Microtubule polyglutamylation is important for regulating cytoskeletal architecture and motility in Trypanosoma brucei

Abstract

The shape of kinetoplastids, such as Trypanosoma brucei, is precisely defined during the stages of the life cycle and governed by a stable subpellicular microtubule cytoskeleton. During the cell cycle and transitions between life cycle stages this stability has to transiently give way to a dynamic behaviour to enable cell division and morphological rearrangements. How these opposing requirements of the cytoskeleton are regulated is poorly understood. Two possible levels of regulation are activities of cytoskeleton-associated proteins and microtubule posttranslational modifications (PTMs). Here, we investigate the functions of two putative tubulin polyglutamylases in T. brucei, TTLL6A and TTLL12B. Depletion of both proteins leads to a reduction in tubulin polyglutamylation in situ and is associated with disintegration of the posterior cell pole, loss of the microtubule plus end-binding protein EB1 and alterations of microtubule dynamics. We also observe a reduced polyglutamylation of the flagellar axoneme. Quantitative motility analysis reveals that the PTM disbalance correlates with a transition from directional to diffusive cell movement. These data show, that microtubule polyglutamylation has an important role in regulating cytoskeletal architecture and motility in this parasite.

TheTrypanosoma bruceisubpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins

“…While the PAVE complex does not appear to bind to post-translational modifications on the C-terminal tail of tubulin, other subpellicular components may use them to localize to different regions of the array and then form associations with core crosslink-associated proteins such as TbAIR9. Tubulin post-translational modifications such as polyglutamylation and tyrosination have different distribution patterns along the array and may serve as landmarks for recruiting specific MAPs (16,39,59,60).…”

Kinetoplastid-specific X2-family kinesins interact with a kinesin-like pleckstrin homology domain protein that localizes to the trypanosomal microtubule quartet

“…The intracellular contents of these protrusions appear to be comparable to the rest of the cell and they are also surrounded by subpellicular MTs. Interestingly, these protrusions are reminiscent of the 'glove phenotype' observed upon depletion of two TTLL enzymes, which are responsible for post-translational modifications appending the C-terminal tails of α-and β-tubulin (Jentzsch et al, 2020). However, we can now only speculate about the nature of these posterior extrusions.…”

“…5C and S3C); the latter antibody was used previously to visualize the basal bodies, where it additionally cross-reacts with TbRP2 (Andre et al, 2014). Upon depletion of either TTLL, polyglutamylation was decreased and α-tubulin tyrosination increased in the posterior end, from which the protrusions emerged (Jentzsch et al, 2020). We did not observe either of these changes in post-translational modifications upon TbPH1/TbKifX2 silencing by observing them on individual cells by IFA (Fig.…”

“…5C). Upon depletion of either TTLL, polyglutamylation was decreased and α-tubulin tyrosination increased in the posterior end, from which the protrusions emerged [53]. We did not observe either of these changes in posttranslational modifications upon TbPH1/TbKifX2 silencing by observing them on individual cells by IFA (S5D Fig.…”

“…). This effect of TbPH1/TbKifX2-depletion resembled that of two tubulin-tyrosine-ligase-like (TTLL) proteins, which post-translationally modify the C-terminal tails of α-and β-tubulin by addition of polyglutamate side chains [53]. Upon depletion of either TTLL, protrusions were observed emanating from the posterior end, which prompted the authors to name this morphological change the 'glove' phenotype.…”