MBC Supplements

This article contains the following material:

• Video1Fig5A.mov Control mitosis in a GFP? injected cell (Video 1, Fig 5A.mov). PtK2 cells in prophase were injected with GFP? and mitosis was imaged using time-lapse phase contrast microscopy as described in Materials and Methods. Images were acquired at 30 s intervals and are displayed here at a rate of 6 frames/s. This movie demonstrates normal timing and morphology of mitosis in the control injected cell shown in Fig. 5A.
• Video2Fig5B.mov Depletion of centromeric MCAK leads to defects in chromosome congression and segregation (Video 2, Fig 5B.mov). PtK2 cells in prophase were injected with GFP-CEN and mitosis was imaged using time-lapse phase contrast microscopy as described in Materials and Methods. Images were acquired at 30 s intervals and are displayed here at a rate of 6 frames/s. This movie demonstrates the GFP-CEN injected cell featured in Fig. 5B, which had multiple defects in chromosome congression and segregation, including delayed mitosis, chromosomes with difficulties congressing, and a lagging chromatid at anaphase. This movie was stopped before the completion of mitosis in order to view the lagging chromatid. Although it is not shown, cytokinesis proceeded normally.
• Video 3 Depletion of centromeric MCAK results in chromosomes that do not congress before anaphase (Video 3.mov). PtK2 cells in prophase were injected with GFP-CEN and mitosis was imaged using time-lapse phase contrast microscopy as described in Materials and Methods. Images were acquired at 30 s intervals and are displayed here at a rate of 6 frames/s. This movie demonstrates a GFP-CEN injected cell with multiple defects in chromosome congression, such as many chromosomes lingering at spindle poles, chromosomes with difficulty congressing, and a chromosome near the left spindle pole that never congresses before anaphase onset. In addition, this cell has two lagging chromatids and a lagging chromosome at anaphase. This movie was stopped before the completion of mitosis in order to view the lagging chromatids and chromosome. Although it is not shown, cytokinesis proceeded normally.
• Video4Fig8C-K.mov Depletion of centromeric MCAK leads to combined merotelic-syntelic kinetochore-microtubule attachments (Video 4, Fig 8C-K.mov). PtK2 cells in prophase were injected with GFP-CEN and mitosis was imaged using time-lapse phase contrast microscopy as described in Materials and Methods. Images were acquired at 15 s intervals and are displayed here at a rate of 6 frames/s. This movie demonstrates a GFP-CEN injected cell with multiple chromosomes lingering at the spindle poles. This cell was fixed 10 s after the final frame of the movie in order to analyze the near-pole chromosomes by serial-section EM. These chromosomes possessed combined merotelic-syntelic kinetochore-microtubule attachments, which are shown in Fig. 8C-K.
• Video 5 Depletion of centromeric MCAK leads to syntelic attachments that are not resolved before anaphase (Video 5.mov). PtK2 cells in prophase were injected with GFP-CEN and mitosis was imaged using time-lapse phase contrast microscopy as described in Materials and Methods. Images were acquired at 15 s intervals and are displayed here at a rate of 6 frames/s. This movie demonstrates a GFP-CEN injected cell with a chromosome at the right spindle pole that never congresses before anaphase onset. The cell was fixed 10 s after the final frame of the movie to perform serial-section EM for ultrastructural analysis of this chromosome, which had syntelic kinetochore-MT attachments as discussed in the text.

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