Research Article: TBCD Links Centriologenesis, Spindle Microtubule Dynamics, and Midbody Abscission in Human Cells

Date Published: January 22, 2010

Publisher: Public Library of Science

Author(s): Mónica López Fanarraga, Javier Bellido, Cristina Jaén, Juan Carlos Villegas, Juan Carlos Zabala, Daniel Lew.

Abstract: Microtubule-organizing centers recruit α- and β-tubulin polypeptides for microtubule nucleation. Tubulin synthesis is complex, requiring five specific cofactors, designated tubulin cofactors (TBCs) A–E, which contribute to various aspects of microtubule dynamics in vivo. Here, we show that tubulin cofactor D (TBCD) is concentrated at the centrosome and midbody, where it participates in centriologenesis, spindle organization, and cell abscission. TBCD exhibits a cell-cycle-specific pattern, localizing on the daughter centriole at G1 and on procentrioles by S, and disappearing from older centrioles at telophase as the protein is recruited to the midbody. Our data show that TBCD overexpression results in microtubule release from the centrosome and G1 arrest, whereas its depletion produces mitotic aberrations and incomplete microtubule retraction at the midbody during cytokinesis. TBCD is recruited to the centriole replication site at the onset of the centrosome duplication cycle. A role in centriologenesis is further supported in differentiating ciliated cells, where TBCD is organized into “centriolar rosettes”. These data suggest that TBCD participates in both canonical and de novo centriolar assembly pathways.

Partial Text: Understanding how the centrosomal components function and are organized during the cell cycle could shed light on many human diseases, from ciliary syndromes to cancer. The centrosome is the major microtubule-organizing center (MTOC) in animal cells, consisting of two centrioles surrounded by proteinaceous pericentriolar material (PCM), from which microtubules nucleate and are released. Microtubules are built of αβ-tubulin heterodimers organized in a head-to-tail fashion. This confers polarity on these polymers, with two different microtubule ends: the plus end, which is more dynamic and oriented towards the periphery of the cells, and the minus end, generally embedded in the centrosome. At the centrosomal core, a centriole pair organizes the surrounding PCM. Centrioles, which are required to assemble the axonemes of cilia and flagella, are structurally very complex. In mammals, they are 500 nm long cylinders with a 200 nm diameter consisting of nine blades arranged in a circle, each containing three highly specialized microtubule segments. Both the exact composition and the assembly of these peculiar microtubules are still unknown. There is also imprecise information regarding the composition and structure of the PCM, in which recent studies have identified hundreds of proteins, most of which play as yet unknown roles [1]. However, one of the most widely accepted concepts is that MTOCs, and in particular the centrosome, accumulate αβ-tubulin polypeptides as part of the PCM for microtubule nucleation. This suggests that there is some tubulin supply at the MTOCs. Yet, the assembly of αβ-tubulin heterodimers is not a trivial matter. The process of association of one α-tubulin with one β-tubulin molecule requires the co-ordinated interaction of a series of tubulin-specific partners, designated tubulin cofactors (TBCs) A–E [2]–[5]. TBCs also play roles in tubulin dissociation [5]–[8], transitory tubulin storage [5], [9]–[12], and tubulin degradation processes [13]–[15], all of which suggest that these proteins, in addition to their original role in tubulin biogenesis, participate in microtubule dynamics by controlling the amount of tubulin available for polymerization. There is considerable evidence in the literature supporting a role for TBCs in this centrosome. Mutations in TBCD, in particular, have been shown to produce aberrations in chromosome numbers in Saccharomyces cerevisiae [16], Schizosaccharomyces pombe [17], Arabidopsis thaliana [18], [19], and Caenorhabditis elegans [20], [21]. TBCD mutations also induce a G1/S blockage and spindle pole body separation in S. pombe [22], [23] and abnormal cytokinesis [19]. In C. elegans, TBCD silencing results in a reduced rate of microtubule nucleation and produces abnormal spindle lengths [21]. Recently, human TBCD (HsTBCD) has been shown to play a role in the organization of the mitotic spindle, and has been hypothesized to recruit from among cytosolic centrosomal proteins, such as pericentrin or γ-tubulin [24].

Since the discovery of TBCs, research into them has largely focused on tubulin biogenesis. This study implicates TBCD in several crucial cell processes. We have shown that TBCD is concentrated at the centrioles and basal bodies, where it plays roles in centriologenesis, ciliogenesis, and spindle organization. TBCD is also found at the Fleming bodies, ring-like structures localized at the midbody during cytokinesis, where it is implicated in cell abscission. In view of all the existing biochemical data, these findings must be interpreted in the context of tubulin-related processes, such as tubulin supply or dissociation.



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