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
Department of Cell and Developmental Biology
Biochemistry | Cell and Developmental Biology | Cell Biology
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.
Chlamydomonas, Cilia, Clonable EM Tag, Cryo-electron Microscopy, DRC3 and DRC4, Dynein, Electron Tomography, FAP134, Nexin-Dynein Regulatory Complex, PF2
DOI of Published Version
J Biol Chem. 2015 Feb 27;290(9):5341-53. doi: 10.1074/jbc.M114.626556. Link to article on publisher's site.
The Journal of biological chemistry
Song K, Awata J, Tritschler D, Bower R, Witman GB, Porter ME, Nicastro D. (2015). 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. Cell and Developmental Biology Publications. https://doi.org/10.1074/jbc.M114.626556. Retrieved from https://escholarship.umassmed.edu/cellbiology_pp/135