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First published online 20 June 2006
doi: 10.1242/jcs.03023

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Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest

¹ Department of Biology/Cell and Developmental Biology, University of Fribourg, Chemin du Musée 10, CH-1700 Fribourg, Switzerland
² Institute of Parasitology, University of Zurich, Winterthurerstr. 266a, CH-8057 Zurich, Switzerland
³ Ludwig-Maximilians-Universität, Department Biologie I, Genetik, Maria-Ward-Str. 1a, München 80638, Germany

View larger version (43K): [in a new window]   Fig. 1. TbDLP is required for Bax-induced mitochondrial fission. (A) Growth curve of the inducible (–Tet, +Tet) T. brucei TbDLP-RNAi cell line. RNAi was confirmed by northern blot (middle panel). Right panel: Immunofluorescence of induced cells using an inner mitochondrial membrane-specific F1-ATPase antiserum. The fraction of cells showing a fragmented mitochondrion is indicated. Bottom panel: Kinetics of TbDLP ablation tested by co-expression of a HAtagged TbDLP (TbDLP-HA). The tagged protein was detected on immunoblots using anti HA-tag antiserum Y-11 (Santa Cruz Biotechnology). Hours of induction are indicated at the top. The lower half of the panel shows the Coomassie-stained tubulin region and serves as a loading control. (B) Same as A, but data are from a T. brucei cell line allowing inducible expression of human Bax (Crausaz-Esseiva et al., 2004). Bax expression was verified by immunoblot. (C) Same as A, but data are from a cell line allowing inducible expression of Bax and RNAi of TbDLP at the same time. The growth curve for the Bax-expressing cell line, as in B, is shown in grey for comparison. Standard errors (n=3-7) are indicated. Bar, 25 µm.

View larger version (58K): [in a new window]   Fig. 2. TbDLP is required for endocytosis. (A) Ablation of TbDLP by RNAi results in enlarged FPs. FP in living cells were visualized by Fluorescein-conjugated tomato lectin (TLect). Nomarski (Nom) images and the merged pictures of the tomato lectin and the DAPIstaining of uninduced (0 h) and induced cells (24 h) are shown. Bars, 5 µm. (B), Analysis of the TbDLP-RNAi cell line by transmission electron microscopy. Bars, 1 µm. (C) Kinetics of appearance of enlarged FPs and loss of endocytic activity during induction of TbDLP-RNAi. FPs were visualized by AMCA sulfo-NHS labeling of surface proteins as described (Engstler et al., 2004). Enlarged FPs in uninduced (–Tet, ) and induced (+Tet, ) TbDLP-RNAi cells were automatically detected using a series of scripted digital image segmentation steps. Total endocytic activity was measured in the same culture by quantifying the internalized AMCA-labeled surface proteins (+Tet, grey symbols). All values were normalized to the corresponding total cell numbers (n>300 cells) and expressed relative to that of the corresponding uninduced culture.

View larger version (75K): [in a new window]   Fig. 3. TbDLP shows a dual localization. A transgenic T. brucei cell line expressing C-terminally HA-tagged TbDLP was analyzed by immunofluorescence. HA-tagged TbDLP was detected using anti HAtag antiserum Y-11 (Santa Cruz Biotechnology) (red). Nuclear (N) and kDNA (K) were visualized by DAPI-staining (blue). The images show 3D-reconstructions from optical sections obtained by confocal microscopy. (A) Co-staining of HA-tagged TbDLP (red) with an antiserum recognizing Hsp60 (green), a marker protein for the mitochondrial matrix. (B) Same as A, but only the nucleus-kDNA region is shown and co-staining was done with the FP marker tomato lectin (TLect) (green). (C) Same as B, but co-staining was done with the antibody YL1/2 (green), which recognizes tyrosinated -tubulin and serves as a marker for the basal body (BB) (Sherwin et al., 1987). (D) Same as B, but co-staining was done with the monoclonal mouse antibody L3B2 (shown in green), which recognizes the flagellar attachment zone (FAZ) (Kohl et al., 1999). (E) Schematic drawing of the nuclear-kDNA regions of T. brucei. The localization of the fraction of TbDLP that is required for endocytosis is indicated relative to intracellular structures and organelles. CM, cell membrane; FL, flagellum; FP, flagellar pocket; MT, mitochondrion. Bars, 1 µm.

View larger version (52K): [in a new window]   Fig. 4. Lack of TbDLP leads to a specific arrest of cytokinesis. (A) Analysis of nuclei and kDNA configurations of DAPI-stained cells during induction of TbDLP-RNAi. The graph indicates the percentages of cells containing the indicated numbers of nuclei and kDNAs (1K1N, 2K1N, 2K2N and others; n>1000 cells). Percentages of NKKN-cells, a subgroup of 2K2N cells where the two kDNAs are localized between the two nuclei, are also indicated. (B) NKKN-cells have a single mitochondrion. 3D-reconstruction from optical sections obtained by confocal microscopy of an anti-Hsp60 (green) and DAPI co-stained (blue) NKKN cell from the induced TbDLP-RNAi cell line. (C) NKKN cells have enlarged FPs. Visualization of FPs was done as in Fig. 2A. (D) Same as B, but staining was with the antibody L3B2 (green), which recognizes the flagellar attachment zone (FAZ) (Kohl et al., 1999). Bars, 2.5 µm.

View larger version (18K): [in a new window]   Fig. 5. Lack of TbCLH impairs endocytosis but not cytokinesis. (A) Growth curve of the uninduced and induced TbCLH-RNAi cell line. (B) Kinetics of appearance of enlarged FPs and loss of endocytic activity during induction of TbCLH-RNAi. Analysis was performed as in Fig. 2C. (C) Analysis of nuclei and kDNA configurations of DAPI-stained cells during induction of TbCLHRNAi carried out as in Fig. 4A.