High-throughput single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking
- Oregon Health and Science University, United States
Abstract
Membrane nanodomains have been implicated in Ras signaling, but what these domains are and how they interact with Ras remain obscure. Here, using single particle tracking with photoactivated localization microscopy (spt-PALM) and detailed trajectory analysis, we show that distinct membrane domains dictate KRasG12D (an active KRas mutant) diffusion and trafficking in U2OS cells. KRasG12D exhibits an immobile state in ~70 nm domains, each embedded in a larger domain (~200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. Moreover, KRasG12D is continuously removed from the membrane via the immobile state and replenished to the fast state, reminiscent of Ras internalization and recycling. Importantly, both the diffusion and trafficking properties of KRasG12D remain invariant over a broad range of protein expression levels. Our results reveal how membrane organization dictates membrane diffusion and trafficking of Ras and offer new insight into the spatial regulation of Ras signaling.
Data availability
We have provided a complete set of model parameters derived from all raw single-particle tracking videos in the supplementary information.
Article and author information
Author details
Daniel M Zuckerman
Funding
National Institutes of Health (U54 CA209988)
- Young Hwan Chang
- Joe W Gray
- Xiaolin Nan
National Science Foundation (MCB1715823)
- Daniel M Zuckerman
Damon Runyon Cancer Research Foundation
- Xiaolin Nan
M J Murdock Charitable Trust
- Joe W Gray
- Xiaolin Nan
Prospect Creek Foundation
- Joe W Gray
- Xiaolin Nan
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Lee et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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High-throughput single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking
eLife 8:e46393.
https://doi.org/10.7554/eLife.46393
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