Defining mechanisms of Ras clustering and signaling in membrane nanodomains with multiplexed superresolution and correlative microscopies

1.项目总结/摘要 总结 膜结合型Ras GTP酶是多种细胞功能的关键调节因子。尽管经过数十年的 研究表明，Ras如何作用于细胞膜以激活其效应子的机制仍然不清楚。 最近的高分辨率成像研究表明，动态的纳米级脂质-蛋白质簇的形成 膜上的纳米团簇可能对Ras功能至关重要。根据主要来自 免疫EM进一步假设不同的Ras亚型占据非重叠的膜结构域 以形成空间和功能不同的纳米团簇。然而，免疫EM的技术局限性 排除了对参与Ras聚类的膜结构域的分子组成的彻底分析 以及Ras如何与这些结构域相互作用。到目前为止，Ras集群和信号传导是否能够 放置在专门的膜结构域，如果是这样，这些结构域是什么，以及它们的组成和 结构影响Ras聚集和信号传导性质。为了解决这些问题，PI的实验室使用 超分辨率显微镜（SRM），相关SRM-EM和高通量单粒子跟踪（SPT）， 在模型细胞系中研究Ras。这些新的成像工具允许对分子位置进行定量分析， 化学计量、扩散和活细胞或固定细胞中的相互作用沿着纳米级细胞环境。使用 通过这些工具，我们确定了膜的区域，称为Ras锚定纳米结构域（RAND）， 瞬时捕获Ras以潜在地促进聚类。初步数据还表明存在RAND， 不同的组成和结构，这可能在Ras调控中发挥关键作用，并解释了不同的 和Ras的环境依赖性细胞功能。根据这些初步研究结果，我们建议系统地 分析RAND的组成，结构和角色的Ras集群和信号在三个特定的目标。第一、 我们将定义RAND中Ras聚类的机制。我们将检验Ras形成簇的假设 在RAND中，通过HVR依赖性定位，然后通过使用G-结构域介导的Ras-Ras相互作用， 单分子FRET（smFRET）和计算机模拟。其次，我们将使用多路复用SRM， 相关SRM-EM来确定RAND的分子和结构特性，并检验以下假设： Ras定位于不同的RAND，这取决于生物学背景。第三，我们将定义RAND的作用， Ras信号传导，并检验Raf在Ras-GTP依赖的RAND中被募集和激活的假设。 以这种方式，Raf在动力蛋白依赖性RAND中被H-Ras激活，在肌动蛋白驱动的RAND中被K-Ras激活。 RAND，Ras-PI 3 K信号转导涉及与Ras-Raf信号转导不同的RAND，最后， 相关RAND的大小决定Ras激活Raf或PI 3 K或两者的能力。综合起来，这些研究将产生 详细了解Ras活性如何在膜上调节以实现功能特异性 和多样性。Ras在癌症等疾病中经常被异常激活，我们预计这一结果将有助于 为治疗目的操纵Ras活性的新策略。

1. PROJECT SUMMARY/ABSTRACT Summary The membrane-bound Ras GTPases are key regulators of diverse cellular functions. Despite decades of research, mechanisms of how Ras operates on the membrane to activate its effectors remain poorly defined. Recent high-resolution imaging studies suggest that formation of dynamic, nanoscopic lipid-protein clusters termed nanoclusters on the membrane may be critical to Ras function. Based on evidence primarily from immuno-EM, it was further hypothesized that different Ras isoforms occupy non-overlapping membrane domains to form spatially and functionally distinct nanoclusters. However, technical limitations of immuno-EM have precluded thorough analyses of the molecular compositions of the membrane domains involved in Ras clustering and how Ras interacts with these domains. To date, it is still debated whether Ras clustering and signaling take place in specialized membrane domains, and if so, what these domains are, and how their composition and structure impact Ras clustering and signaling properties. To address these questions, the PI’s lab uses superresolution microscopy (SRM), correlative SRM-EM, and high-throughput single-particle tracking (SPT) to study Ras in model cell lines. These new imaging tools allow quantitative analysis of molecular location, stoichiometry, diffusion, and interaction in live or fixed cells along with the nanoscopic cellular context. Using these tools, we identified regions of the membrane, referred to as Ras anchoring nano-domains (RANDs), that transiently trap Ras to potentially facilitate clustering. Preliminary data also suggest the presence of RANDs with diverse compositions and structures, which could play a key role in Ras regulation and account for the diverse and context-dependent cellular functions of Ras. Prompted by these initial findings, we propose to systematically analyze RANDs in composition, structure, and roles in Ras clustering and signaling in three specific Aims. First, we will define the mechanisms of Ras clustering in RANDs. We will test the hypothesis that Ras forms clusters in RANDs through HVR-dependent localization followed by G-domain mediated Ras-Ras interaction by using single-molecule FRET (smFRET) and computer simulations. Second, we will use multiplexed SRM and correlative SRM-EM to determine the molecular and structural identities of RANDs and test the hypothesis that Ras localizes to diverse RANDs depending on the biological context. Third, we will define the role of RANDs in Ras signaling and test the hypotheses that Raf is recruited to and activated in RANDs in a Ras-GTP dependent manner, that Raf is activated by H-Ras in dynamin-dependent RANDs and by K-Ras in actomyosin-driven RANDs, that Ras-PI3K signaling involves distinct RANDs from Ras-Raf signaling, and lastly, that the abundance of relevant RANDs determines the ability of Ras to activate Raf or PI3K or both. Together, these studies will yield detailed molecular insight into how Ras activities are regulated on the membrane to achieve functional specificity and diversity. Ras is often aberrantly activated in diseases such as cancer, and we anticipate the results to lend new strategies for manipulating Ras activity for therapeutic purposes.