Novel roles for the flagellum in cell morphogenesis and cytokinesis of trypanosomes

Abstract

Flagella and cilia are elaborate cytoskeletal structures conserved from protists to mammals, where they fulfil functions related to motility or sensitivity. Here we demonstrate novel roles for the flagellum in the control of cell size, shape, polarity and division of the protozoan Trypanosoma brucei. To investigate the function of the flagellum, its formation was perturbed by inducible RNA interference silencing of com ponents required for intraflagellar transport, a dynamic process necessary for flagellum assembly. First, we show that down-regulation of intraflagellar transport leads to assembly of a shorter flagellum. Strikingly, cells with a shorter flagellum are smaller, with a direct correlation between flagellum length and cell size. Detailed morphogenetic analysis reveals that the tip of the new flagellum defines the point where cytokinesis is initiated. Secondly, when new flagellum formation is completely blocked, non-flagellated cells are very short, lose their normal shape and polarity, and fail to undergo cytokinesis. We show that flagellum elongation controls formation of cytoskeletal structures (present in the cell body) that act as molecular organizers of the cell.

Fig. 1. TbDHC1b and TbIFT88 are required for flagellum assembly. (A) Alignment of protein sequences of the DHC1b central domain from T.brucei (Tb), Chlamydomonas reinhardtii (Cr), Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Homo sapiens (Hs) and the cytoplasmic dynein from Saccharomyces cerevisae (Sc). Conserved residues are shown in blue, and green and red blocks indicate dynein and DHC1b signatures, respectively. (B) RT–PCR analysis performed on total RNA extracted from non-induced and 48 h-induced (TbDHC1b) RNAi or (TbIFT88) RNAi mutants using specific primers for TbDHC1b, TbIFT88 or an unrelated RNA as a control. Knockdown of TbDHC1b (C) or of TbIFT88 (D) lead to production of non-flagellated cells over time. Cells were induced for the indicated periods and stained with the anti-PFRA antibody L8C4 as a marker of the flagellum (green), and with DAPI (blue) showing nucleus and kinetoplast. (E) RNAi was induced in (TbDHC1b) RNAi or (TbIFT88) RNAi cell lines, and cells were fixed at the indicated times and processed for immunofluorescence with anti-PFRA. Cells possessing at least one flagellum were scored as positive. Arrows indicate when non-flagellated cells were first detected. (F) In the same experiment, cell growth was monitored in the presence (+) or absence (–) of tetracycline.

Fig. 2. Non-flagellated trypanosomes lose cell shape and polarity. (A and B) Cells were stained with the L8C4 antibody (green) and with DAPI (blue). (A) A non-induced (TbDHC1b) RNAi cell showing the typical trypanosome polarity. The asterisk indicates the posterior end of the cell. (B) (TbDHC1b) RNAi trypanosome induced for 48 h without a flagellum did not exhibit normal cell shape. (C) Electron micrograph of a (TbDHC1b) RNAi non-flagellated cell (48 h induction), revealing the absence of polarity and partial microtubule disorganization. (D and E) Phase contrast images of trypanosomes stained with DAPI (blue, left panels) and with anti-clathrin antibodies (red, right panels). Non- induced (TbIFT88) RNAi cells (D) showed a defined signal at the posterior end, whereas non-flagellated (TbIFT88) RNAi cells induced for 72 h (E) showed dispersion of clathrin vesicles throughout the cytoplasm. (F–H) Non-flagellated trypanosomes exhibit abnormal cell growth. Phase contrast images of (TbIFT88) RNAi trypanosomes stained with DAPI (blue, left panels) and with the anti-tyrosinated tubulin antibody YL1/2 (yellow, right panels). (F) Non-induced trypanosome, showing normal polarized cell growth that takes place by microtubule elongation at the posterior end (arrows). The arrowheads indicate the position of the new flagellum. (G and H) (TbIFT88) RNAi cells induced for 72 h: incorporation of tyrosinated tubulin was seen throughout the cytoskeleton (G) or at opposite ends (H).

