FIGURE 3.
Plasmodiumhomologue of the Mini-Chromosome Maintenance Complex Binding Protein (PfMCMBP) depletion causes abnormal nuclear morphology with additional MTOC ectopic foci and formation of extended mitotic spindle microtubules. (A) Representative pictures from [+]/[−] Shld1 PfMCMBP3HADDparasites (40-44 h.p. i.) after PFA-fixation and probing with mouse anti-CrCen1 (1:500, green) and mouse anti-αTubulin (1:200, red) followed by isotype-specific goat anti-mouse antibodies. Three different z-slices from a single representative [+] Shld1 parasite are shown in rows i-iii. Panels iv-vi represent three different PfMCMBP-deficient parasites cultured [−] Shld1. Images were taken with LSM880 Airyscan using the 63× Objective. Yellow arrows indicate bipolar spindle and yellow asterisk indicate short microtubule between duplicated MTOC in WT condition. Scale bar at 1 μm. (B) The length of mitotic microtubules was obtained using ImageJ software and (C) the count of bipolar spindles per cell was done using the Nikon E800 microscope. Schizonts with three or four nuclei and at least one bipolar microtubule were selected for analysis. Two biological replicates with 50 schizonts per replicate were counted. Values that were significantly different (p < .0001) by unpaired t-test are indicated by asterisk (n = 2, mean with ±SD error bars)