Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures
Abstract
Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nanocages, and other macromolecular assemblies, are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures’ subcellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs’ primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon reaction with the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. Importantly, this was achieved by replacing only a fraction of GvpA with the tagged version. We used fluorescence images of the tagged GVs to study the GV size and distance distributions within these cells. This bioorthogonal and fractional labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.
Copyright and License
© 2024 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY 4.0.
Acknowledgement
Imaging was performed in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. The authors particularly thank Dr. Giada Spigolon of the Biological Imaging Facility for her assistance with the confocal microscopy and 3D rendering of the z-stacks. This work was supported by the Chan Zuckerberg Initiative (2020-225370 to M.G.S.) and the National Institutes of Health (DP1 EB033154 to M.G.S.). Related work in the Shapiro lab was supported by the David and Lucile Packard Foundation. M.G.S. is an investigator of the Howard Hughes Medical Institute.
Conflict of Interest
The authors declare no competing financial interest.
Data Availability
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Sequence alignment, bacterial screening data, fluorescence images, and concentration calculations (PDF)
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Additional details
- ISSN
- 1520-4812
- PMCID
- PMC10961726
- DOI
- 10.1021/acs.bioconjchem.3c00518
- Beckman Institute
- California Institute of Technology
- Arnold and Mabel Beckman Foundation
- 2020-225370
- Chan-Zuckerberg Initiative
- DP1 EB033154
- National Institutes of Health
- David and Lucile Packard Foundation
- Howard Hughes Medical Institute
- Caltech groups
- Tianqiao and Chrissy Chen Institute for Neuroscience