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

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.

Structure of the ciliary axoneme at nanometer resolution reconstructed by TYGRESS

“…The tensile strength could be important for N-DRC function while resisting considerable mechanical stress during ciliary beating. The stability against "pulling" forces that would separate neighboring doublets from each other is also consistent with the importance of the N-DRC for maintaining the axoneme integrity (Alford et al 2016). Consistent with this function, lack of either one of the three subunits DRC1, 2 and 4 leads to severe motility defects and is linked to the ciliary disease Primary Ciliary Dyskinesia (PCD) (Austin-Tse et al , Horani et al 2013, Wirschell et al 2013, Olbrich et al 2015.…”

“…The N-DRC is separated in two major regions, the base plate that is important for the N-DRC binding to the DMT, and the linker region that connects to the neighboring DMT ( Fig. 3F), which is critical for both axoneme integrity and ciliary motility (Alford et al 2016). In contrast to previous cryo-ET studies that observed the N-DRC base plate as a single rod-shaped density , in the TYGRESS reconstruction three N-DRC subunits are well-resolved as three long filamentous structures that slowly twist around each other (shown in purple, yellow, and dark green in Fig.…”

Posttranslational Modifications of Tubulin and Cilia

“…Alternatively, the dynein e/B-tubule links could increase the microtubule curvature by generating a drag force on the microtubules that are simultaneously pushed by other "faster" dyneins (Kotani et al 2007;Suryavanshi et al 2010). In addition to the effects on dynein e, TTLL9 also affects the axoneme integrity by influencing the DRC-nexin/B-tubule links between the adjacent doublets (Kubo et al 2012;Alford et al 2016).…”

Electrostatic interaction between polyglutamylated tubulin and the nexin–dynein regulatory complex regulates flagellar motility

“…Furthermore, previous studies suggested that tubulin polyglutamylation regulates flagellar motility by controlling specific axonemal dynein and the nexin–dynein regulatory complex (N-DRC; Kubo et al. , 2012 ; Alford et al. , 2016 ).…”