Title
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
UMMS Affiliation
Department of Cell and Developmental Biology
Publication Date
2015-02-27
Document Type
Article
Disciplines
Biochemistry | Cell and Developmental Biology | Cell Biology
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.
Keywords
Chlamydomonas, Cilia, Clonable EM Tag, Cryo-electron Microscopy, DRC3 and DRC4, Dynein, Electron Tomography, FAP134, Nexin-Dynein Regulatory Complex, PF2
DOI of Published Version
10.1074/jbc.M114.626556
Source
J Biol Chem. 2015 Feb 27;290(9):5341-53. doi: 10.1074/jbc.M114.626556. Link to article on publisher's site.
Journal/Book/Conference Title
The Journal of biological chemistry
Related Resources
PubMed ID
25564608
Repository Citation
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