Abstract
Acetylation of the lysine 40 of α-tubulin (K40) is a post-translational modification occurring in the lumen of microtubules (MTs) and is controlled by the α-tubulin acetyl-transferase αTAT1. How αTAT1 accesses the lumen and acetylates α-tubulin there has been an open question. Here, we report that acetylation starts at open ends of MTs and progressively spreads longitudinally from there. We observed acetylation marks at the open ends of in vivo MTs re-growing after a Nocodazole block, and acetylated segments growing in length with time. Bias for MTs extremities was even more pronounced when using non-dynamic MTs extracted from HeLa cells. In contrast, K40 acetylation was mostly uniform along the length of MTs reconstituted from purified tubulin in vitro. Quantitative modelling of luminal diffusion of αTAT1 suggested that the uniform acetylation pattern observed in vitro is consistent with defects in the MT lattice providing lateral access to the lumen. Indeed, we observed that in vitro MTs are permeable to macromolecules along their shaft while cellular MTs are not. Our results demonstrate αTAT1 enters the lumen from open extremities and spreads K40 acetylation marks longitudinally along cellular MTs. This mode of tip-directed microtubule acetylation may allow for selective acetylation of subsets of microtubules.
Highlights
We observed that acetylated K40 segments became detectable as early as 4 minutes after Nocodazole washout (Fig. 1A)
Careful examination of the distribution of acetylated segments visible at 5 minutes after washout revealed that acetylated K40 marks were frequently, but not always, associated with MT extremities (Fig. 1C)
Measurements of the fluorescence intensity distribution along the long axis of MTs showed an average enrichment of acetylated K40 marks at the extremities of MTs (Fig. 1D)
Summary
Microtubules (MTs) are dynamic polymers composed of αβ-tubulin dimers that assembled into hollow tubes. When lateral access was not allowed, we observed that acetylation marks spreads longitudinally from the open MT extremities (Fig. 3D) This is in qualitative agreement with our ex vivo results. We observed by Atomic Force Microscopy that αTAT1 binds to the exterior face of in vitro MTs (Supplementary Figure 4) This exterior binding could enhance αTAT1 entry to the lumen through holes or defects in the lattice of MTs polymerized in vitro, or to the open ends of MTs polymerized in vivo. Microtubule-associated proteins (MAPs) might block lateral holes in cellular MTs, preventing αTAT1 access to the lumen at these sites In this case, appropriate MAPs would need to be included in in vitro acetylation assays to recapitulate in vivo results. This selective, tip-oriented acetylation mechanism has important consequences since cell-front oriented acetylated MTs are instrumental in controlling directional cell migration[12]
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