Centriole assembly and the role of Mps1: defensible or dispensable?

Abstract

The Mps1 protein kinase is an intriguing and controversial player in centriole assembly. Originally shown to control duplication of the budding yeast spindle pole body, Mps1 is present in eukaryotes from yeast to humans, the nematode C. elegans being a notable exception, and has also been shown to regulate the spindle checkpoint and an increasing number of cellular functions relating to genomic stability. While its function in the spindle checkpoint appears to be both universally conserved and essential in most organisms, conservation of its originally described function in spindle pole duplication has proven controversial, and it is less clear whether Mps1 is essential for centrosome duplication outside of budding yeast. Recent studies of Mps1 have identified at least two distinct functions for Mps1 in centriole assembly, while simultaneously supporting the notion that Mps1 is dispensable for the process. However, the fact that at least one centrosomal substrate of Mps1 is conserved from yeast to humans down to the phosphorylation site, combined with evidence demonstrating the exquisite control exerted over centrosomal Mps1 levels suggest that the notion of being essential may not be the most important of distinctions.

Figure 1

Centriole assembly as a modular process. Centriole assembly in human cells proceeds through a pathway analogous to that described in C. elegans, but requires several additional proteins not present in worms, and proceeds through a cartwheel template as opposed to a central tube. Centrioles are not templates per se, but rather provide a surface for the assembly of cartwheels, depicted as a hub composed of hSas6 (red) and 9 symmetrical spokes (blue). We propose that cartwheels then serve as a platform onto which additional centriole modules are assembled in a proximal to distal, or "bottom-up" fashion. Yellow and green rectangles are used to depict proximal (yellow) and distal (green) centriole modules. The frame surrounding these modules is meant to depict maturation into the final structure rather than centriolar microtubules.

Figure 2

Cell cycle profile of Mps1 and its control by degradation. The top panel shows the cell cycle profile of hMps1 protein levels (broken grey line) and protein kinase activity (solid black line), adapted from the data of Hogg et al., 1994 [64]. After a sharp peak of hMps1 activity at G1/S that is not accompanied by a rise in whole cell protein levels, both protein and activity peak in mitosis. After completing its function in the spindle checkpoint, Mps1 is targeted for degradation at mitotic exit by both Cdc20- and Cdh1-associated APC/C complexes through the hMps1 D-box. Cdh1-dependent hMps1 degradation keeps cytoplasmic hMps1 levels low in G1, while OAZ targets the centrosomal pool of Mps1 for degradation through the MDS. Phosphorylation of T468 within the MDS transiently suppresses OAZ-mediated degradation, allowing accumulation of a centrosomal pool coincident with centrosome duplication. The lower panel shows a schematic of the 853 amino acid hMps1 protein indicating the positions of the D-box (amino acids 256-263) and MDS (amino acids 420-507) in yellow as the binding sites for Cdh1/Cdc20 (blue) and OAZ (red), respectively, and the kinase domain in black.

Figure 3

Proposed functions for Mps1 in centriole assembly. Studies with OAZ and Cetn2 suggest two functions for Mps1 in centriole assembly, an early function in procentriole assembly and a later function in centriole maturation that is mediated by Cetn2. We propose that the single centriolar focus of hSas6 observed upon overexpression of OAZ or hMps1KD represents an intermediate in centriole assembly (depicted as a red and blue sunburst, red indicating hSas6 and blue representing other cartwheel proteins). The observation that this structure is observed upon attenuation of Mps1 activity [29] suggests that Mps1 activity remodels this intermediate into cartwheels, onto which procentrioles are subsequently assembled. Because hMps1^Δ12/13 generates excess hSas6-containing structures in the absence of Cetn2 [28], this early function of Mps1 is presumably Cetn2-independent. The observation that Mps1 is required for the ability of Cetn2 to organize distal centriole elements [28] suggests that Mps1 has a second, Cetn2-dependent function in the maturation of centrioles. Structures and colors are as in Figure 1, with the exception of carets used to depict the centriolar appendages of the maternal centriole, and the sunburst depicting the hSas6-containing intermediate.

