A Spindle Checkpoint Functions during Mitosis in the EarlyCaenorhabditis elegansEmbryo

Abstract

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis. INTRODUCTIONDuring mitosis, some microtubules emanating from bipolar microtubule organizing centers grow toward chromosomes and attach to specialized chromosomal regions called kinetochores (Skibbens and Hieter, 1998;Cleveland et al., 2003). Once captured, sister chromatids are segregated, one to each of two daughter cells. Eukaryotic cells have evolved a mechanism called the spindle checkpoint, to monitor chromosomal segregation and increase the fidelity of mitosis (reviewed in Gardner and Burke, 2000;McIntosh et al., 2002). Until spindle microtubules from both poles capture and align all sister chromatid pairs at a metaphase plate, the spindle checkpoint produces a delay in the onset of anaphase. If this delay is bypassed by reducing checkpoint function, anaphase starts prematurely, and daughter cells may receive unequal complements of chromosomes. Such genomic instability can result in lethality and in tumorigenesis (Hartwell and Kastan, 1994;Basu et al., 1999).Originally identified and characterized in Saccharomyces cerevisiae, seven spindle assembly checkpoint genes (MAD1, MAD2, MAD3, BUB1, BUB2, BUB3, and MPS1) are known to function at kinetochores to inhibit anaphase onset, or to mediate mitotic exit in the presence of spindle abnormalities (Hoyt et al., 1991;Li and Murray, 1991). Mutations in these genes reduce or eliminate the cell cycle delays that normally occur after treatment with microtubule depolymerizing drugs (Wang and Burke, 1995;Pangilinan and Spencer, 1996). Bub1 and Bub3 form a protein kinase complex that, in concert with Mps1, functions at kinetochores upstream of Mad1 and Mad2 (Roberts et al., 1994;H...

Cell cycle progression requires the CDC-48 UFD−1/NPL−4 complex for efficient DNA replication

“…S3 and Table S1). Similarly, downregulation of the mitotic regulator genes mdf-1 and air-2 (21,22), which encode homologues of the spindle checkpoint factor MAD1 and the mitotic progression kinase Aurora B, did not suppress the cell division delay ( Fig. S2 B and C, and Table S1).…”

Chromosomal analysis of blastocysts from balanced chromosomal rearrangement carriers

“…Several checkpoints control the proper alignment of chromosomes during mitosis and meiosis and block entry into anaphase when the chromosomes are not appropriately attached to the spindle (Encalada et al 2005, Wei et al 2011. However, these mechanisms may be deficient to avoid all the errors, supported by findings of lagging chromosomes in human embryos (Coonen et al 2004).…”

“…Although the efficiency of the in-house locus-specific probes was pre-validated using mitotic metaphase chromosomes of cultured peripheral blood lymphocytes, they may be suboptimal with interphase nuclei of single blastomeres. Alternatively, chromosomal repair processes such as mitotic checkpoints (Encalada et al 2005, Wei et al 2011) and reabsorption of chromosomecontaining cytoplasmic debris (Chavez et al 2012), which are active during embryonic development to the blastocyst stage, may be involved. The percentage of chromosomally balanced blastocysts observed in the present study (w60%) exceeds the predicted incidence of Rob carriers (1/6 normal, 1/6 carrier) (Bint et al 2011).…”

Suppressors of Spindle Checkpoint Defect (such) Mutants Identify New mdf-1/MAD1 Interactors in Caenorhabditis elegans

“…All images were taken with a Retiga 2000R (Qimaging) digital camera and Openlab 4.0.2 software (Improvision). Time points measured were from the nuclear envelope breakdown (NEBD) to the nuclear envelope reformation (NER) as determined by the analysis of differential interference contrast (DIC) images, as described previously (Encalada et al 2005). To measure the time points from NEBD to anaphase and from anaphase to NER, embryos from hermaphrodites carrying an integrated H2BTGFP transgene were prepared and visualized using the same procedure.…”

“…MDF-1/Mad1, MDF-2/Mad2, and BUB-1/Bub1 are required in both yeast and C. elegans to promote mitotic delays in the presence of either chemical or mutational disruptions of the microtubule cytoskeleton (Kitagawa and Rose 1999;Encalada et al 2005). During anoxiainduced suspended animation, embryos lacking functional SAN-1/Mad3 or MDF-2/Mad2 fail to arrest the 1 cell cycle (Nystul et al 2003).…”