Journal
CYTOSKELETON
Volume 73, Issue 7, Pages 331-340Publisher
WILEY
DOI: 10.1002/cm.21301
Keywords
cilia; axoneme; doublet microtubule; nexin; dynein; dynein regulatory complex; DRC; N-DRC
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Funding
- NIH (Emory University FIRST Postdoctoral Career Development Award) [K12 GM000608]
- American Heart Association
- Department of Pediatrics
- Pediatric Research Center
- Children's Hospital of Atlanta (CHOA) [NIAAA P50 AA013757 10 pilot project 00006748]
- NIH [NIH GM051173, GM-055667, GM083122]
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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 approximate to 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. (c) 2016 Wiley Periodicals, Inc.
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