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

doi:10.1117/12.2547699

20 February 2020 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

Author Affiliations +

Proceedings Volume 11250, High-Speed Biomedical Imaging and Spectroscopy V; 112500N (2020) https://doi.org/10.1117/12.2547699
Event: SPIE BiOS, 2020, San Francisco, California, United States

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.

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Yerim Lee, Kai Tao, Carey Phelps, Tao Huang, Barmak Mostofian, Daniel Zuckerman, and Xiaolin Nan "Probing the spatiotemporal dynamics of Ras-associated membrane nanodomains with high-throughput single particle tracking via photoactivated localization microscopy (spt-PALM)", Proc. SPIE 11250, High-Speed Biomedical Imaging and Spectroscopy V, 112500N (20 February 2020); https://doi.org/10.1117/12.2547699

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KEYWORDS

Diffusion

Radium

Molecules

Nanodomains

Particles

Data modeling

Microscopy

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