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.
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.
authors.affiliation: From the Biology Department, Brandeis University, Waltham, Massachusetts 02454.
authors.forename: Kangkang
authors.initials: K
authors.lastname: Song
authors.affiliation: the Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, and.
authors.forename: Junya
authors.initials: J
authors.lastname: Awata
authors.affiliation: the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Douglas
authors.initials: D
authors.lastname: Tritschler
authors.affiliation: the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Raqual
authors.initials: R
authors.lastname: Bower
authors.affiliation: the Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, and.
authors.forename: George B
authors.initials: GB
authors.lastname: Witman
authors.affiliation: the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Porter
authors.affiliation: From the Biology Department, Brandeis University, Waltham, Massachusetts 02454, nicastro@brandeis.edu.
authors.forename: Daniela
authors.initials: D
authors.lastname: Nicastro
mesh_terms: D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D054468:Axoneme / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D015153:Blotting, Western; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004398:Dyneins / Q000235:genetics / Q000378:metabolism*; D055032:Electron Microscope Tomography / Q000379:methods*; D005407:Flagella / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009068:Movement; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D020418:Algal Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D004398:Dyneins
keywords: Chlamydomonas; Cilia; Clonable EM Tag; Cryo-electron Microscopy; DRC3 and DRC4; Dynein; Electron Tomography; FAP134; Nexin-Dynein Regulatory Complex; PF2