Fig. 3. Flagellum length controls cell size. (A and B) Field of (TbDHC1b) RNAi trypanosomes induced for 48 h. Flagellum was labelled with L8C4 (green), FAZ filament with L6B3 (red lines), basal body with BBA4 (red spots) and DNA was stained with DAPI (blue). (A) DIC image; (B) merged fluorescence. Cells with a shorter flagellum appeared smaller (arrows) than cells with a normal flagellum (arrowheads). (C, D and F) (TbDHC1b) RNAi trypanosomes were non-induced (black squares), or induced for 24 h (blue) or 48 h (red). (C) Mononucleated cells. Cell body size was measured and plotted versus the measured flagellum length. Each point represents an individual cell. (D) In the same uniflagellated trypanosomes, the distance between the anterior tip of the cell and the kinetoplast was measured and plotted versus flagellum length. Non-flagellated cells were not included, as recognition of the posterior/anterior end is virtually impossible. (E) Two models for the generation of small cells with short flagella. The nucleus is shown in blue, the flagellum in green, the FAZ in red and the basal body/kinetoplast (shown as a single circle for simplicity) in black. The asterisk indicates the posterior end. Normal cell size of a binucleated (‘2K2N’) or of a uninucleated (‘1K1N’) trypanosome is delimited by the dashed lines. Point of cleavage initiation is indicated by the thick arrow and probable cleavage progression by the dashed arrow (see text). (F) Binucleated cells. Cell size was measured and plotted versus new flagellum length.

Fig. 4. New and old flagella are required for cell morphogenesis. (TbDHC1b) RNAi cells are shown by DIC and (TbIFT88) RNAi cells are shown by phase contrast. Cell cycle of normal, non-induced, (TbDHC1b) RNAi trypanosomes (A–C), of 48-h-induced (TbDHC1b) RNAi (D–F) and (TbIFT88) RNAi (G–I). Flagellum was labelled with L8C4 (green), FAZ filament with L6B3 (red lines), basal body with BBA4 (red spots) and DNA was stained with DAPI (blue). Top panels, DIC merged with flagellum staining (green); bottom image, merged fluorescence. Yellow arrows indicate kinetoplast DNA position. After 48 h of induction, trypanosomes cannot assemble a new flagellum, but nevertheless formation of a short new FAZ (tip indicated by the white arrowhead) is observed. (J and K) Non-flagellated (TbIFT88) RNAi cells (induced for 72 h) stained with the anti-basal body BBA4 and anti-FAZ L6B3 (both in red), and with DAPI (blue). Kinetoplasts segregation failed, even after completion of nuclear mitosis (K). (L) Non-flagellated (TbDHC1b) RNAi cells (induced for 72 h) stained with the anti-basal body BBA4 and anti-FAZ L6B3 (both in red), and with DAPI (blue). Although basal body duplication and segregation occurred, no new FAZ formation was detected. (M and N) After 72 h of induction, flagellated cells were immunolabelled using antibody 1B41 (red) and stained with DAPI (blue), showing lack of assembly of FAZ-associated microtubules, accumulation of patches of material around the basal bodies area and absence of cytokinesis.

Fig. 5. The FC in wild-type and in induced (TbIFT88) RNAi cells. Normal wild-type (A–D) and (TbIFT88) RNAi trypanosomes induced for 36 h (E–F) were stained with an anti-FC antiserum. Left panels, phase contrast image merged with DAPI staining (blue). Central panels, FC staining only; right panels, magnification of the area surrounding the FC. Top, phase contrast image; bottom, phase contrast image merged with FC staining (green). Stars, tip of the old flagellum; arrows, tip of the new flagellum. (A) The left cell has only one flagellum, and its tip is not stained. The right cell has an elongating new flagellum and its distal tip shows defined staining. In contrast, the distal tip of the old flagellum is not stained. Similar observations were made as the new flagellum elongated (B–D) and stained with the anti-FC antiserum (green). (E) Binucleated cell whose new flagellum is too short (compare with D). (F) Binucleated cell without a new flagellum. FC is still present on the old flagellum of ∼30% of these cells. (G) Electron micrograph of a negatively stained cytoskeleton of (TbDHC1b) RNAi non-induced trypanosome. The discrete pyramidal structure of the FC is shown in the enlarged box. (H) Electron micrograph of a negatively stained cytoskeleton of (TbDHC1b) RNAi trypanosome induced for 48 h. The old basal body lies adjacent to the flagellum (enlarged left box), whereas no flagellum at all is visible on the new basal body (enlarged right box). In these conditions, the FC is not detected.