Figure 5. Kinetochore defects in condensin-depleted cells lead to a high incidence of syntelic and merotelic mis-attachments of kinetochore microtubules, which are not corrected prior to anaphase onset.

A) Condensin-less anaphase has an abnormally high frequency of lagging chromosomes. An example of anaphases in condensin-depleted HeLa cells with centromeres labeled with CREST antibodies and spindle microtubules with anti-α-tubulin antibodies. The estimated frequency of lagging and/or non-disjoined centromeres is more than 20%, as compared to less than 1% in wild type. The lagging centromeres were defined as falling outside the distance to the pole for the bulk of centromeres in untreated cells. As many centromeres were extremely stretched, and were not counted as a result, the actual lagging number should be substantially higher. B) A high incidence of syntelic mis-attachments of kinetochore microtubules in metaphase upon condensin depletion. HeLa cells were treated as in Fig. 2A, except that they were incubated on ice for 10 min prior to fixation and staining as in (A). The magnified region shows syntelic mis-attachment of individual chromosomes. Many attachments appear to be merotelic, as indicated by centromere stretching. C) Normal bipolar attachment of sister kinetochores to microtubules in condensin-proficient HeLa cells. HeLa cells treated with mismatched siRNA (mis RNAi) were processed for and imaged by transmissive electron microscopy (see Methods). Microtubules (MT) interact with sister kinetochores (KIN) from opposite poles in these control cells. No incidence of merotelic attachments or stretched centromere chromatin was detected (more than 100 kinetochores imaged). The insert schematic depicts the arrangement of sister kinetochores with attached microtubules. Lower magnification (left) shows the entire metaphase plate. D) Condensin depletion results in stretched centromeric chromatin and deformed kinetochore layers, possibly caused by merotelic mis-attachments. An example of two neighboring kinetochores in SMC2-depleted cells (SMC2 RNAi) is shown (right panel). The extreme stretching of centromere chromatin is accompanied by a deformed and rounded kinetochore structure, which possibly indicates some degree of merotelic attachments to both kinetochores. Lower magnification (left panel) shows the typical view of metaphase in condensin-less cells. E) Merotelic mis-attachment is pervasive in condensin-depleted human cells. Two examples of moderate merotelic mis-attachments of kinetochores in hSMC2-depleted cells are shown (right panel). At least half (arrows) of the kinetochore in each case is attached to the opposite pole. The insert schematic shows the arrangement of a merotelic kinetochore with attached microtubules. It is likely only a small fraction of merotelic kinetochores are identifiable in each metaphase, as most are extremely stretched (i.e. probably double-merotelic) based on light microscopy and immuno-EM data (Fig. S3).