In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

Kangkang Song; Junya Awata; Douglas Tritschler; Raqual Bower; George B. Witman; Mary E. Porter; Daniela Nicastro

doi:10.1074/jbc.M114.626556

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography*

From the ^‡Biology Department, Brandeis University, Waltham, Massachusetts 02454,
the ^§Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, and
the ^¶Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455

↵2 To whom correspondence should be addressed: Biology Dept., Rosenstiel Center, MS029, Brandeis University, 415 South St., Waltham, MA 02454. Tel.: 781-736-2408; Fax: 781-736-2419; E-mail: nicastro{at}brandeis.edu.

↵1 Both authors contributed equally to this work.

Background: Techniques to localize proteins in situ at high resolution are important but limited.

Results: Combining SNAP tag technology with cryo-electron tomography, we precisely localized proteins within the N-DRC that are important for ciliary motility.

Conclusion: The developed method was applied to localize proteins with ∼3 nm resolution without interfering with the complex function.

Significance: The method is a powerful tool for studies of proteins in situ.

Abstract

Cryo-electron tomography (cryo-ET) has reached nanoscale resolution for in situ three-dimensional imaging of macromolecular complexes and organelles. Yet its current resolution is not sufficient to precisely localize or identify most proteins in situ; for example, the location and arrangement of components of the nexin-dynein regulatory complex (N-DRC), a key regulator of ciliary/flagellar motility that is conserved from algae to humans, have remained elusive despite many cryo-ET studies of cilia and flagella. Here, we developed an in situ localization method that combines cryo-ET/subtomogram averaging with the clonable SNAP tag, a widely used cell biological probe to visualize fusion proteins by fluorescence microscopy. Using this hybrid approach, we precisely determined the locations of the N and C termini of DRC3 and the C terminus of DRC4 within the three-dimensional structure of the N-DRC in Chlamydomonas flagella. Our data demonstrate that fusion of SNAP with target proteins allowed for protein localization with high efficiency and fidelity using SNAP-linked gold nanoparticles, without disrupting the native assembly, structure, or function of the flagella. After cryo-ET and subtomogram averaging, we localized DRC3 to the L1 projection of the nexin linker, which interacts directly with a dynein motor, whereas DRC4 was observed to stretch along the N-DRC base plate to the nexin linker. Application of the technique developed here to the N-DRC revealed new insights into the organization and regulatory mechanism of this complex, and provides a valuable tool for the structural dissection of macromolecular complexes in situ.

Footnotes

↵* This work was supported, in whole or in part, by National Institutes of Health Grants R01 GM083122 (to D. N.), R01 GM055667 (to M. E. P.), and R37 GM030626 (to G. B. W.). This work was also supported by Robert W. Booth Endowment at University of Massachusetts Medical School (to G. B. W.) and by the March of Dimes Foundation (to D. N.).
↵ This article contains supplemental Videos 1–3.