The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme

• Type of Material: Article
• Authors: Lea Alford, Emory University; Daniel Stoddard, Brandeis University; Jennifer H. Li, Emory University; Emily L. Hunter, Emory University; Douglas Tritschler, University of Minnesota; Raqual Bower, University of Minnesota; Daniela Nicastro, University of Texas Southwestern Medical School; Mary E. Porter, University of Minnesota; Winfield Sale, Emory University
• Language: English
• Date: 2016-06-01
• Publisher: Wiley
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2016 Wiley Periodicals, Inc. This is the peer reviewed version of the following article, which has been published in final form. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
• Final Published Version (URL): http://dx.doi.org/10.1002/cm.21301
• Title of Journal or Parent Work: Cytoskeleton
• ISSN: 1949-3584
• Volume: 73
• Issue: 7
• Start Page: 331
• End Page: 340
• Grant/Funding Information: E.L.H. was supported by a predoctoral fellowship from American Heart Association.; The work was also supported by Department of Pediatrics and the Pediatric Research Center, Children’s Hospital of Atlanta (CHOA) Pilot Grant NIAAA P50 AA013757 10 pilot project 00006748, and grants from the NIH (W.S.S., NIH GM051173; M.E.P., GM-055667 and D.N. GM083122).; L.M.A. was supported by a training grant from the NIH (Emory University FIRST Postdoctoral Career Development Award K12 GM000608).
• Keywords: ARMS; Life Sciences & Biomedicine; DYNEIN-REGULATORY-COMPLEX; RADIAL SPOKES; CHLAMYDOMONAS-FLAGELLA; MOTILITY; axoneme; WILD-TYPE; nexin; Science & Technology; DRC; dynein regulatory complex; FLAGELLAR AXONEMES; dynein; CRYOELECTRON TOMOGRAPHY; Cell Biology; SEA-URCHIN SPERM; cilia; doublet microtubule; N-DRC
• Research Categories: Biology, Molecular; Biology, Cell

Abstract

We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed “reactivated cell models.” ATP-induced reactivation of wild-type cells resulted in the forward swimming of ∼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function.