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

• Published in: The Journal of biological chemistry 2015-02, Vol.290 (9), p.5341-5353
• Main Authors: Song, Kangkang, Awata, Junya, Tritschler, Douglas, Bower, Raqual, Witman, George B., Porter, Mary E., Nicastro, Daniela
• Format: Article
• Language: English
• Subjects: Algal Proteins - genetics; Algal Proteins - metabolism; Axoneme - genetics; Axoneme - metabolism; Axoneme - ultrastructure; Blotting, Western; Cell Biology; Chlamydomonas; Chlamydomonas reinhardtii - genetics; Chlamydomonas reinhardtii - metabolism; Chlamydomonas reinhardtii - physiology; Cilia; Clonable EM Tag; Cryo-electron Microscopy; DRC3 and DRC4; Dynein; Dyneins - genetics; Dyneins - metabolism; Electron Microscope Tomography - methods; Electron Tomography; FAP134; Flagella - genetics; Flagella - metabolism; Flagella - ultrastructure; Microscopy, Fluorescence; Models, Molecular; Movement; Multiprotein Complexes - chemistry; Multiprotein Complexes - metabolism; Mutation; Nexin-Dynein Regulatory Complex; PF2; Protein Structure, Quaternary; Protein Structure, Tertiary; Protein Subunits - genetics; Protein Subunits - metabolism; Reproducibility of Results
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.626556

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. Techniques to localize proteins in situ at high resolution are important but limited. Combining SNAP tag technology with cryo-electron tomography, we precisely localized proteins within the N-DRC that are important for ciliary motility. The developed method was applied to localize proteins with ∼3 nm resolution without interfering with the complex function. The method is a powerful tool for studies of proteins in situ.