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

J Biol Chem. 2015 Feb 27;290(9):5341-53. doi: 10.1074/jbc.M114.626556. Epub 2015 Jan 6.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algal Proteins / genetics
  • Algal Proteins / metabolism*
  • Axoneme / genetics
  • Axoneme / metabolism
  • Axoneme / ultrastructure
  • Blotting, Western
  • Chlamydomonas reinhardtii / genetics
  • Chlamydomonas reinhardtii / metabolism*
  • Chlamydomonas reinhardtii / physiology
  • Dyneins / genetics
  • Dyneins / metabolism*
  • Electron Microscope Tomography / methods*
  • Flagella / genetics
  • Flagella / metabolism*
  • Flagella / ultrastructure
  • Microscopy, Fluorescence
  • Models, Molecular
  • Movement
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / metabolism*
  • Mutation
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Reproducibility of Results

Substances

  • Algal Proteins
  • Multiprotein Complexes
  • Protein Subunits
  • Dyneins