FIG 5.

Overexpression of TbKAP6 arrested cell growth and caused kDNA loss and segregation defects in some cells. (A) Effects on cell growth. The cumulative cell count on the y axis is the measured value times the dilution factor. Inset, Northern blot of TbKAP6 mRNA isolated from uninduced cells or cells induced for overexpression for 1 day and 2 days. Tubulin mRNA is probed as the loading control. (B) Effects on kDNA morphology changes following overexpression, shown by DAPI staining of uninduced cells (day 0) and cells induced for overexpression for 2, 4, and 6 days. N, nucleus; k, kinetoplast DNA. Star, kinetoplasts that underwent asymmetric segregation. Bar, 5 μm. (C) Kinetics of kDNA loss during a 6-day course of overexpression. At each time point, 400 to 600 randomly chosen DAPI-stained cells were evaluated. (D) Effects on kDNA segregation, shown by DAPI staining of uninduced cells (day 0) and cells induced for overexpression for 2 and 4 days. Arrows, the larger daughter kinetoplast from asymmetric segregation; arrowheads, the smaller daughter kinetoplast from asymmetric segregation. Bar, 5 μm. (E) Bar graphs showing increasing kDNA segregation defects during a 6-day course of overexpression. At different time points, 40 to 100 2K cells were analyzed. White bar, cells with normal symmetric segregation of sister kDNAs; black bar, cells with kDNA segregation defects.