Rubin T et al. (2014), Cenp-meta is required for sustained spindle che...

XB-ART-49095

Biol Open 2014 May 29;36:522-8. doi: 10.1242/bio.20148490.

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Cenp-meta is required for sustained spindle checkpoint.

Rubin T , Karess RE , Rahmani Z .

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Cenp-E is a kinesin-like motor protein required for efficient end-on attachment of kinetochores to the spindle microtubules. Cenp-E immunodepletion in Xenopus mitotic extracts results in the loss of mitotic arrest and massive chromosome missegregation, whereas its depletion in mammalian cells leads to chromosome segregation defects despite the presence of a functional spindle assembly checkpoint (SAC). Cenp-meta has previously been reported to be the Drosophila homolog of vertebrate Cenp-E. In this study, we show that cenp-metaΔ mutant neuroblasts arrest in mitosis when treated with colchicine. cenp-metaΔ mutant cells display a mitotic delay. Yet, despite the persistence of the two checkpoint proteins Mad2 and BubR1 on unattached kinetochores, these cells eventually enter anaphase and give rise to highly aneuploid daughter cells. Indeed, we find that cenp-metaΔ mutant cells display a slow but continuous degradation of cyclin B, which eventually triggers the mitotic exit observed. Thus, our data provide evidence for a role of Cenp-meta in sustaining the SAC response.

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Species referenced: Xenopus
Genes referenced: btg3 bub1 bub1b bub3 ccnb1.2 mad2l1 mxd1 neb rps27 spc25 tbx2

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	Fig. 1. The cenp-metaΔ gene and gene product.(A) The Cenp-meta gene locus contains 11 exons and shares 55 nucleotides of 3′ untranslated region (utr) with the 3′ utr of the abrupt gene, which is transcribed in the opposite direction. Exon structure, direction of transcription (arrows), site of P-element insertion, and extent of the 5 Kb deletion in cenp-metaΔ mutation are indicated. (B) Western with Cenp-meta antibody shows one band above 250 kD in wild-type third instar larval brain protein extracts (WT) not found in larval brain protein extracts from cenp-metaΔ homozygous mutant (cenp-metaΔ). This band is consistent with the previously reported predicted size of Cenp-meta protein, which is 257 kD (Yucel et al., 2000). The same blot was stripped and reprobed with tubulin antibody to verify that equal amount of protein extracts were loaded for each lane. Asterisk indicates non-specific bands present in WT and cenp-metaΔ protein extracts.
	Fig. 2. Mitotic timing in cenp-metaΔ mutant and cenp-metaΔ mad2 double mutant neuroblasts.(A) Comparative mitotic timing of cenp-metaΔ and cenp-metaΔ mad2 double mutant cells. NEB is defined as when RFP-Spc25 begins to be visible on kinetochores. cenp-metaΔ (blue diamonds) are delayed, with an average 15.1 minutes vs 9.6 minutes in WT (yellow diamonds). cenp-metaΔ mad2 (pink diamonds) enter anaphase earlier than WT cells (7.2 minutes vs 9.6 minutes, P<0.005). cenp-metaΔ mad2 double mutant cells (pink diamonds) show the same timing as the one that was previously reported for mad2 alone (Buffin et al., 2007; Rahmani et al., 2009), thereby indicating that the prometaphase delay in cenp-metaΔ is SAC-dependent. (B–D) Still frames extracted from typical movies used for the determination of mitotic timing (from NEB to anaphase). (B) WT, (C) cenp-metaΔ, (D) cenp-metaΔ mad2 double mutant. All cells are marked with RFP-Spc25. See also supplementary material Movies 1, 2, 3. Scale bars: 5 µm.
	Fig. 3. The checkpoint proteins Mad2 and BubR1 remain associated with the kinetochores of unaligned chromosomes in cenp-metaΔ mutant cells entering anaphase.(A–C) Selected frames from a WT (A) or two different cenp-metaΔ mutant cells (B,C) marked with GFP-Mad2 showing three mitotic stages (Prometaphase, Anaphase, Late anaphase). See the corresponding supplementary material Movies 4, 5, 6. Mad2 streaming during prometaphase is not affected in cenp-metaΔ mutant cells (B,C). While Mad2 signal is totally gone by the time the WT cell enters anaphase (A), it is still visible on kinetochores (arrowhead) of unaligned chromosomes at the poles and lasts until late anaphase in cenp-metaΔ mutant cells (B,C). (D–F) Selected frames from a WT (D) or two different cenp-metaΔ mutant cells (E,F) marked with GFP-Rod (green) and RFP-BubR1 (red). Note that GFP-Rod is used to monitor the anaphase onset since BubR1 signals are gone at this stage. BubR1 signal is no longer visible at metaphase (Met) in the WT cell (D) whereas it persists on unaligned kinetochores (arrowhead) seen in cenp-metaΔ mutant cells (E,F) entering anaphase (Ana). See also supplementary material Movies 7, 8, 9. Scale bars: 5 µm.
	Fig. 4. Cyclin B degradation profiles in WT and cenp-metaΔ mutant neuroblasts.Typical onset of cyclin B degradation (OCBD) in a single neuroblast. (A) WT, (B) cenp-metaΔ mutant neuroblast. (A,B) The frames showing RFP-Rod (red) and GFP-cyclin B (green) at various time points (indicated as a,b,c, on both graphs) in WT (A) and cenp-metaΔ mutant (B) cells were taken from supplementary material Movies 10 and 11, respectively. (C) Mean time of OCBD versus anaphase (ANA) onset. OCBD begins 2.9 minutes before anaphase onset in WT cells (see also Rahmani et al., 2009) whereas it is a slow continuous process in cenp-metaΔ mutant cells. See also supplementary material Movies 10, 11. Scale bars: 5 µm.

References [+] :

Abrieu, CENP-E as an essential component of the mitotic checkpoint in vitro. 2000, Pubmed, Xenbase