Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex

Long Gui, Kangkang Song, Douglas Tritschler, Raqual Bower, Si Yan, Aguang Dai, Katherine Augspurger, Jason Sakizadeh, Magdalena Grzemska, Thomas Ni, Mary E. Porter, Daniela Nicastro

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

The nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the "functional subunits," namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.

Original languageEnglish (US)
Pages (from-to)23152-23162
Number of pages11
JournalProceedings of the National Academy of Sciences of the United States of America
Volume116
Issue number46
DOIs
StatePublished - Nov 12 2019

Bibliographical note

Funding Information:
We thank Chen Xu (Brandeis University) and Zhenguo Chen and Daniel Stoddard (University of Texas [UT] Southwestern Medical Center) for management of the electron microscope facilities and training. The UT Southwestern Cryo-Electron Microscopy Facility is supported in part by Cancer Prevention and Research Institute of Texas (CPRIT) Core Facility Support Award RP170644. We also thank Gang Fu for assisting with tomogram reconstruction. We are grateful to Cai Kai for critical reading of the manuscript. We also thank LeeAnn Higgins and Todd Markowski in the Center for Mass Spectrometry and Proteomics at the University of Minnesota for assistance with mass spectrometry of the iTRAQ-labeled samples. This center is supported by multiple grants including National Science Foundation (NSF) Major Research Instrumentation Grants 9871237 and NSF-DBI-0215759 as described at https://cbs.umn.edu/cmsp/about. We also acknowledge Matt Laudon and the Chlamydomonas Genetics Center (University of Minnesota) for strains. This facility is supported by the NSF Living Stock Collections for Biological Research Program Grants 0951671 and 00017383. The antibody to RSP16 was generously provided by Pinfen Yang (Marquette University) and the antibody to DRC1 by Win Sale (Emory University). This study was funded by the following grants: National Institutes of Health Grants R01GM083122 (to D.N.) and R01GM055667 (to M.E.P.), and CPRIT Grant RR140082 (to D.N.).

Funding Information:
ACKNOWLEDGMENTS. We thank Chen Xu (Brandeis University) and Zhenguo Chen and Daniel Stoddard (University of Texas [UT] Southwestern Medical Center) for management of the electron microscope facilities and training. The UT Southwestern Cryo-Electron Microscopy Facility is supported in part by Cancer Prevention and Research Institute of Texas (CPRIT) Core Facility Support Award RP170644. We also thank Gang Fu for assisting with tomogram reconstruction. We are grateful to Cai Kai for critical reading of the manuscript. We also thank LeeAnn Higgins and Todd Markowski in the Center for Mass Spectrometry and Proteomics at the University of Minnesota for assistance with mass spectrometry of the iTRAQ-labeled samples. This center is supported by multiple grants including National Science Foundation (NSF) Major Research Instrumentation Grants 9871237 and NSF-DBI-0215759 as described at https://cbs.umn.edu/cmsp/ about. We also acknowledge Matt Laudon and the Chlamydomonas Genetics Center (University of Minnesota) for strains. This facility is supported by the NSF Living Stock Collections for Biological Research Program Grants 0951671 and 00017383. The antibody to RSP16 was generously provided by Pinfen Yang (Marquette University) and the antibody to DRC1 by Win Sale (Emory University). This study was funded by the following grants: National Institutes of Health Grants R01GM083122 (to D.N.) and R01GM055667 (to M.E.P.), and CPRIT Grant RR140082 (to D.N.).

Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.

Keywords

  • Axoneme
  • Cilia
  • Cryo-electron tomography
  • Flagella
  • N-DRC

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