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First published online 29 January 2008
doi: 10.1242/jcs.016741

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Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

¹ Departments of Pathology and Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
² Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, USA
³ BioMedical Research Centre, School of Medicine, Health Policy and Practice, University of East Anglia Norwich, Norfolk, NR4 7TJ, UK

View larger version (30K): [in this window] [in a new window]   Fig. 1. Inhibition of TbSPT2 causes growth arrest in T. brucei. (A) TbSPT2 expression in bloodstream (BF) and procyclic (PC) stage parasites was analyzed by northern blot hybridization. RNAi was induced in a procyclic cell line containing an integrated copy of the RNAi construct pZJM-TbSPT2 by the addition of 1 µg/ml Tet. TbSPT2 RNA levels were compared at day 1 post induction (TbSPT2 +Tet d1) with those in cells of the 29-13 cell line and in uninduced transfectants (TbSPT2–Tet d1). Ethidium bromide (EtBr)-stained gels are shown beneath the northern blots and the positions of molecular size standards are indicated (in kb). (B) The in vitro growth of the TbSPT2 RNAi cell line was assessed in the presence or absence of Tet induction and compared with that of the 29-13 line. Cultures of the three parasite lines were initiated in SDM79 medium at a density of 106 cells/ml and culture densities were determined daily for 6 days.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Sphingolipid biosynthesis is reduced by myriocin treatment or TbSPT2 RNAi. The T. brucei 29-13 cell line was grown in the absence or presence of myriocin (1.5 µM) for 1 day; TbSPT2 RNAi cells were grown for 3 days in the presence or absence of Tet and pulse-labeled with [3H]serine for 40 minutes. Lipids were extracted using the Blight-Dyer method and resolved by TLC and visualized by autoradiography. Each lane contains material from 5x108 cells. Positions of phosphatidylethanolamine (PE) and phosphatidylserine (PS) and ceramide standards are indicated.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Sphingolipid levels are reduced in the TbSPT2 RNAi cell line and in the 29-13 T. brucei cell line treated with myriocin (Myr). Negative-ion mode ESI-MS spectra of total lipids obtained from procyclic T. brucei are shown. 29-13, procyclic 29-13 cell line; 29-13 + Myr, 29-13 cell line treated with 1.5 µM myriocin; TbSPT2 + Tet, SPT2-RNAi-induced transfectant cells; TbSPT2–Tet, SPT2-RNAi-uninduced transfectant cells. m/z, mass to charge ratio; IPCs, inositolphosphorylceramides; PIs, phosphatidylinositols.

View larger version (60K): [in this window] [in a new window]   Fig. 4. Polymorphic phenotype of the TbSPT2 RNAi line. (A) Scanning electron micrographs of TbSPT2 RNAi cells grown for either 3 or 6 days in the presence of Tet. Compared with 29-13 (WT) cells, many TbSPT2 RNAi +Tet cells showed incomplete cytokinesis (Days 3a and 3b). In some TbSPT2 RNAi +Tet cells, the parental and daughter flagella both emerged from the same flagellar pocket, with the daughter flagellum partially detached from the parent flagellum and the cell body (Day 3c). At day 6 post Tet induction, many enlarged TbSPT2 RNAi cells containing polyflagellated extensions were observed (Days 6a and b). (B) DNA content analysis of the TbSPT2 RNAi line with (+) or without (–) Tet over 5 days of treatment. The x-axis shows the DNA content, measured by intensity of fluorescence emission at 440 nm. The y-axis shows the number of cells. The number of cells with a DNA content of 4C increased on days 1-2; number of cells with a DNA content of >4C and <2C increased progressively on days 3-5. The same was observed upon treating the cells with increasing concentrations of myriocin for 24 hours. (C) TbSPT2 RNAi –Tet and +Tet cells at day 3, and 29-13 cells treated with 1.5 µM myriocin for 24 hours were analyzed by immunofluorescence microscopy by using antiserum specific for the paraflagellar rod (ROD1). Cells were also visualized by phase microscopy and by using DAPI staining. (i) control TbSPT2 RNAi–Tet. (ii-v) TbSPT2 RNAi + Tet. (vi-vii) 29-13 cells treated with 1.5 µM myriocin. Both treatment conditions produced similar phenotypes, with TbSPT2 RNAi +Tet cell images being representative of myriocin-treated cells and vice versa. (ii) A cell stuck partway through cytokinesis, undergoing another round of DNA replication. Note that the kinetoplast has already been re-segregated in the upper part of the cell. (iii) Double (0N2K) zoids (left) and single (0N1K) zoids (right) – anucleate cells with two or one kinetoplasts, respectively; the 0N2K-zoid has a partially detached daughter flagellum. (iv) 2N1K* cell (containing an undivided kinetoplast) with a detached daughter flagellum. (v) A 2N1K cell (left) and a 4K2N cell with a detached flagellum (right). (vi) A 2N2K cell with a mispositioned cleavage furrow that can give rise to a zoid and a 2N1K cell. (vii) A 4N3K cell with one undivided kinetoplast, one detached flagellum, and a cytoplasmic tether. Arrows indicate cleavage furrows and cytoplasmic tethers.

