Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells

Abstract

Centrioles are composed of long-lived microtubules arranged in nine triplets.However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

CEP44 ensures the formation of bona fide centriole wall, a requirement for the centriole-to-centrosome conversion

“…Structural changes of centriolar MTs induced upon CEP295 binding to the procentriole probably allow the recruitment of CEP44. Consistent with this notion, depletion of TUBE1 and TUBD1 that affect C-MT biogenesis 14 also reduced CEP44 recruitment to centrioles.…”

“…6e-g). However, the siRNA depletion phenotype was milder than the reported centriole defect in εand δ-tubulin knockout human cell lines that assemble centrioles by the de novo pathway 14 . Residual amounts of εand δ-tubulin probably buffer the strength of the defect under siRNA conditions.…”

“…Structural integrity of the centrioles ensures their CCC. TUBE1 (ε-tubulin isoform) and TUBD1 (δ-tubulin) were described to be important for MT triplet formation of centrioles 14 . As a proof of the concept that the formation of bona fide proximal end of the centriole structure is a requirement for the CCC, we depleted TUBE1 and TUBD1 via siRNA and analysed cross-sections of the dCs proximal end by EM.…”

“…The newly born daughter centriole (dC) grows perpendicularly on the mother centriole (mC) and is kept engaged on it until the cell enters the next cell cycle 13 . During the process of the dC elongation, its structure grows radially and builds MT triplets via TUBE1 and TUBD1 14 .…”

Procentriole microtubules as drivers of centriole reduplication

“…In ensuing experiments, we explored the contribution of PC maturation on distancing. The ability of PCs to mature and accumulate PCM components requires the presence of MTs (Atorino et al, 2020;Wang et al, 2017). Thus, we decided to generate PCs that lack MT walls and explore how they behave in the presence of high levels of active Plk1.…”