Figure 4.

Kinetochores with depleted levels of Nuf2 and Hec1 move erratically and in a microtubule-dependent manner. HeLa cells transiently expressing CENPB-GFP were mock siRNA transfected (A and A′) or transfected with Nuf2 siRNA (B and B′). Forty-eight hours after siRNA transfection, cells were imaged on a spinning disk confocal microscope. Images were collected every 10 s over a period of 10-40 min. For microtubule depolymerization experiments, mock-transfected cells were incubated with 20 μM nocodazole for 2 h before imaging (C and C′). Individual kinetochores and their corresponding sisters were tracked using the “track points” function accessed using the Metamorph software analysis system (Universal Imaging), and points were plotted on X and Y axes (A-C). Insets show a maximum projection of a z-series stack through a cell from each corresponding condition (scale bars, 5 μm). Montages in A′-C′ are frames from the time-lapse movies at 10-s intervals showing a single representative sister kinetochore pair from each condition (scale bars, 1 μm).