Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores

Abstract

Mps1 is an upstream component of the spindle assembly checkpoint, which, in human cells, is required for checkpoint activation in response to spindle damage but not apparently during an unperturbed mitosis. Mps1 also recruits Mad1 and Mad2 to kinetochores. However, whether the enzymatic activity of Mps1 is required for these processes is unclear. To address this question, we established an RNA interference (RNAi) complementation assay. Repression of Mps1 triggers premature anaphase, often with unaligned or maloriented chromosomes. This phenotype is rescued by an RNAi-resistant wild-type Mps1 transgene but not by a catalytically inactive mutant. An analogue-sensitive allele, Mps1M602A, also rescues the RNAi-induced defect, but not when inhibited by the adenosine triphosphate analogue 1-NM-PP1. Thus, Mps1 activity does restrain anaphase during an unperturbed mitosis. Furthermore, although catalytically inactive Mps1 can restore kinetochore localization of Mad1, only the active kinase restores Mad2 localization. Thus, in human cells, Mps1 catalytic activity is required for spindle checkpoint function and recruitment of Mad2.

Characterization of novel MPS1 inhibitors with preclinical anticancer activity

“…Even in the absence of SAC activators, both classes of MPS1 inhibitors markedly increased the rate of chromosome misalignments resulting from erroneous MT-KT attachments and promoted a premature anaphase entry (i.e., before the formation of a correct equatorial metaphase plate). These results are in line with previous findings obtained with other MPS1-specific inhibitors, [8][9][10]12,15,47 upon MPS1 depletion 16,24 or following the conditional knockout of TTK, 11 confirming the central implication of this mitotic kinase in SAC function and chromosome congression. Upon exposure to MPS1 inhibitors, colorectal carcinoma cells displayed severely abnormal anaphases, and, as they progressed in mitosis, they underwent two alternative catastrophic fates: (i) they divided in a bipolar (often asymmetrical) manner, generating two aneuploid cells that most often died in the following interphase, or (ii) they failed to complete cytokinesis and hence became hyperploid.…”

“…10,16 Consistent with these observations, both the inhibition/depletion and the overexpression of MPS1 abrogate SAC functions, leading to aneuploidy/polyploidy and eventually cell death. 9,16,[22][23][24] MPS1 appears to play additional, more controversial, roles in mitosis or interphase. For instance, MPS1 not only may influence mitotic exit and cytokinesis 25 but also seems to promote the assembly of a cytosolic anaphase-promoting complex/cyclosome inhibitory complex during interphase to define the overall timing of the M phase.…”

Spindly switch controls anaphase: Spindly and RZZ functions in chromosome attachment and mitotic checkpoint control

“…Whether Spindly, which is a mitotic phosphoprotein, 32 is controlled by protein phosphorylation is currently unknown. Intriguingly, similar to Spindly RNAi, disruption of the mitotic checkpoint kinases Bub1 and BubR1, [96][97][98] as well as Mps1, [99][100][101][102][103][104][105] results in severe chromosome alignment defects. Moreover, when anchored to kinetochores via fusion to Mis12, Mps1 was prevented to leave the kinetochore, and again, similar to Spindly mutants, chromosomes were unable to segregate due to an unsatisfied SAC.…”

Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae

“…For generation of Flp-In TRex HeLa cell line, caveolin1 was amplified by PCR and cloned into a pcDNA5/FRT/TO vector modified to contain a Cterminal GFP tag and mutagenized to create the vector pcDNA5/FRT/TO/ caveolin1-GFP by PCR cloning. The vector was cotransfected into Flp-In TRex tetracycline inducible HeLa cells (a kind gift from Stephen S. Taylor, University of Manchester, UK) with the Flp-recombinase-encoding plasmid pOG44 (Invitrogen) as described previously (Tighe et al, 2008). The cells were grown in DMEM (GIBCO) supplemented with 10% foetal bovine serum (Invitrogen), 100 mg/ml hygromycin B and 5 mg/ml blasticidin S HCl (both GIBCO) for plasmid selection.…”