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First published online September 3, 2008
doi: 10.1242/10.1242/jcs.030643

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Centrin4 coordinates cell and nuclear division in T. brucei

Jie Shi¹, Joseph B. Franklin², Jordan T. Yelinek², Ingo Ebersberger^3,4, Graham Warren^2,* and Cynthia Y. He^2,,

¹ Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543
² Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
³ Centre for Integrative Bioinformatics Vienna, Max F. Perutz Laboratories, Dr Bohr-Gasse 9, A-1030 Vienna, Austria
⁴ University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria

View larger version (27K): [in this window] [in a new window]   Fig. 1. Characterization of TbCentrin4. (A) Characterization of antibodies against TbCentrin4. Total lysates containing equal amounts of protein (12 µg) from control procyclic cells (lanes labeled 1) or cells stably expressing TbCentrin4-YFP (lanes labeled 2) were fractionated, separated by electrophoresis and immunoblotted with the indicated antibodies. Note that antibody against GFP (which also recognizes YFP; second panel) recognized the same fusion protein as polyclonal antibodies against TbCentrin4 (third panel), which also recognized two proteins at 14 and 16 kDa (arrows, fourth panel, longer exposure of third panel). These represent endogenous TbCentrin4 and were not recognized by the monoclonal antibody 20H5 (which recognizes co-migrating TbCentrin1 and TbCentrin2; first panel). (B) Maximum-likelihood sequence tree of centrins and calmodulins. Numbers on branches represent bootstrap support values. Branches supported by less than 50% of the bootstrap replicates were collapsed to form a multifurcation. L_major, Leishmania major; L_donovani, Leishmania donovani; CALM, calmodulin; Cen, centrin. We used the centrin nomenclature suggested by Selvapandiyan et al. (Selvapandiyan et al., 2007) with the exception of the TbCentrins (He et al., 2005), which are suffixed `_He' to highlight our results. The numbers on the right indicate the number of predicted EF-hand domains in those proteins grouped by the numbered square brackets.

View larger version (53K): [in this window] [in a new window]   Fig. 2. Localization of TbCentrin4. (A-D) Cells stably expressing TbCentrin4-YFP (green) were fixed and labeled for Golgi (anti-GRASP; red), kinetoplasts (DAPI; small blue dots) and nuclei (DAPI; large blue structures). The outline of each cell is indicated by a white line. Note the presence of TbCentrin4 on both basal bodies (open arrowheads) and the bi-lobe structure (filled arrowheads; large arrowheads indicate the lobe next to the old Golgi, small arrowheads the lobe that becomes associated with the new Golgi), which was closely associated with the Golgi throughout the cell cycle, as previously observed for TbCentrin2. The same cells are shown at higher magnification in supplementary material Fig. S2. (E-G) TbCentrin4-YFP cells were also fixed for cryo-iEM and labeled with an anti-GFP antibody followed by protein-A–gold (10 nm) labeling. TbCentrin4 was found at both the basal bodies (indicated by open arrowheads in E; shown also in F) and the Golgi/ERES region (bracketed region in G). K, kinetoplast; G, Golgi; ERES, ER export site. (H-M) Representative confocal images of a 1K1N (cell containing one kinetoplast and one nucleus) (H-J) and a 2K2N cell (containing duplicated kinetoplasts and nuclei) (K-M) representing early and late cell cycle stages showing that 20H5 (H,K) and anti-TbCentrin4 (I,L) both label basal bodies (open arrowheads) and the bi-lobe structure (filled arrowheads). Scale bars, 5 µm (A-D); 0.5 µm (E,G); 0.2 µm (F); 1 µm (H-M).

