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

1.项目总结/摘要 摘要 膜结合的RAS GTP酶是多种细胞功能的关键调节因子。尽管几十年来 研究表明，RAS如何在膜上作用以激活其效应器的机制仍未明确。 最近的高分辨率成像研究表明，动态纳米级脂蛋白簇的形成 膜上的纳米团簇可能是RAS功能的关键。基于证据主要来自 免疫-EM，进一步推测不同的RAS亚型占据不重叠的膜结构域 以形成空间上和功能上不同的纳米团簇。然而，免疫-EM的技术限制有 排除了对涉及RAS聚集的膜域的分子组成的彻底分析 以及RAS如何与这些域相互作用。到目前为止，RAS集群和信令是否采用 放在专门的膜结构域中，如果是的话，这些结构域是什么，它们的组成和 结构影响RAS集群和信令属性。为了解决这些问题，PI的实验室使用了 超分辨显微镜(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。这些新的成像工具可以对分子位置进行定量分析， 在活细胞或固定细胞中的化学计量学、扩散和相互作用以及纳米细胞环境。vbl.使用 这些工具，我们确定了膜的区域，称为RAS锚定纳米结构域(RAND)， 瞬间捕获RAS，以潜在地促进集群。初步数据也表明兰特的存在与 不同的组成和结构，这可能在RAS监管中发挥关键作用，并解释不同的 和RAS的上下文相关的细胞功能。在这些初步发现的推动下，我们建议有系统地 分析RAND的组成、结构和在RAS集群中的角色，并在三个特定目标中发送信号。第一, 我们将定义RAS集群在RAND中的机制。我们将检验RAS形成星团的假设 在RAND中通过依赖于HVR的定位和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型SRM和 相关的SRM-EM来确定RAND的分子和结构同一性，并检验假设 RAS根据生物学背景定位于不同的RAN。第三，我们将定义兰德在 RAS信号和测试RAF在RAS-GTP依赖的RAS中被招募和激活的假设 在动力蛋白依赖的RAN中，Raf被H-RAS激活，在肌动球蛋白驱动的RAN中，Raf被K-RAS激活 RAS-PI3K信号涉及与RAS-Raf信号不同的RAD，最后，丰度 相关RAT的大小决定了RAS激活RAF和/或PI3K的能力。总而言之，这些研究将产生 对RAS活性如何在膜上调节以实现功能特异性的详细分子洞察 和多样性。RAS在癌症等疾病中经常被异常激活，我们预计结果将有助于 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.