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Enzymatic Labeling of Bacterial Proteins for Super-resolution Imaging in Live Cells

Ho, Samuel H. and Tirrell, David A. (2019) Enzymatic Labeling of Bacterial Proteins for Super-resolution Imaging in Live Cells. ACS Central Science, 5 (12). pp. 1911-1919. ISSN 2374-7943. PMCID PMC6935894. https://resolver.caltech.edu/CaltechAUTHORS:20191121-100808414

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Abstract

Methods that enable the super-resolution imaging of intracellular proteins in live bacterial cells provide powerful tools for the study of prokaryotic cell biology. Photoswitchable organic dyes exhibit many of the photophysical properties needed for super-resolution imaging, including high brightness, photostability, and photon output, but most such dyes require organisms to be fixed and permeabilized if intracellular targets are to be labeled. We recently reported a general strategy for the chemoenzymatic labeling of bacterial proteins with azide-bearing fatty acids in live cells using the eukaryotic enzyme N-myristoyltransferase. Here we demonstrate the labeling of proteins in live Escherichia coli using cell-permeant bicyclononyne-functionalized photoswitchable rhodamine spirolactams. Single-molecule fluorescence measurements on model rhodamine spirolactam salts show that these dyes emit hundreds of photons per switching event. Super-resolution imaging was performed on bacterial chemotaxis proteins Tar and CheA and cell division proteins FtsZ and FtsA. High-resolution imaging of Tar revealed a helical pattern; imaging of FtsZ yielded banded patterns dispersed throughout the cell. The precision of radial and axial localization in reconstructed images approaches 15 and 30 nm, respectively. The simplicity of the method, which does not require redox imaging buffers, should make this approach broadly useful for imaging intracellular bacterial proteins in live cells with nanometer resolution.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/acscentsci.9b00617DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935894PubMed CentralArticle
ORCID:
AuthorORCID
Ho, Samuel H.0000-0001-7647-0752
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2019 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: June 24, 2019; Published: November 21, 2019. The authors thank members of the Tirrell laboratory for insightful discussions. S.H.H. thanks Y. Hui, A. Collazo, and R. Stanciauskas for helpful advice. This work was supported by the Joseph J. Jacobs Institute for Molecular Engineering for Medicine and the Center for Environmental Microbial Interactions at the California Institute of Technology. The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
CaltechUNSPECIFIED
Issue or Number:12
PubMed Central ID:PMC6935894
Record Number:CaltechAUTHORS:20191121-100808414
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20191121-100808414
Official Citation:Enzymatic Labeling of Bacterial Proteins for Super-resolution Imaging in Live Cells. Samuel H. Ho and David A. Tirrell. ACS Central Science 2019 5 (12), 1911-1919. DOI: 10.1021/acscentsci.9b00617
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:99981
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:21 Nov 2019 18:25
Last Modified:07 Jan 2020 22:34

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