The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme.
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. © 2016 Wiley Periodicals, Inc.
Department of Cell Biology, Emory University, 465 Whitehead Biomedical Research Building, 615 Michael Street, Atlanta, Georgia.
Lea M
LM
Alford
Biology Department, Brandeis University, Rosenstiel Basic Medical Science Research Center, 415 South Street, Waltham, Massachusetts.|Departments Of Cell Biology and Biophysics, University of Texas Southwestern Medical School, 6000 Harry Hines Blvd. Dallas, Texas.
Daniel
D
Stoddard
Department of Cell Biology, Emory University, 465 Whitehead Biomedical Research Building, 615 Michael Street, Atlanta, Georgia.
Jennifer H
JH
Li
Department of Cell Biology, Emory University, 465 Whitehead Biomedical Research Building, 615 Michael Street, Atlanta, Georgia.
Emily L
EL
Hunter
Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, Minnesota.
Douglas
D
Tritschler
Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, Minnesota.
Raqual
R
Bower
Departments Of Cell Biology and Biophysics, University of Texas Southwestern Medical School, 6000 Harry Hines Blvd. Dallas, Texas.
Daniela
D
Nicastro
Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, Minnesota.
Mary E
ME
Porter
Department of Cell Biology, Emory University, 465 Whitehead Biomedical Research Building, 615 Michael Street, Atlanta, Georgia.
Winfield S
WS
Sale
D054468:Axoneme / Q000235:genetics / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D002923:Cilia / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*
D008869:Microtubule-Associated Proteins; D010940:Plant Proteins; C484652:microtubule nexin
DRC; N-DRC; axoneme; cilia; doublet microtubule; dynein; dynein regulatory complex; nexin