 细胞极性对于细胞行为的几乎所有方面都是必不可少的，包括细胞形态、细胞运动性和由不对称分裂引起的细胞身份。细胞极性在遗传系统中得到了最广泛的研究，虽然这导致了许多调节因子的发现，但仍有更多的调节因子有待确定。非经典Wnt信号传导（即非连环蛋白依赖性）控制细胞极性的许多情况，例如在细胞分裂期间或建立神经元的定向形态。通过基因筛选已经鉴定出许多与细胞极性相关的基因，包括细胞受体、细胞骨架调节因子和Wnt信号分子。然而，目前尚不清楚这些基因是如何协同工作，机械地调节细胞极性。 我们的目标是将生物化学方法与EM断层扫描相结合，以建立细胞极性如何控制的机制模型。Wnt在许多发育系统中调节极化受体定位，但这是否发生在细胞极化的早期或晚期尚不清楚。我们开发了一种非正统的策略，使用已知浓度的配体在受控的时间段内对Wnt信号进行急性刺激后对细胞进行成像，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如何响应于通过Wnt 5a的信号而被控制。 这些被应用于黑色素瘤细胞，其中Wnt 5a已被证明促进细胞侵袭（1）。 通过用纯化的Wnt 5a处理黑色素瘤细胞系，我们发现了由Wnt 5a诱导的新的极化结构（2）。该复合物由IgG家族细胞粘附分子（黑素瘤细胞粘附分子，或“MCAM”）、F-肌动蛋白和肌球蛋白IIB组成。这种独特的复合物，我们称之为Wnt-受体-肌动蛋白-肌球蛋白（WRAM）复合物，在暴露于Wnt 5a后30分钟内形成，反映了细胞更广泛的极化。Wnt 5a诱导WRAM复合物在细胞后部的极化定位，并且其形成导致膜的快速回缩，从而导致细胞向前部方向移动。 因此，WRAM复合物的形成通过调节后膜收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Wnt 5a在趋化性提示的背景下细胞极化。 这种对急性Wnt 5a暴露的新反应允许研究启动细胞极化的早期事件，这不容易通过经典的遗传方法完成。相关的电子显微镜（EM）和EM层析成像被用来映射在WRAM复杂的形成过程中细胞的结构变化。我们对表达MCAM-GFP 1的活细胞进行成像，并在Wnt 5a处理后的不同时间通过快速冷冻进行冷冻，从而捕获WRAM复合物形成中的不同步骤。这将使我们有可能将超微结构的变化与WRAM复合体的变化联系起来。相关EM将使我们能够观察MVBs在特定的步骤区分的内化，膜运输，与细胞骨架和质膜蛋白的相互作用。