Kinetochores accelerate centrosome separation to ensure faithful chromosome segregation

Abstract

SummaryAt the onset of mitosis, cells need to break down their nuclear envelope, form a bipolar spindle and attach the chromosomes to microtubules via kinetochores. Previous studies have shown that spindle bipolarization can occur either before or after nuclear envelope breakdown. In the latter case, early kinetochore-microtubule attachments generate pushing forces that accelerate centrosome separation. However, until now, the physiological relevance of this prometaphase kinetochore pushing force was unknown. We investigated the depletion phenotype of the kinetochore protein CENP-L, which we find to be essential for the stability of kinetochore microtubules, for a homogenous poleward microtubule flux rate and for the kinetochore pushing force. Loss of this force in prometaphase not only delays centrosome separation by 5-6 minutes, it also causes massive chromosome alignment and segregation defects due to the formation of syntelic and merotelic kinetochore-microtubule attachments. By contrast, CENP-L depletion has no impact on mitotic progression in cells that have already separated their centrosomes at nuclear envelope breakdown. We propose that the kinetochore pushing force is an essential safety mechanism that favors amphitelic attachments by ensuring that spindle bipolarization occurs before the formation of the majority of kinetochore-microtubule attachments.

Effects of aging in catastrophe on the steady state and dynamics of a microtubule population

“…In PtK1 cells, the to and fro motion in mono-oriented chromosomes were observed to last for ∼ 1200s [40], but the observed motion does not show the same degree of regularity as in the previous example. In HeLa cells, the autocorrelation function of translational movement of sister-kinetochores was experimentally measured more recently; the half-period of oscillations, measured as the position of the first minimum was found to be in the range ∼ 10s − 40s [41,42]. With the available information, it is unclear whether aging and related effects in microtubule catastrophe plays a role in these phenomena; however, given the fact that the observed period of oscillations of mono-oriented chromosomes [38,40] is close to the what was found in our simulations (Fig.10), we feel that further investigations in this direction could be worthwhile.…”

CRL4RBBP7 is required for efficient CENP-A deposition at centromeres

“…For RNAi experiments, cells were transfected with 50 nM siRNAs (Qiagen, Hilden, Germany or Microsynth, Balgach, Switzerland) for 72 h using Oligofectamine (Invitrogen) or Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. The target sequences of double-stranded RNA used in this study were as follows: siControl, 59-GGACCTGGAGGTCTGCTGT-39 (McHedlishvili et al, 2012); siCENP-A, 59-AACACAGTCGGCGGAGACAAG-39 (Carroll et al, 2009);siHJURP, 59-CTACTGGGCTCAACTGCAA-39 (Dunleavy et al, 2009); siCUL4A, 59-AAGAATCCTACTGCTGATCGA-39 (Piwko et al, 2010); siCUL4B, 59-CACCGTCTCTAGCTTTGCTAA-39 (Piwko et al, 2010); siDDB1, 59-CCACTAGATCGCGATAATAAA-39 (Piwko et al, 2010); siRBBP7, 59-GCGGATAAGACCGTAGCTTTA-39 (Piwko et al, 2010); siRBBP4, 59-GCCACTCAGTTGATGCTCA-39 (Hayashi et al, 2004); siCDC20, 59-AACCTTGTGGATTGGAGTTCT-39 (Piwko et al, 2010); and siMAD2L1, 59-CAGAAAGCTATCCAGG-ATGAA-39 (Piwko et al, 2010). Cells were synchronized at the G1/S transition by 16 h incubation in 2 mM thymidine (Sigma, St. Louis, MO), washed twice with pre-warmed phosphate-buffered saline (PBS) solution, incubated for 9 h in culture medium and then incubated for another 16 h in 2 mM thymidine.…”

“…Immunofluorescence, SNAP tag labelling and microscopy Cells were fixed as described previously (McHedlishvili et al, 2012). The following antibodies were used: anti-CREST antisera (1:250, Antibodies Incorporated), rabbit anti-cyclin-A (1:500, Santa Cruz Biotechnology) and mouse anti-CENP-A (1:2000, Abcam, Cambridge, UK).…”

The TRAF-interacting protein (TRAIP) is a regulator of the spindle assembly checkpoint

“…In siCTRL-transfected cells, the median duration of mitosis was 116.7 min [95% confidence interval (CI)5111.7-123.3 min; range, 75 to 275 min]. The high variability of duration of mitosis was mainly caused by the time between NEB to anaphase onset (Lim et al, 2013;Mchedlishvili et al, 2012;Rieder et al, 1994;Toso et al, 2009), which varied from 20 to 200 min (median, 53.3 min; 95% CI546.7-61.7 min). The time from anaphase to cytokinesis fluctuated only moderately from 45 to 85 min (median, 63.3 min; 95% CI560-66.7 min) ( Fig.…”