Figure 4. Kinesin^CaaX localizes near the nucleus and kinetoplast and is enriched in post-mitotic cells.

(A) Left Western blot of whole cell lysates of bloodstream form (BSF) T. brucei tagged HA- Kinesin^CaaX cell line either induced (+) or non-induced (−) for 48 hours. While more material is loaded in the (−) non-induced lane, much more HA-Kinesin is observed in the (+) induced lane. Right Quantitative PCR (qPCR) showed induced cells had 4.2 times more mRNA for Kinesin^CaaX than non-induced controls (error bars are SD). (B) DAPI staining and fluorescence microscopy reveals a larger proportion of cells with 2 kinetoplasts and one nucleus (2K1N) and a reduction in 1K1N cells in tetracycline-induced Kinesin^CaaX T. brucei cells compared with non-induced cells, with the effect becoming prominent at 48 hours (C) The mean and SD of 2 experiments at 48 hours. Statistical analysis was performed using a Student T test. (D) Ectopic expression of Kinesin^CaaX results in a slight decrease in G1 cells and a slight increase in cells between the G1 and G2/M peaks at 48 hours after tetracycline induction. (E) Cells were stained with DAPI to stain nuclear and kinetoplast DNA (blue), anti-HA rat mAb primary and polyclonal anti-Rat IgG Cy5.5 (red) secondary 24 hours post-tetracycline induction. Cells with 2K1N and 2K2N DNA content have an increased amount of Kinesin^CaaX relative to 1K1N cells. (F) Cells were stained with DAPI to stain nuclear and kinetoplast DNA (blue), FITC-anti-mouse IgG to localize the KMX mouse monoclonal antibody (mAb) that binds β- tubulin (green) and anti-HA rat mAb primary and polyclonal anti-rat IgG Cy5.5 (red) secondary that localize HA-Kinesin^CaaX 24 hours post tetracycline induction. β-tubulin localization demonstrates these cells have normal morphology. The cell marked 1Kdiv1N has an elongated kinetoplast consistent with a kinetoplast that is undergoing division.