Article Figures & Data
Figures
- FIG. 1.
Effects of double CRK knockdowns on the growth of procyclic-form T. brucei cells. Cloned procyclic trypanosome cells harboring the double CRK RNAi plasmid constructs were each incubated in culture medium containing 1.0-μg/ml tetracycline (+Tet) at 26°C. Cell growth was monitored daily, and the cell numbers plotted in a logarithmic scale. The insets show the intracellular mRNA levels in the cells after a 3-day RNAi induction monitored by semiquantitative RT-PCR. Quantitation of α-tubulin mRNA (TUB) was included as a sampling control.
- FIG. 2.
Effects of double CRK knockdowns on the growth of bloodstream-form T. brucei cells. Cloned bloodstream trypanosome cells harboring the double CRK RNAi plasmid constructs were each incubated at 37°C in culture medium containing 1.0-μg/ml tetracycline (+Tet) at 37°C. Cell growth was monitored daily, and the cell numbers plotted in a logarithmic scale. The insets show the intracellular mRNA levels in the cells after a 3-day RNAi induction monitored by semiquantitative RT-PCR. Quantitation of α-tubulin mRNA (TUB) was included as a sampling control.
- FIG. 3.
FACS analysis of double CRK-deficient procyclic-form T. brucei cells. Time samples of RNAi-induced procyclic-form T. brucei cells were stained with PI and subjected to FACS analysis for DNA content. The histograms from FACScan are presented on the left-hand side of each panel. The percentages of cells in G1, S, and G2/M phases were determined with ModFitLT software and plotted on the right-hand side of each panel.
- FIG. 4.
FACS analysis of double CRK-deficient bloodstream-form T. brucei cells. Time samples of RNAi-induced bloodstream-form T. brucei cells were stained with PI and subjected to FACS analysis for DNA content. The histograms from FACScan are presented on the left-hand side of each panel. The percentages of cells in G1, S, and G2/M phases were determined with ModFitLT software and plotted on the right-hand side of each panel.
- FIG. 5.
The morphological phenotype of CRK3 + CRK2-deficient procyclic-form T. brucei cells. The procyclic-form T. brucei cells 5 days after RNAi induction for knocking down expression of CRK3 + CRK2 were stained with PI and examined under a fluorescence microscope. (A) Quantification of cells with different numbers of nuclei and kinetoplasts. N, nucleus: K, kinetoplast. (B) Upper panel, control 1N1K, 1N2K, and 2N2K cells without RNAi induction. Lower panel, CRK3 + CRK2-knockdown cells showing the 1N*1K, 1N*2K, and 0N1K (zoid) phenotypes.
- FIG. 6.
Morphological phenotype of CRK3 + CRK2-deficient bloodstream-form T. brucei cells. The bloodstream-form T. brucei cells 3 days after RNAi induction for knocking down expression of CRK3 + CRK2 were stained with PI and examined under a fluorescence microscope. (A) Quantification of cells with different numbers of nuclei and kinetoplasts. N, nucleus: K, kinetoplast. (B) Upper panel, control 1N1K, 1N2K, and 2N2K cells without RNAi induction. Lower panel, the CRK3 + CRK2-knockdown cells showing 1N*2K and XNXK phenotypes.
- FIG. 7.
Double immunofluorescence assay of CRK3 + CRK2-deficient bloodstream-form T. brucei cells. The bloodstream-form T. brucei cells 3 days after CRK3 + CRK2 RNAi induction were stained with DAPI for DNA, YL1/2 for tyrosinated α-tubulin, and ROD1 for the PFR and examined under a fluorescence microscope. (A) A 1N1K control cell without RNAi induction. (B) CRK3 + CRK2-deficient XNXK cells. ROD1 stained the flagellum, whereas YL1/2 stained the basal body and the newly assembled microtubules.
- FIG. 8.
Double immunofluorescence assay of CRK3 + CRK2-deficient procyclic-form T. brucei cells. The procyclic-form T. brucei cells 5 days after CRK3 + CRK2 RNAi induction were stained with DAPI for DNA, YL1/2 for tyrosinated α-tubulin, and ROD1 for the PFR and examined under a fluorescence microscope. (A) A 1N1K control cell without RNAi induction. (B) CRK3 + CRK2-deficient 1N*1K and 1N*2K cells and zoids. (C) A CRK1 + CRK2-deficient procyclic-form 1N1K cell with elongated and branched posterior ends. ROD1 stained the flagellum, whereas YL1/2 stained the basal body and the newly assembled microtubules.
- FIG. 9.
A model showing distinctive responses to G2/M arrest by the procyclic and the bloodstream forms of T. brucei. SN, nuclear S phase; M, mitosis; C, cytokinesis. Note the location of kinetoplasts in the midregion of the procyclic form but in the far posterior end of the bloodstream form. Note also in the controls that the two nuclei are partitioned between the two kinetoplasts prior to cytokinesis in the procyclic form. No such partition occurs in the bloodstream form.
