Cell-Specific α-Tubulin Isotype Regulates Ciliary Microtubule Ultrastructure, Intraflagellar Transport, and Extracellular Vesicle Biology

Abstract

Summary Cilia are found on most non-dividing cells in the human body and, when faulty, cause a wide range of pathologies called ciliopathies. Ciliary specialization in form and function is observed throughout the animal kingdom, yet mechanisms generating ciliary diversity are poorly understood. The “tubulin code” – a combination of tubulin isotypes and tubulin post-translational modifications – can generate microtubule diversity. Using C. elegans, we show that α-tubulin isotype TBA-6 sculpts 18 A- and B-tubule singlets from nine ciliary A-B doublet microtubules in cephalic male (CEM) neurons. In CEM cilia, tba-6 regulates velocities and cargoes of intraflagellar transport (IFT) kinesin-2 motors kinesin-II and OSM-3/KIF17 without affecting kinesin-3 KLP-6 motility. In addition to their unique ultrastructure and accessory kinesin-3 motor, CEM cilia are specialized to produce extracellular vesicles. tba-6 also influences several aspects of extracellular vesicle biology, including cargo sorting, release, and bioactivity. We conclude that this cell-specific α-tubulin isotype dictates the hallmarks of CEM cilia specialization. These findings provide insight into mechanisms generating ciliary diversity and lay a foundation for further understanding the tubulin code.

Glutamylation Regulates Transport, Specializes Function, and Sculpts the Structure of Cilia

“…In the ccpp-1;ttll-11 double mutant, GFP∷KLP-6 velocity was similar to wild type. To date, perturbing glutamylation is the only genetic manipulation found to affect KLP-6 motility in vivo [24, 35]. …”

“…EVs shed and released by the CEMs and RnBs contain polycystins, and can be visualized by PKD-2∷GFP (Figure 4A, B; [7]). The kinesin-3 KLP-6 and α-tubulin TBA-6 regulate release of ciliary EVs, with mutants releasing fewer PKD-2∷GFP labeled EVs and also accumulating excessive amounts of EVs in the cephalic lumen surrounding the CEM cilium, as visualized by the PKD-2∷GFP reporter in living animals and transmission electron microscopy (TEM) in fixed animals [7, 24]. …”

“…The structure of the wild-type CEM axoneme is distinctive (Figure 6A; [24]). Like most ciliary axonemes, the CEM ciliary transition zone contains a ring of nine doublet MTs, connected to the membrane by Y-link structures, followed by a proximal region with nine doublet MTs without Y-links [24].…”

“…CEM cilia display an ultrastructural specialization in which nine MT doublets splay into nine A-tubule and nine B-tubule singlets in middle regions of the axoneme, but remain joined in distal and proximal regions [24]. The α-tubulin isotype TBA-6 is essential for B-tubule singlet formation, hence the tubulin code is implicated in generating this specialized EV-releasing cilium [24].…”

“…The α-tubulin isotype TBA-6 is essential for B-tubule singlet formation, hence the tubulin code is implicated in generating this specialized EV-releasing cilium [24]. …”