View larger version (55K): [in this window] [in a new window]   Fig. 3. Depletion of TbCentrin4 inhibits cell growth. (A,B) Cells with a stably integrated Centrin4-RNAi construct were grown with tetracycline to induce RNAi or without. Samples were taken for cell counting (A; results are given as the mean ± s.d., n=3) and immunoblotting (B) using the indicated antibodies to confirm selective depletion of TbCentrin4, but not TbCentrin1 and TbCentrin2. 12 µg total protein were loaded in each lane.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Generation of zoids through depletion of TbCentrin4, but not TbCentrin2. (A,B) Cells were induced for TbCentrin2-RNAi (A) or TbCentrin4-RNAi (B), and the DNA content of fixed cells was monitored using DAPI staining. Cells were divided into six categories: one kinetoplast and no nucleus (1K0N; zoids), one kinetoplast and one nucleus (1K1N), two kinetoplasts and one nucleus (2K1N), two kinetoplasts and two nuclei (2K2N), one kinetoplast and two nuclei (1K2N), and cells with multiple nuclei (Multinucleated). Results from two independent experiments were averaged for n=500 cells/time point. The number of zoids (1K0N) increased in cells depleted of TbCentrin4 but not of TbCentrin2. (C,D) Cells analyzed by flow cytometry using forward scatter to measure the cell size. 100,000 events were analyzed for each time point. (E,F) TbCentrin2-depletion led to larger cells, whereas depletion of TbCentrin4 generated two populations, the smaller cells mostly zoids (E), the larger cells mostly multinucleated (F). Scale bar, 5 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Zoids and 1K2N cells appear to be produced as siblings. Cells induced for TbCentrin4-RNAi for 24 hours were fixed and labelled with DAPI (nucleus, kinetoplast), 20H5 (basal bodies and bi-lobe) and anti-GRASP (Golgi). (A) A cell near the end of cytokinesis, separating into one daughter cell that contains two nuclei, and the other daughter cell without a nucleus. (B) A cell just after division, one daughter cell with two nuclei, the other with none. (C,D) Both siblings contain basal bodies (open arrowheads), a kinetoplast (arrows), a bi-lobe (closed arrowheads; the large arrowhead indicating the lobe that is associated with the single Golgi stack, the small arrowhead indicating the other lobe) and a Golgi complex (red). Scale bar, 5 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 6. (A-I) Duplication of the FAZ is unaffected by depletion of TbCentrin4. Control (A,B), TbCentrin4-RNAi (C-G) and TbCentrin2-RNAi (H,I) cells were fixed and labeled for FAZ (L3B2; red) and DNA (DAPI; blue). Note that the new FAZ (indicated by triple arrows) in control cells that undergo nuclear division remained attached to the cell body and that cell division initiated only after full segregation of the duplicated nuclei (distance 6 µm; not shown) (see also Robinson et al.,1995). In cells depleted of TbCentrin4 (C,D), the FAZ was the same length in both the 1K1N (control) and 1K0N (zoid) cells (E; n=100, mean ± s.d.). However, cell division initiated even before full segregation of the nuclei (F,G). By contrast, in cells depleted of TbCentrin2, formation of the new FAZ was inhibited (truncated new FAZ indicated by arrow), consistent with the known inhibition of cytokinesis (H,I). Scale bar, 5 µm.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Association of the bi-lobe with the FAZ. Fixed cells were labeled with L3B2 for FAZ (red), anti-TbCentrin4 for bi-lobe (filled arrowheads; green) and basal bodies (open arrowheads; green), and DAPI for nucleus and kinetoplast (blue). At all stages of the cell cycle, the posterior tip of the FAZ overlapped with the bi-lobe, in particular the anterior lobe (large filled arrowheads). Scale bar, 5 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Model describing the effects of depletion of TbCentrin2 or TbCentrin4. In normal cells (middle), duplication and segregation of basal bodies (green), flagella (gray) and kinetoplasts (small blue squares) are followed by division of the nuclei (large blue circles). Cytokinesis initiates at the anterior end of the cell only after full segregation of the nuclei, with the more-posterior daughter nucleus positioned between the segregated kinetoplasts. The cytokinetic furrow forms in between the segregated organelles and partitions them equally into daughter cells. The FAZ (red) is believed to have an important role in the formation of the cytokinetic furrow. In cells depleted of TbCentrin2 (left), duplication/segregation of basal bodies and kinetoplasts is inhibited, whereas the flagellum and nucleus duplicate normally. Formation of the new FAZ is also inhibited, possibly explaining the subsequent inhibition of cell division. In TbCentrin4-depleted cells (right), duplication and segregation of basal bodies, kinetoplasts, flagella and FAZ appear normal. Nuclear division, however, appears delayed relative to cell division, leading to formation of 1K2N and zoid daughters.

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