ABSTRACT Bacteria use surface-exposed, proteinaceous fibers called pili for diverse behaviors, including horizontal gene transfer, surface sensing, motility, and pathogenicity. Visualization

of these filamentous nanomachines and their activity in live cells has proven challenging, largely due to their small size. Here, we describe a broadly applicable method for labeling and

imaging pili and other surface-exposed nanomachines in live cells. This technique uses a combination of genetics and maleimide-based click chemistry in which a cysteine substitution is made

in the major pilin subunit for subsequent labeling with thiol-reactive maleimide dyes. Large maleimide-conjugated molecules can also be used to physically interfere with the dynamic activity

choice. Visualization of extracellular nanomachines such as pili using this approach can provide a more comprehensive understanding of the role played by these structures in distinct

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CONTENT BEING VIEWED BY OTHERS ADHESION PILUS RETRACTION POWERS TWITCHING MOTILITY IN THE THERMOACIDOPHILIC CRENARCHAEON _SULFOLOBUS ACIDOCALDARIUS_ Article Open access 14 June 2024

_ACINETOBACTER BAYLYI_ REGULATES TYPE IV PILUS SYNTHESIS BY EMPLOYING TWO EXTENSION MOTORS AND A MOTOR PROTEIN INHIBITOR Article Open access 18 June 2021 LIVE-PAINT ALLOWS SUPER-RESOLUTION

MICROSCOPY INSIDE LIVING CELLS USING REVERSIBLE PEPTIDE-PROTEIN INTERACTIONS Article Open access 20 August 2020 DATA AVAILABILITY The data that support this study are available from the

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ACKNOWLEDGEMENTS We thank M. D. Koch, A. M. Randich, and M. Jacq for critical feedback on the manuscript. This work was supported by grant R35GM122556 from the National Institutes of Health

and by a Canada 150 Research Chair in Bacterial Cell Biology to Y.V.B., by grants R35GM12867 and AI118863 from the National Institutes of Health to A.B.D., and by National Science

Foundation Fellowship 1342962 to C.K.E. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Biology, Indiana University, Bloomington, IN, USA Courtney K. Ellison, Triana N. Dalia, 

Ankur B. Dalia & Yves V. Brun * Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Quebec, Canada Yves V. Brun Authors * Courtney K. Ellison

View author publications You can also search for this author inPubMed Google Scholar * Triana N. Dalia View author publications You can also search for this author inPubMed Google Scholar *

Ankur B. Dalia View author publications You can also search for this author inPubMed Google Scholar * Yves V. Brun View author publications You can also search for this author inPubMed 

Google Scholar CONTRIBUTIONS C.K.E. and Y.V.B conceived the study. C.K.E. and T.N.D. performed the experiments. C.K.E., A.B.D., and Y.V.B. analyzed the data. C.K.E. wrote the manuscript with

help from A.B.D. and Y.V.B. CORRESPONDING AUTHOR Correspondence to Yves V. Brun. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION

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https://www.nature.com/articles/s41564-018-0174-y SUPPLEMENTARY INFORMATION REPORTING SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ellison,

C.K., Dalia, T.N., Dalia, A.B. _et al._ Real-time microscopy and physical perturbation of bacterial pili using maleimide-conjugated molecules. _Nat Protoc_ 14, 1803–1819 (2019).

https://doi.org/10.1038/s41596-019-0162-6 Download citation * Received: 30 November 2018 * Accepted: 05 March 2019 * Published: 26 April 2019 * Issue Date: June 2019 * DOI:

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