Supplemental Material

This article contains the following supporting material:

• Figure 1 - Cellular localization of endogenous CLASP2 during mitosis in human HeLa cells. CLASP2 was detected with a rabbit polyclonal antibody raised against the recombinant C-terminal fragment of CLASP2 and in the merged image is shown in green. MTs are shown in red and were revealed with a mouse monoclonal antibody against α-tubulin. The DNA in blue was counterstained with DAPI. CLASP2 starts being recruited to kinetochores during prometaphase and throughout metaphase, where significant staining along the spindle and at the poles can be seen. During anaphase and telophase CLASP2 accumulates at the spindle midzone and midbody (small green arrows in the merged column). During cytokinesis, CLASP2 associates with the inner fraction of the midbody and is present in visicle-like structures around the centrosome. In the far right column it is represented a higher magnification of the corresponding region in the merged column on the left or in the case of the cells in anaphase and telophase the DNA channel is shown alone. To confirm localization of CLASP2 in the outer kinetochore (large green arrows) during prometaphase, we performed co-immunolocalization with anti-centromere antibodies (ACA; only merged in the higher magnification picture; large blue arrows). Scale bar is 10 μm.

• Figure 2 - Cellular localization of EGFP-CLASP2 in HeLa cells. EGFP-CLASP2 is shown in green, MTs in red and DNA in blue. At the prophase/prometaphase transition EGFP-CLASP2 localizes to some kinetochores, indicating that its recruitment to this structure is an asynchronous process that accompanies kinetochore maturation. During metaphase and anaphase A, EGFP-CLASP2 can clearly be found at kinetochores, centrosomes, and somewhat distributed along the spindle. The kinetochore accumulation of EGFP-CLASP2 drops down significantly during anaphase B while re-distributing to the spindle midzone. During telophase and cytokinesis, EGFP-CLASP2 strongly accumulates at the midbody. Centrosome localization of EGFP-CLASP2 persisted throughout mitosis and cytokinesis. Yellow arrows indicate localization of CLASP2 in some MT plus-ends (shown as an insert in the merged column on the left). Selected regions showing kinetochore accumulation of EGFP-CLASP2 throughout mitosis are represented as small insertions on the right column. Scale bar is 10 μm.

• Figure 3 - Western blot analysis of the expression levels of EGFP-CLASP1 in the stable HeLa cell line. Protein extracts from 1x10⁶ cells were prepared from Parental HeLa and HeLa cells stably expressing GFP-CLASP1 (clones 1 and 2) and proteins separated by SDS-PAGE according to their molecular weight. Proteins were then transferred to nitrocellulose membranes, which were incubated with antibodies against CLASP1, GFP and α-tubulin (as a loading control). After quantification of the expression levels of ectopic proteins, Clone 1 was selected and used for all FRAP and co-localization experiments.

• Figure 4 - Quantification of EGFP-CLASP2 levels at kinetochores. Stable expression of EGFP-CLASP2 (green) was induced in 3T3 fibroblasts to levels near the endogenous and cells analysed by immunofluorescence microscopy with ACA (red). DNA was counterstained with DAPI (blue). (A-A’’’) Prometaphase cell. (B-B’’’) Chromosome spreads from a cell treated with 10 μM nocodazole to depolymerize MTs. (C-C’’’) Metaphase cell. (D-D’’’) Cell treated with 100 nM taxol to stabilize MTs. Selected regions showing kinetochore accumulation of EGFP-CLASP2/ACA on each experimental situation are represented as small insertions on the right column. Scale bar is 10 μm. (E) Quantification of the EGFP-CLASP2 fluorescence intensity at kinetochores during metaphase and in cells treated with nocodazole or taxol. The values on each experimental set indicate the average and standard deviations for fluorescence intensity ratios between EGFP-CLASP2 and ACA at individual kinetochores (N=10 kinetochores from 3 cells for each situation).

• Figure 5 - Determination of the turnover rates of mammalian CLASPs by FRAP. (A-B) Representative frames for bleaching and recovery of centrosomal EGFP-CLASP1 during interphase and mitosis, respectively. (C) The same experiment was performed for kinetochores during prometaphase. (D-E) Representative frames for bleaching and recovery of centrosomal EGFP-CLASP2 during interphase and mitosis, respectively. (A’, B’, C’, D’, and E’) The corresponding fluorescence recovery signal after photobleaching was fit to a single-exponential curve as described in Experimental Procedures. Inset: half-time of the exponential curve. Scale bar is 5 μm. (F) Statistic analysis of the EGFP-CLASP1 and EGFP-CLASP2 turnover rates at centrosomes for each experimental condition and cell cycle stage. The respective p-values are shown (Student’s t-test), including those that were not statistically significant (red). Each line connects the conditions that do not vary in the particular pair being compared. As an example, a p-value of 0.662 was obtained when comparing CLASP1 and CLASP2 during mitosis in the absence of nocodazole.

