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Spatially mapping the diffusivity of proteins in live cells based on cumulative area analysis

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Abstract

Molecular motion provides a way for biomolecules to mix and interact in living systems. Quantifying their motion is critical to the understanding of how biomolecules perform its function. However, it has been a challenged task to spatially map the fast diffusion of unbound proteins in the heterogenous intracellular environment. Here we reported a new imaging technique named cumulative area based on single-molecule diffusivity mapping (CA-SMdM). The strategy is based on the comparison of single-molecule images between a shorter and longer exposure time. With longer exposure time, molecules will travel further, thus giving more blurred single-molecule images, hence implying its local diffusion rates. We validated our technique through measuring the fast diffusion rates (10–40 µm2/s) of fluorescent dye in glycerol-water mixture, and found the values fit well with Stokes-Einstein equation. We further showed that the spatially mapping of diffusivity in live cells is plausible through CA-SMdM, and it faithfully reported the local diffusivity heterogeneity in cytosol and nucleus. CA-SMdM provides an efficient way to mapping the local molecular motion, and therefore will have profound applications in probing the biomolecular interactions for living systems.

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Acknowledgements This work was supported by the National Key R&D Program of China (2022YFA1305400) and the National Natural Science Foundation of China (22104113, 22274122).

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Authors and Affiliations

  1. College of Chemistry and Molecular Sciences & Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, 430072, China

    Huihui Gao, Chu Han & Limin Xiang

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  1. Huihui Gao
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  2. Chu Han
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  3. Limin Xiang
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Correspondence to Limin Xiang.

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Supporting information The supporting information is available online at http://chem.scichina.com and http://link.springer.com/journal/11426. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

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Gao, H., Han, C. & Xiang, L. Spatially mapping the diffusivity of proteins in live cells based on cumulative area analysis. Sci. China Chem. 66, 3307–3313 (2023). https://doi.org/10.1007/s11426-023-1764-x

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  • DOI: https://doi.org/10.1007/s11426-023-1764-x

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