The Tubulin Code: A Navigation System for Chromosomes during Mitosis

Abstract

Before chromosomes segregate during mitosis in metazoans, they align at the cell equator by a process known as chromosome congression. This is in part mediated by the coordinated activities of kinetochore motors with opposite directional preferences that transport peripheral chromosomes along distinct spindle microtubule populations. Because spindle microtubules are all made from the same α/β-tubulin heterodimers, a critical longstanding question has been how chromosomes are guided to specific locations during mitosis. This implies the existence of spatial cues/signals on specific spindle microtubules that are read by kinetochore motors on chromosomes and ultimately indicate the way towards the equator. Here, we discuss the emerging concept that tubulin post-translational modifications (PTMs), as part of the so-called tubulin code, work as a navigation system for kinetochore-based chromosome motility during early mitosis.

Keywords: CENP-E; Dynein; detyrosination; motors; post-translational modifications; tubulin code.

Figure 1. Key figure – Microtubule (de)tyrosination selectively guides kinetochore-based motor proteins required for accurate chromosome congression to the spindle equator.

Schematic representation of the impact of tubulin tyrosination/detyrosination on the regulation of motor proteins responsible for chromosome movements in mitosis. Dynein/Dynactin have a preference for tyrosinated astral microtubules and counteract chromokinesins-mediated PEFs. By bringing chromosomes to the vicinity of the spindle pole-localized Aurora A kinase, Dynein/Dynactic prevents premature stabilization of erroneous end-on attachments and contributes to the local activation of CENP-E. At the pole, CENP-E interacts with detyrosinated microtubules, becoming dominant over dynein to move chromosomes towards the equator. Thus, the levels of microtubule (de)tyrosination provide the directional bias for CENP-E-mediated chromosome transport and regulate the Dynein/CENP-E activity switch that facilitates chromosome congression.

Figure 2. Tubulin PTMs with potential roles in mitosis.

Illustrative model depicting different post-translational modifications of the tubulin dimers within a microtubule (modified from ref. (7)) with potential roles during mitosis. Acetylation of lysine 40 of α–tubulin occurs on the microtubule lumen and is mediated by the tubulin acetyl transferase αTAT1, whereas deacetylation is performed by at least two deacetylases, HDAC6 and Sirt2. Detyrosination of α–tubulin and polyglutamylation of both α– and β–tubulin take place at the outer surface of the microtubule lattice, which is in direct contact with motor proteins. Tubulin detyrosination is driven by a yet-unidentified tubulin carboxypeptidase (TCP), while retyrosination requires action of the tubulin tyrosine ligase (TTL). Polyglutamylation is catalyzed by multiple TTL-like (TTLL) glutamylases and reversed by several deglutamylases. Phosphorylation of Ser172 of β-tubulin occurs specifically during mitosis and is mediated by Cdk1 kinase.

Figure 3. Microtubule detyrosination on a subset of mitotic spindle microtubules is required for accurate chromosome congression.

a) Point-scanning confocal immunofluorescence images of U2OS cells stained for DNA [4′,6- diamidino-2-phenylindole (DAPI), blue], anti-centromere antibody (ACA, white), tyrosinated tubulin (green), and detyrosinated tubulin (red). Note that the astral microtubules are immuno-stained exclusively with tyrosinated tubulin antibody, as detyrosination occurs only on more stable kinetochore (and possibly interpolar) microtubules. Scale bar, 10 μm. b-e) Spinning-disk confocal imaging of live U2OS cells stably expressing CENP-A–GFP and mCherry-tubulin. Pole-proximal chromosomes are unable to congress and remain locked at the poles when either CENP-E activity is inhibited (c) or microtubule detyrosination is prevented using the TCP inhibitor parthenolide (d). In contrast, TTL depletion prevents pole-proximal chromosomes to reach the pole causing their ejection in random directions (e). Arrowheads highlight chromosomes that cannot reach the pole, likely due to the Dynein loss of function on now detyrosinated astral microtubules and/or are ejected away from the pole by the enhanced CENP-E activity on detyrosinated astrals. Time, hours:min. Scale bar, 10 μm.