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

<jats:p>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 <jats:italic>drc</jats:italic> 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 <jats:italic>drc</jats:italic> cell models, despite forward motility in live <jats:italic>drc</jats:italic> cells. Dark‐field light microscopic observations of <jats:italic>drc</jats:italic> cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild‐type reactivated cell models remained intact. The <jats:italic>sup‐pf4</jats:italic> and <jats:italic>drc3</jats:italic> mutants, unlike other <jats:italic>drc</jats:italic> mutants, retain most of the N‐DRC linker that interconnects outer doublet microtubules. Reactivated <jats:italic>sup‐pf4</jats:italic> and <jats:italic>drc3</jats:italic> 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 <jats:italic>tpg</jats:italic> 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.</jats:p>