View larger version (136K): [in this window] [in a new window]   Fig. 5. Ultrastructural abnormalities of the TbSPT2 RNAi cell line. (A-H) Transmission electron microscopy images of (A) T. brucei 29-13 cells and of (B-H) cells of the TbSPT2 RNAi cell line grown for either 3 days (B,C) or 6 days (D-H) in the presence of Tet. Scale bars, 1 µm. (A) T. brucei 29-13 cell showing an undivided kinetoplast (K) next to the flagellar pocket (FP), a normal nucleus (N) and acidocalcisomes (A). (B) Cell with an abnormally large misshapen kinetoplast. (C) Cell with multiple nuclei. (D) Two cytoplasmic lipid deposits (*), one surrounded by the endoplasmic reticulum and one surrounded by a coiled mitochondrion (M). Mitochondrial distention is observed at the top and bottom aspects of the cell. Acidocalcisomes (center left) appear normal. (E) Multilamellar structure (arrowhead) contiguous with the kinetoplast (K) membrane. (F) Fragmented Golgi complex next to the ER, and several cross-sections of a distended mitochondrion. (G) Coiled multilamellar structure (arrowhead) next to the nucleus above the Golgi. The Golgi cross-section appears normal. (H) Distended flagellar pocket.

View larger version (21K): [in this window] [in a new window]   Fig. 6. TbSPT2 RNAi growth defect and cytokinesis defect can be rescued with 3-KDS, but not ceramide. (A) TbSPT2 RNAi cell lines grown in the presence of Tet were supplemented with either 1 µM 3-KDS or 5 µM C-18 ceramide (Cer) and compared with uninduced (–Tet) RNAi cells. Cultures were initiated in SDM79 medium at a density of 106 cells/ml and culture densities were determined daily for 6 days using a Coulter counter. TbSPT2 RNAi +Tet cells complemented with 3-KDS grow to ten times the number of those without 3-KDS. Ceramide complementation has no effect on cell number. (B) Flow cytometric analysis of DNA content of the TbSPT2 RNAi cell line complemented with 3-KDS and ceramide as described above, shown 3 days post Tet induction. To assess DNA content, timed samples were collected and stained with 20 µg/ml Hoechst dye solution containing 0.1% Triton X-100 and 0.5% formaldehyde, and analyzed using BD LSRII Flow Cytometer. Cell numbers are given at the x-axis, DNA content as measured by intensity of emission at 440 nm is given at the y-axis. A clear decrease in 2C DNA content and an increase in >4C and <2C DNA content is seen with addition of 3-KDS but not ceramide to TbSPT2 RNAi cell line induced with Tet.

View larger version (39K): [in this window] [in a new window]   Fig. 7. Sphingolipid depletion does not affect the localization of calflagin to the flagellum or DRMs in procyclic T. brucei. (A) Procyclic T. brucei were incubated with 1.5 µM myriocin for 24 hours, and TbSPT2 RNAi cell lines were incubated with or without Tet for 3 days. Bloodstream T. brucei were left untreated or treated with 0.5 µM myriocin for 24 hours. The cells were analyzed by DIC for cell morphology, stained with DAPI for DNA content and analyzed by immunofluorescence using an anti-calflagin antibody to visualize calflagin localization. Calflagin localized to the flagellum under all treatment conditions. (B,C) Procyclic 29-13 and TbSPT2 RNAi lines were treated as described above. Bloodstream T. brucei were left untreated or treated with 0.15 µM myriocin for 24 hours. 2x108 cells were harvested and extracted with 1% ice-cold Triton X-100, loaded on the bottom of an OptiPrep gradient, and ultracentrifuged as described in Materials and Methods. OptiPrep fractions were analyzed by western blotting using calflagin-specific serum. Fractions 2 and 3 represent the buoyant DRM fractions of the cell. Calflagin remains associated with the DRM fractions in both myriocin-treated 29-13 and RNAi +Tet cells as well as the controls, but is lost in bloodforms upon treatment with myriocin.

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