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First published online 6 September 2005
doi: 10.1242/jcs.02567

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Distinct cytoskeletal modulation and regulation of G1-S transition in the two life stages of Trypanosoma brucei

¹ Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, CA 94143-2280, USA
² Department of Molecular and Cell Biology, University of California, 142 LSA #3200, Berkeley, CA 94720-3200, USA

View larger version (181K): [in a new window]   Fig. 1. Ultrastructural analysis of a CRK1+CRK2-deficient procyclic-form T. brucei cell. (A) The ultrastructure of an elongated and branched posterior end examined by transmission electron microscopy. Cortical microtubule arrays can be seen in the elongated posterior end shown in the rectangle. The extended and branched mitochondrial structure is marked by asterisks. (B) An enlarged view of cytoskeleton in the rectangle in A, showing a bundle of microtubule corset. Bars, 100 nm (A); 0.5 nm (B).

View larger version (51K): [in a new window]   Fig. 2. Mitochondrial staining of CRK1+CRK2-deficient procyclic-form cells. CRK1+CRK2-deficient procyclic form and control cells were stained by MitotrackerTM Green FM (Mitotracker) and DAPI. The single mitochondrion extends from one end to the other of the wild-type cell. An elongated and/or branched mitochondrion is seen filling the elongated/branched posterior end of the CRK1+CRK2-deficient cell.

View larger version (46K): [in a new window]   Fig. 3. Rhizoxin shortens the posterior ends of CRK1+CRK2-deficient procyclic-form cells. Rhizoxin (1 nM) was added to the CRK1+CRK2-deficient procyclic-form cells at the same time as tetracycline. The cells were cultured for 5 days, collected and stained with DAPI, YL1/2 antibody (A) and MitotrackerTM Green FM (B).

View larger version (23K): [in a new window]   Fig. 4. Effects of double knockdowns of CRK1 and CRK2 on the growth and cell cycle progression in bloodstream-form cells. (A) Cloned bloodstream trypanosome cells harboring the CRK1+CRK2 RNAi plasmid construct were incubated in culture medium at 37°C without (-Tet) or with 1.0 µg/ml tetracycline (+Tet). Cell growth was monitored daily and cell numbers plotted on a logarithmic scale. The inset shows the semi-quantitative RT-PCR assessment of intracellular mRNA levels after a 3-day RNAi induction. -Tubulin mRNA (TUB) was included as a sampling control. (B) Samples of the CRK1+CRK2 knockdown cells over 4 days were stained with propidium iodide and subjected to FACS analysis for DNA content. The histograms from the FACScan are presented on the left and the percentages of cells in G1, S and G2-M phases determined by the ModFitLT software are plotted on the right.

View larger version (19K): [in a new window]   Fig. 5. Effect of knocking down CRK1 and CRK2 on the numbers of nuclei and kinetoplasts and BrdU incorporation in bloodstream-form cells. (A) 3 days after RNAi induction, cells were stained with propidium iodide and examined with a fluorescence microscope for tabulation of cells with different numbers of nuclei (N) and kinetoplasts (K). Data are presented as the mean percentage (±s.e.) of total cells counted (>200) from three independent experiments. (B) BrdU incorporation analysis. The double knockdown cells were labeled with BrdU 3-5 days after the initiation of RNAi and examined by immunofluorescence assay.

View larger version (41K): [in a new window]   Fig. 6. Effects from double knockdowns of CRK1 and CRK2 on the morphology of bloodstream-form cells. (A) Cells 3 days after RNAi induction were stained with propidium iodide and examined with a fluorescence microscope. Upper panel, 1N1K, 1N2K and 2N2K control cells without RNAi induction. Lower panel, CRK1+CRK2-depleted 1N1K, 1N2K and 2N2K cells. (B) A CRK1+CRK2-depleted cell stained with YL1/2 antibody, showing the absence of newly synthesized microtubule from the posterior end.

View larger version (31K): [in a new window]   Fig. 7. Effect of triple knockdowns of CRK1, CRK2 and CycE1/CYC2 on the cell cycle progression and morphology of bloodstream-form cells. (A) Cloned cells harboring the CycE1/CYC2+CRK1+CRK2 RNAi plasmid construct were incubated at 37°C without (-Tet) or with 1.0 µg/ml tetracycline (+Tet). Cell growth was monitored daily and cell numbers plotted on a logarithmic scale. The inset shows the semi-quantitative RT-PCR estimation of intracellular mRNA 3 days after RNAi induction with -tubulin mRNA (TUB) included as a sampling control. (B) Cells sampled on day 1 to 4 were stained with propidium iodide and subjected to FACS analysis for DNA content. The histograms from the FACScan are presented on the left, and the percentages of cells in G1, S and G2-M phases determined by the ModFitLT software are plotted on the right. (C) Cells with different numbers of nuclei (N) and kinetoplasts (K). Data are presented as the mean percentage (±s.e.) of total cells counted (>200) from three independent experiments. (D) BrdU incorporation into cells 3-5 days after RNAi induction. Arrows indicate where BrdU is not incorporated into the nuclei. (E) A triple knockdown cell stained with YL1/2 antibody for newly synthesized microtubules: none was found at the posterior end.

View larger version (56K): [in a new window]   Fig. 8. The in vitro differentiation of CRK1+CRK2-deficient and CycE1/CYC2+CRK1+CRK2-deficient bloodstream-form cells into the procyclic form. Cells with RNAi induced for 3 days were initiated for in vitro differentiation. (A) Cell samples were collected at different time points after differentiation initiation and stained on western blot with anti-VSG221 and anti-procyclin antibodies to monitor the disappearance of VSG221 and appearance of procyclin during differentiation. Anti--tubulin antibody was included as a sampling control. (B) Differentiated double knockdown cells (middle panel) and triple knockdown cells (lower panel) were stained with YL1/2 antibody and compared with the procyclic-form CRK1+CRK2 knockdown cells (top panel). Note that only the cells in the top panel were stained by the antibody at the posterior ends. (C) BrdU incorporation into the differentiated double and triple knockdown cells. Note the similar outcomes to those presented in Fig. 5B and Fig. 7D on the bloodstream-form cells.

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