Figure 4

Modes of centriole over production. We propose that hMps1 and Cetn2 cooperate to generate centriole overproduction by two distinct mechanisms. First, in Mps1^Δ12/13-induced centriole overproduction, hMps1^Δ12/13 promotes the assembly of multiple hSas6 containing precursors (analogous to the proposed hSas6-containing intermediate in procentriole assembly described in Figure 3). As in the canonical centriole assembly pathway, we assume that hMps1 is required for the remodeling of these precursors into cartwheels, onto which procentrioles are assembled. The figure reflects the possibility that not all of the precursors become cartwheels, based on the observation that the percentage of cells with excess hSas6 foci is greater than the percentage of cells with excess γ-Tubulin or CP110 foci [28]. Second, hMps1 is also required for initiation of Cetn2-induced centriole overproduction. We propose that overexpression of wild type Cetn2 leads to the assembly of Cetn2-containing precursors that organize distal centriole modules in a top-down fashion that is independent of hSas6. Because Mps1 is required for the initiation of these structures, we cannot assess a role for Mps1 in their maturation. However, while the initiation of these structures is hSas6-independent, some aspects of their maturation require hSas6, as suggested by the observation that a subset of centriole proteins are not recruited to these structures in the absence of hSas6 [28]. Because these structures recruit hSas6, it is possible that they can recruit cartwheels (indicated by a question mark). Structures and colors are as in Figure 3.

Figure 5

IN-1 is not equally effective against all Mps1 substrates in vitro. In vitro kinase assays were performed as described by Yang et al. (2010) [28], with GST-Mps1 (0.4 mM) and either 6hisCetn2 (Cetn2) or Myelin Basic Protein (MBP) as substrate (10 mM). The IN-1 Mps1 inhibitor described by Kwiatkowski et al. (2010) [60] was included at the indicated concentrations in μM. The top and bottom panels show autoradiographs of kinase assays with Cetn2 as substrate (subst.), the bottom cropped to show just Cetn2. The middle panel shows a similar kinase assay using MBP as substrate, cropped to show just MBP. Arrows to the left indicate the signals corresponding to Mps1 autophosphorylation (which is attenuated in the presence of Cetn2), Cetn2 phosphorylation, and MBP phosphorylation. While enzyme and substrate concentrations differ from those used by Kwiatkowski et al. (2010) [60], Cetn2 phosphorylation and Mps1 autophosphorylation were observed at IN-1 concentrations that blocked MBP phosphorylation (5 and 10 μM IN-1), and residual Cetn2 phosphorylation was observed even at 100 μM IN-1.

Figure 6

The many roles of Mps1 in the maintenance of genomic integrity. Many functions for hMps1 have been described in human cells, in both the centriole and nuclear cycles. Although the described functions for hMps1 are quite varied, when properly coordinated they ensure the integrity of the genome. Recent work discussed extensively in this review suggests that hMps1 functions in both procentriole assembly and centriole maturation. While it is possible that these functions are dispensable, failure to properly regulate them leads to centriole overproduction that can generate aberrant mitotic spindles. hMps1 has also been implicated in cytokinesis [59], responsible for partitioning of one copy each of the duplicated genome and centrosome into daughter cells. hMps1 regulates the p53-dependent post-mitotic checkpoint that prevents cell cycle entry after failed mitosis, and defects in this function can allow aneuploid cells to proliferate [57]. hMps1 regulates the Chk2-dependent DNA damage response, and defects in this function lead to defective arrest in the presence of damaged DNA [58]. Finally, hMps1 regulates the spindle assembly checkpoint [55], and defects in this function lead to chromosome segregation errors. The function of hMps1 in SMAD signaling represents a transcriptional input into the spindle assembly checkpoint [56]. In this figure, a circle represents the cell cycle, with phases labeled and arrows representing transitions. The centriole cycle is depicted on the inside, with carets indicating centriolar appendages (which are assembled onto the two oldest centrioles during mitosis) and cartwheels (which are degraded during mitosis) as in Figures 1, 3, and 4. The nuclear cycle is depicted on the outside, with the nucleus as a grey oval, and chromosomes in blue. The mitotic spindle is indicated by green lines representing microtubules, mitotic chromosomes in blue, kinetochores in cyan, and red lines indicating an activated spindle checkpoint.