• Figure 6 - Live cell imaging of mitosis in Clasp2 KO embryonic fibroblasts. (A-E) Selected frames from 5 time-lapse recordings of Clasp2 KO MEFs showing several examples of chromosome missegregation and aneuploidy. (A) This cell shows a chromosome rosette conformation typical of monopolar spindles where chromosomes were found to be highly static with no apparent oscillation. After approximately 1 h, ectopic cleavage furrows (arrowheads) started to form without chromosome segregation. (B) This cell was recorded from prometaphase through cytokinesis, developing a tripolar spindle (asterisks) and resulting in aneuploid chromosome segregation. (C) This cell is shown at the onset of anaphase where an extra chromosomal mass segregates outside the main spindle axis resulting in the formation of a micronucleus after chromosome decondensation in telophase. (D) This cell is also shown at the onset of anaphase where a lagging chromosome remains at the cleavage furrow. The presence of chromatin in the spindle midzone may have resulted in cytokinesis failure. (E) This cell is shown during the process of adaptation to a mitotic delay with apparent sister-chromatid separation followed by chromosome decondensation without segregation. (F) Wild type MEF entering mitosis in the presence of 10 μM nocodazole. The cell remained arrested for more than 2 hours. Time is shown in hrs:mins:secs. Scale bar is 25 μm.

• Movie 1 - Time-lapse sequence from a wild type MEF with a normal mitotic behaviour, forming a normal metaphase plate with normal oscillating chromosomes and entering anaphase in less than 30 min from NEB.

• Movie 2 - Time-lapse sequence from a Clasp2 KO MEF progressing normally throughout mitosis. This cell entered anaphase in less than 25 min after NEB. Note the behaviour of chromosomes that never formed a well-defined metaphase plate.

• Movie 3 - Time-lapse sequence from a Clasp2 KO MEF entering mitosis from NEB. Note that chromosomes never formed a metaphase plate and were organized in a rosette configuration typical of monopolar spindles, without entering anaphase for ~50 min.

• Movie 4 - Time-lapse sequence from a Clasp2 KO MEF in prometaphase showing chromosomes in a rosette configuration typical of monopolar spindles. The chromosomes remained highly static, with no apparent oscillations over a period of 3h. After approximately 1h of recording without the occurrence of chromosome segregation, ectopic cleavage furrows started to form.

• Movie 5 - Time-lapse sequence from a Clasp2 KO MEF that developed a tripolar spindle and entered anaphase, resulting in aneuploid chromosome segregation.

• Movie 6 - Time-lapse sequence from a Clasp2 KO MEF where an extra chromosomal mass segregates outside the main spindle axis, resulting in the formation of a micronucleus after chromosome decondensation in telophase.

• Movie 7 - Time-lapse sequence from a Clasp2 KO MEF progressing normally throughout mitosis. This cell entered anaphase in less than 30 min after NEB with the presence of lagging chromosomes that formed chromatin bridges during telophase. Note the highly dynamic behaviour of chromosomes that never formed a well-defined metaphase plate.

• Movie 8 - Time-lapse sequence from a Clasp2 KO MEF during anaphase showing lagging chromosomes at the time and position of the cleavage furrow, which resulted in a cytokinesis failure and formation of a bi-nucleated cell.

• Movie 9 - Time-lapse sequence from a Clasp2 KO MEF from a metaphase-like state undergoing adaptation. Note that chromatids seem to become resolved minutes before chromatin started decondensing without chromosome segregation. This suggests that sister-chromatid separation may have occurred during the adaptation process.

• Movie 10 - Time-lapse sequence from a Clasp2 KO MEF entering mitosis in the presence of 10 μM nocodazole. The cell remained in c-mitosis for more than 2 h indicating a functional spindle assembly checkpoint.

• Movie 11 - Time-lapse sequence from a Clasp2 WT MEF entering mitosis in the presence of 10 μM nocodazole. The cell remained in c-mitosis for more than 2 h indicating a functional spindle assembly checkpoint.

• Movie 12 - Time-lapse sequence from a Clasp2 KO MEF from prometaphase, in the presence of 20 μM of the proteosome inhibitor MG-132. The cell was able to complete and sustain chromosome alignment at the metaphase plate, while anaphase entry was being prevented.