Probing the spatiotemporal dynamics of Ras-associated membrane nanodomains with high-throughput single particle tracking via photoactivated localization microscopy (spt-PALM)

Yerim Lee, Kai Tao, Carey Phelps, Tao Huang, Barmak Mostofian, Daniel Zuckerman, Xiaolin Nan

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Membrane nanodomains have commonly been implicated in biological processes. However, what these nanodomains are and how they participate in the processes of interest are still unclear, primarily due to challenges in probing these nanoscopic and dynamic structures in cells. Using high-throughput single particle tracking via spt-PALM and detailed trajectory analysis, here we demonstrate that membrane nanodomains associated with the small GTPase KRas could be detected and analyzed in live cells. By stochastically activating and tracking single PAmCherry1-KRas molecules on the membrane, spt-PALM yields 5,000 to 100,000 single-molecule diffusion trajectories of KRas. Analysis of these trajectories with variational Bayes SPT (vbSPT) revealed that KRas exhibits an immobile state in domains ∼70 nm in size, each embedded in a larger domain (∼200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. By analyzing the transition kinetics among the three states, we found that KRas is continuously removed from the membrane via the immobile state and replenished to the fast state, likely coupled to internalization and recycling. Our results demonstrate the utility of high-throughput SPT in uncovering the impact of nanoscopic landscape of the membrane on the spatiotemporal dynamics and potentially multimer formation and signaling of membrane-bound biomolecules.

Original languageEnglish (US)
Title of host publicationHigh-Speed Biomedical Imaging and Spectroscopy V
EditorsKevin K. Tsia, Keisuke Goda
PublisherSPIE
ISBN (Electronic)9781510632639
DOIs
StatePublished - Jan 1 2020
EventHigh-Speed Biomedical Imaging and Spectroscopy V 2020 - San Francisco, United States
Duration: Feb 1 2020Feb 4 2020

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume11250
ISSN (Print)1605-7422

Conference

ConferenceHigh-Speed Biomedical Imaging and Spectroscopy V 2020
CountryUnited States
CitySan Francisco
Period2/1/202/4/20

Keywords

  • membrane domains
  • Ras
  • single-particle tracking
  • vbSPT

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging

Fingerprint Dive into the research topics of 'Probing the spatiotemporal dynamics of Ras-associated membrane nanodomains with high-throughput single particle tracking via photoactivated localization microscopy (spt-PALM)'. Together they form a unique fingerprint.

  • Cite this

    Lee, Y., Tao, K., Phelps, C., Huang, T., Mostofian, B., Zuckerman, D., & Nan, X. (2020). Probing the spatiotemporal dynamics of Ras-associated membrane nanodomains with high-throughput single particle tracking via photoactivated localization microscopy (spt-PALM). In K. K. Tsia, & K. Goda (Eds.), High-Speed Biomedical Imaging and Spectroscopy V [112500N] (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 11250). SPIE. https://doi.org/10.1117/12.2547699