Figure 1.
Enzymatic activity and intracellular localization of TbHslVU.
(A). TbHslV contains the conserved threonine and lysine residues (arrows) found essential for the activities of HslV in E. coli [6] and the β-subunits of 20S CP in T. brucei [54]. (B). The ATP-dependent peptidase activity of TbHslV. Wild type and three TbHslV mutants T20A, T21A and K53A were expressed as HA-tagged proteins in T. brucei, immunoprecipitated and assayed for hydrolysis of Cbz-Gly-Gly-Leu-AMC. (C). Cells stably expressing TbHslVU-HA were labeled for mitochondria with Mitotracker green dye (green), immunostained with anti-HA mAb for HA-tagged proteins (red) and counterstained with DAPI for DNA (blue). Arrows indicate the focal points of HA-staining corresponding to the positions of kinetoplasts (arrowheads). (D). Cells expressing TbHslVU-HA with the putative mitochondrial targeting sequences deleted were stained with anti-HA antibody (red) and counterstained with DAPI. Bars: 2 µm.
Figure 2.
RNAi knockdown of TbHslVU affects cell growth and kinetoplast morphology.
(A). Cells were grown in the presence (+) of tetracycline to induce RNAi for 7 days, and cell growth was monitored daily. Northern blots were performed to assess levels of TbHslV, TbHslU1 and TbHslU2 mRNA before (−) and after (+) 2 days of RNAi (insets). (B–E). Un-induced control cells (B) and cells after RNAi induction for 7 days (C) were labeled with YL1/2 antibody for basal bodies (BB, arrowheads) and counterstained with DAPI for nucleus (N) and kinetoplast (arrows). (D). Two TbHslVU RNAi cells at the final stage of cell division were still connected by a thin thread of kinetoplast DNA (arrows) between two basal bodies (arrowheads) in two well-separated cells. Bars: 2 µm. (E). Tabulation of RNAi cells with kinetoplasts in varying sizes and morphologies. Approximately 200 cells were counted at each time point and the data represent averages from three independent experiments.
Figure 3.
TbHslVU RNAi led to heterogeneously sized kinetoplasts.
(A). Flow cytometry analysis of DHE stained cells. A total of 25,000 cells were counted in each experiment (Left panel). DHE stains exclusively the kinetoplasts in control and RNAi cells (Right panel). (B). Southern analysis of changes in minicircle and maxicircle DNA content during TbHslU RNAi. The kinetics of minicircle (open circle) and maxicircle (filled square) accumulation are presented to the right of the Southern blots. (C). DAPI staining of the isolated kDNA networks. (D). Surface areas of the isolated kDNA networks stained with DAPI, and measured with the NIH Image software.
Figure 4.
Electron microscopic examination of kDNA networks from the control and TbHslV RNAi cells.
Methods were described by [38]. (A, B, E, F) kDNA networks from cells after 7 days of TbHslV RNAi. (H) A kDNA network from an un-induced control cell. (C, D) Enlargements of the network in A, corresponding to the areas outlined in white framed boxes. (G) Enlargement of the network in F framed in a white box. The arrow in G indicates a maxicircle. Scale bars for A, B, E, F, and H, 2 µm and for C, D, and G, 0.5 µm.
Figure 5.
Effect of TbHslU1+2 double RNAi on free minicircle replication intermediates.
(A). Total DNA was fractionated on an agarose/ethidium gel and a Southern blot was probed for minicircles. N/G, nicked/gapped minicircles; L, linearized minicircles; CC, covalently-closed minicircles, *, nonspecific hybridization to nuclear DNA. The nicked/gapped minicircles form a doublet with the lower component possibly linearized minicircles. Since it is present prior to RNAi induction (day 0), it is likely unrelated to RNAi. (B). Quantitation (by Phosphorimager) of bands from A. The nicked/gapped species includes both components of the doublet.
Figure 6.
In situ TdT-catalyzed Fluorescein-dUTP labeling in cells after 7 days of TbHslV RNAi.
(A). The control cells (a–f). (B, C). The TbHslV RNAi cells (g–p). Percentages of TdT-labeled cells in control and TbHslV RNAi cells are presented (k). Bars: 2 µm.
Figure 7.
In situ detection of minicircles and maxicircles in the kinetoplast by FISH.
Cells were fixed, probed for minicircles (red) or maxicircles (green), and counterstained with DAPI. kDNA is indicated with a K and nucleus with an N. Bar: 2 µm. FISH does not detect covalently-closed DNA minicircles because they are non-denaturable [24]. This is likely also true for maxicircles. Thus the FISH signal may not be proportional to the total populations of minicircles and maxicircles. Bars: 2 µm.