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

> **NIH NIH R01** · OREGON HEALTH & SCIENCE UNIVERSITY · 2020 · $357,280

## Abstract

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 activit...

## Key facts

- **NIH application ID:** 9974087
- **Project number:** 1R01GM132322-01A1
- **Recipient organization:** OREGON HEALTH & SCIENCE UNIVERSITY
- **Principal Investigator:** Xiaolin Nan
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $357,280
- **Award type:** 1
- **Project period:** 2020-07-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9974087

## Citation

> US National Institutes of Health, RePORTER application 9974087, Defining mechanisms of Ras clustering and signaling in membrane nanodomains with multiplexed superresolution and correlative microscopies (1R01GM132322-01A1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9974087. Licensed CC0.

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