Tracking the Biogenesis and Inheritance of Subpellicular Microtubule in Trypanosoma brucei with Inducible YFP-α-Tubulin
Figure 6
GCP2-RNAi affects new FAZ extension. Cells with a stably integrated GCP2-RNAi construct were grown with tetracycline to induce RNAi or without as control. To monitor the efficiency of RNAi, GCP2-RNAi cells were transfected to allow transient transfection of YFP-GCP2 (a). Samples were then taken every 24 hours after induction for growth assay ((a); results shown as mean ± SD, ) and immunoblotting with anti-YFP and anti-BiP (inset). For quantitation of cell cycle effects (b), 400 cells were scored for their DNA contents in each of 3 independent experiments and the results shown as mean ± SD. For motility assays ((c), (d)), uninduced control and cells induced for GCP2-RNAi for 48 hours were diluted in fresh medium, imaged at 2 frames/second for 1 minute, and the movement of individual cells tracked (c) and velocity calculated (d). The 2D-tracks of ~60 cells from three independent experiments were generated by in silico tracking on movies. The velocity results are shown as mean velocity ± SEM of 3 independent experiments with 20–25 cells per experiment. The effect of GCP2 depletion on the new FAZ and flagella elongation was monitored in >100 biflagellated cells in control or cells induced for GCP-RNAi for 48 hours ((e), (f)). The length of new FAZ was plotted against corresponding new flagellum length for each cell measured (e). Alternatively, cells were grouped based on new flagellum length range and FAZ length (shown as mean length ± SEM) was plotted against the flagellum length range (f).
