Microtubule glycylation promotes basal body attachment to the cell cortex

Abstract

STATEMENTBasal bodies position motile cilia at the cell cortex. This study finds tubulin glycylation to promote BB-associated microtubule elongation and structural attachment of basal bodies to the cell's cortical cytoskeleton. ABSTRACTMotile cilia generate directed hydrodynamic flow that is important for the motility of cells and extracellular fluids. To optimize directed hydrodynamic flow, motile cilia are organized and oriented into a polarized array. Basal bodies (BB) nucleate and position motile cilia at the cell cortex. Cytoplasmic BB-associated microtubules are conserved structures that extend from BBs. Using the ciliate, Tetrahymena thermophila, combined with EM-tomography and light microscopy, we show that BB-appendage microtubules assemble coincident with new BB assembly and are attached to the cell cortex. These BB-appendage microtubules are specifically marked with post translational modifications of tubulin, including glycylation. Mutations that prevent glycylation shorten BB-appendage microtubules and disrupt BB positioning and cortical attachment. Consistent with the attachment of BB-appendage microtubules to the cell cortex for BB positioning, mutations that disrupt the cellular cortical cytoskeleton similarly disrupt the cortical attachment and positioning of BBs. In summary, BBappendage microtubules promote the organization of ciliary arrays through attachment to the cell cortex.

Plastic cell morphology changes during dispersal

“…Motility of organisms from one habitat to another is an essential component of dispersal, and the BB and cilia parameters that promote cilia-dependent cell propulsion is important for cell motility 27, 30, 37, 46, 47 . First, we tried to identify innate patterns of trait associations across the 22 strains that could be linked to dispersal propensity based on the set of traits described in the previous section.…”

“…Immunofluorescence imaging procedures were modified from prior studies to maximize the quality of BB, cilia staining and retention of cell shape 37 . For BB and cilia visualization, cells were washed in 10 mM Tris HCl pH 7.4, followed by PHEM buffer (60 mM 1,4-piperazinediethanesulfonic acid, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 mM EGTA, and 2 mM MgCl 2 , pH 6.9), and then fixed in a Triton X-100 / paraformaldehyde solution (0.25% Triton X-100 and 3.2% paraformaldehyde in PHEM buffer) for 5 min.…”

“…Fluorescence imaging was performed as previously described 37 . Briefly, either a Nikon widefield TiE fluorescence microscope stand equipped with a 100x NA 1.4 Plan Apo objective and (which was not certified by peer review) is the author/funder.…”

“…In the case of BB and cilia position, loss of BB stability factors reduces BB and cilia number in ciliary rows and also slows cellular motility [34][35][36] . Furthermore, disrupting BB anchoring structures causes BBs to be mispositioned, which alters cell morphology and reduces the speed and linearity of cell swimming 27,37,38 . In contrast, starvation of T. thermophila results in highly motile cells with behaviors (fast and directed swimming) suggested to be associated with dispersal.…”

C-Terminal Tail Polyglycylation and Polyglutamylation Alter Microtubule Mechanical Properties

“…Glycylation is present on the length of mature cilia and may contribute to overall cilia stability (22). In addition, glycylation is present on microtubule structures that anchor the basal bodies to the cell cortex (23). In contrast, glutamylation in cilia is important for early ciliary maturation (24) and proper ciliary beating (25).…”