LOCALIZATION OF RAS REGULATORS DURING DICTYOSTELIUM CHEMOTAXIS

盘基网柄菌趋化过程中 RAS 调节因子的定位

• 批准号: 8169650
• 负责人: RICHARD A FIRTEL
• 金额: $ 2.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169650
• 关键词: Actins; Cell Line; Cell membrane; Cells; Chemotactic Factors; Chemotaxis; Complex; Computer Retrieval of Information on Scientific Projects Database; Cytoplasm; Cytoskeleton; Data; Dictyostelium; Funding; Grant; Guanine; Guanine Nucleotide Exchange Factors; Institution; Microscope; Microscopy; Protein phosphatase; Proteins; Ras Inhibitor; Ras Signaling Pathway; Research; Research Personnel; Resources; Scaffolding Protein; Signal Transduction; Source; Testing; Total Internal Reflection Fluorescent; United States National Institutes of Health; cell motility; polarized cell; protein complex; ras GTPase-Activating Proteins; ras Guanine Nucleotide Exchange Factors

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. Ras is a key regulator of directed cell migration, controlling directional sensing, cell motility as well as signal relay in Dictyostelium. We showed that Ras is preferentially activated at the plasma membrane at the leading edge of migrating cells, resulting in the localized activation of downstream effectors that regulate the actin cytoskeleton and thereby promote directed cell movement. We recently identified a protein complex made of two Ras guanine exchange factors (RasGEFs), which are activators of Ras, a scaffold protein, and the protein phosphatase 2A (PP2A). We found that this RasGEF complex selectively controls an important Ras signaling pathway that regulates both chemotaxis and signal relay. Since activated Ras is locally enriched at the front of migrating cells, we hypothesized that the RasGEFs would be similarly localized. We tested this hypothesis by fusing GFP to different components of the RasGEF complex and assessing their localization in chemotaxing cells under a confocal microscope. We now have evidence that upon sudden uniform chemoattractant stimulation that the RasGEF complex is transiently translocated from the cytoplasm to the plasma membrane. However, because of the strong cytosolic signal, we were unable to determine if the RasGEF complex is present at the plasma membrane in polarized cells that are migrating in a chemoattractant gradient. We were previously faced with a similar problem when studying the localization of a Ras GTPase activating protein (RasGAP), a Ras inhibitor in this case, and TIRF microscopy allowed us to detect plasma membrane localization of this protein when other types of microscopy did not. We are thus confident that TIRF microscopy would also provide us with an answer concerning any plasma membrane localization of the RasGEF complex in chemotaxing cells. All the conditions to look at chemotaxing cells in TIRF microscopy have been optimized already so we expect we will be able to get all of the needed data in only a few sessions. We propose to look at two different cell lines: one expressing a GFP-fused protein from the RasGEF complex and a control cell line expressing soluble, cytosolic GFP.

这个子项目是许多利用 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。 Ras是网骨藻细胞定向迁移、定向感知、细胞运动和信号传递的关键调节因子。我们发现Ras优先在迁移细胞前缘的质膜上激活，导致下游效应物的局部激活，这些效应物调节肌动蛋白细胞骨架，从而促进定向细胞运动。我们最近发现了一种由两个Ras鸟嘌呤交换因子（RasGEFs）组成的蛋白质复合物，RasGEFs是Ras（一种支架蛋白）和蛋白磷酸酶2A（PP 2A）的激活剂。我们发现，这种RasGEF复合物选择性地控制一个重要的Ras信号通路，调节趋化性和信号中继。由于激活的Ras在迁移细胞的前部局部富集，我们假设RasGEFs也会类似地定位。我们通过将GFP与RasGEF复合物的不同组分融合并在共聚焦显微镜下评估其在趋化细胞中的定位来验证这一假设。我们现在有证据表明，在突然均匀的化学引诱刺激，RasGEF复合物是短暂的从细胞质易位到质膜。然而，由于强烈的胞质信号，我们无法确定RasGEF复合物是否存在于以化学引诱物梯度迁移的极化细胞的质膜上。我们以前面临着一个类似的问题时，研究本地化的Ras GTP酶激活蛋白（RasGAP），Ras抑制剂在这种情况下，和TIRF显微镜使我们能够检测质膜定位这种蛋白质时，其他类型的显微镜没有。因此，我们相信，TIRF显微镜也将为我们提供一个答案，关于任何质膜定位的RasGEF复杂的趋化细胞。在TIRF显微镜中观察化学趋化细胞的所有条件都已经优化，因此我们预计我们将能够在几次会议中获得所有需要的数据。我们建议看两个不同的细胞系：一个表达的GFP融合蛋白的RasGEF复合物和一个控制细胞系表达可溶性，胞质GFP。