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Visualization of Exosome Release and Uptake During Cell Migration Using the Live Imaging Reporter pHluorin_M153R-CD63

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Cell Migration in Three Dimensions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2608))

Abstract

Exosome secretion and uptake regulate cell migration through autocrine and paracrine mechanisms. Monitoring exosome secretion and uptake during cell migration is critical for investigation of these mechanisms. Exosomes can be visualized by direct labeling with fluorescent dyes or by tagging intrinsic markers with fluorescent proteins for live imaging. Due to several limitations of fluorescent dye-labeled exosomes, we created two bright genetically encoded reporters of exosome secretion, pHluorin_M153R-CD63 and pHluorin_M153R-CD63-mScarlet. Here, we describe how to visualize secretion and uptake of exosomes labeled with these pH-sensitive and pH-insensitive fluorescent protein-tagged exosomal markers during cell migration using time-lapse fluorescent microscopy.

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Acknowledgments

This work was supported by NIH grants R01CA206458, R01CA249684, and R01CA249424.

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Authors and Affiliations

  1. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA

    Bong H. Sung

Authors
  1. Bong H. Sung
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  2. Alissa M. Weaver
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Corresponding author

Correspondence to Bong H. Sung .

Editor information

Editors and Affiliations

  1. Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam University Medical Center, Amsterdam, The Netherlands

    Coert Margadant

1 Electronic Supplementary Materials

A migrating HT1080 cell stably expressing pHluorin_M153R-CD63. Time-series images were taken on a fibronectin-coated glass-bottom dish (1 μg/mL) every 30 s. (The movie was rotated by 180° and reproduced under the term of the Creative Commons CC BY 4.0 license [23]. Copyright © 2020, Springer Nature (MP4 11222 kb))

Endocytosis and acidification of extracellular exosome deposits. Live confocal microscopy of pHluorin_M153R-CD63-mScarlet on a fibronectin-coated glass-bottom dish (1 μg/mL) with images taken every minute. Exosome deposits (white arrows) contacted by cells are endocytosed and acidified in endosomal compartments (magenta arrows). (Reproduced under the term of the Creative Commons CC BY 4.0 license [23]. Copyright © 2020, Springer Nature (MP4 2391 kb))

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Sung, B.H., Weaver, A.M. (2023). Visualization of Exosome Release and Uptake During Cell Migration Using the Live Imaging Reporter pHluorin_M153R-CD63. In: Margadant, C. (eds) Cell Migration in Three Dimensions. Methods in Molecular Biology, vol 2608. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2887-4_6

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  • DOI: https://doi.org/10.1007/978-1-0716-2887-4_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2886-7

  • Online ISBN: 978-1-0716-2887-4

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