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: 8170840
• 负责人: NATALIE G. AHN
• 金额: $ 1.25万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170840
• 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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 细胞极性对于细胞行为的几乎所有方面都至关重要，包括细胞形态、细胞运动和不对称分裂引起的细胞身份，细胞极性主要在遗传系统中进行研究，但这导致了许多调节因子的发现。更多的信息有待确定。非经典 Wnt 信号传导（即不依赖于 ¿-连环蛋白）控制细胞极性的许多情况，例如在细胞分裂过程中或在建立神经元的定向形态过程中，许多参与细胞极性的基因已通过遗传鉴定。筛选包括细胞受体、细胞骨架调节剂和 Wnt 信号分子，但尚不清楚这些基因如何机械地协同作用来调节细胞极性。我们的目标是将生化方法与 EM 断层扫描相结合，以建立细胞极性的机制模型。 Wnt 在许多发育系统中调节极化受体定位，但尚不清楚这种情况是否发生在细胞极化过程的早期或晚期。配体在受控的时间段内，并发现了一种前所未有的机制来解释如何通过 Wnt5a 响应信号来控制细胞极性和定向细胞运动。这些研究应用于黑色素瘤细胞，其中 Wnt5a 已被证明可以促进细胞侵袭 (1)。通过用纯化的 Wnt5a 处理黑色素瘤细胞系，我们发现了 Wnt5a 诱导的新型极化结构 (2) 该复合物由 IgG 家族细胞粘附分子（黑色素瘤细胞粘附分子，或“MCAM”）、F-肌动蛋白和肌球蛋白 IIB 这种独特的复合物，我们称为“Wnt-受体-肌动蛋白-肌球蛋白（WRAM）复合物”，在接触 Wnt5a 后 30 分钟内形成，反映了更广泛的分布。 Wnt5a 诱导细胞后部 WRAM 复合物的极化定位，其形成导致细胞膜快速回缩，从而导致细胞向前移动。 WRAM 复合体通过调节后膜回缩来控制定向细胞运动，在存在趋化梯度的情况下，WRAM 复合体相对于高尔基体向远端定向，表明 Wnt5a 在趋化信号的作用下发生细胞极化。 Wnt5a 暴露可以研究引发细胞极化的早期事件，而传统的遗传方法很难完成相关电子显微镜 (EM) 和 EM 断层扫描，以绘制变化图。我们对表达 MCAM-GFP l 的活细胞进行成像，并在 Wnt5a 处理后的不同时间通过快速冷冻来捕获 WRAM 复合物形成过程中的不同步骤，这将使得将超微结构的变化关联起来。随着 WRAM 复合体的变化，相关 EM 将使我们能够观察 MVB 的特定步骤，这些步骤通过内化、膜运输以及与细胞骨架和质膜蛋白的相互作用来区分。