JCS030643 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Protein sequence alignment of nine calmodulins and 21 centrins. Predicted positions of the four EF-hand domains are denoted by red boxes and labeled EF-h1 to EF-h4. For EF-hand domains that could not be predicted in individual proteins, the corresponding sequence in the alignment has been crossed out in red. Cg: Candida glabrata; Ci: Ciona intestinalis; Cr: Chlamydomonas reinhardtii; Hs: Homo sapiens; Kl: Kluyveromyces lactis; Ld: Leishmania donovani; Ll: Lilium longiflorum; Lm: Leishmania major; Mm: Mus musculus; Pf: Plasmodium falciparum; Pt: Paramecium tetrauralia; Sc: Saccharomyces cerevisiae; Tb: Trypanosoma brucei.

• Supplemental Figure S2 -

  Fig. S2. Localization of TbCentrin4-YFP. Cells in Fig. 2 (A-D) are shown at higher magnification to better illustrate the localization of TbCentrin4-YFP (green) on both the basal bodies (open arrowheads) and the bi-lobed structure (filled arrowheads). Golgi, red; DNA, blue. Bar, 5 µm.

• Supplemental Figure S3 -

  Fig. S3. Anti-TbCentrin4 does not crossreact with TbCentrin2. Purified GST-TbCentrin2 and GST-TbCentrin4 proteins (10 µg/ml) were used to compete for anti-TbCentrin4 (red) binding in immunofluorescence assays. Only GST-TbCentrin4 inhibited antibody labeling, GST-TbCentrin2 had no effect. Cells were also stained for DNA using DAPI (blue).

• Supplemental Figure S4 -

  Fig. S4. TbCentrin4-RNAi induced with 10, 1 or 0.2 µg/ml tetracycline. Stable TbCentrin4-RNAi cells were grown in the absence or presence of 10, 1 or 0.2 µg/ml tetracycline, to induce RNAi, and samples taken for (A) cell counting (results presented as mean ± s.d., n=3) (B) immunoblotting using the indicated antibodies to confirm selective depletion of TbCentrin4, but not TbCentrins 1 or 2. Each lane contained equal loading of 12 µg total protein. The level of α-tubulin was also monitored and used as loading control. (C) Same cells induced with 1 µg/ml tetracycline were also fixed and stained with DAPI to monitor DNA content. Rapid production of zoids was also observed, similar to that observed in cells induced by 10 µg/ml tetracycline (see Fig. 4B).

• Supplemental Figure S5 -

  Fig. S5. Efficient depletion of TbCentrin4 in zoids, 1K2N and multinucleated (MN) cells. TbCentrin4-RNAi cells induced with 10 µg/ml tetracycline were fixed at 0, 24, 48 and 72 hours post induction, stained with DAPI for DNA (blue) and anti-TbCentrin4 (red) to monitor TbCentrin4 depletion over the course of induction. A z-stack of ten optical sections (at 0.5-µm intervals) spanning the whole cell depth was captured for both fluorescence channels and the images were collapsed for 2D presentation. Corresponding DIC images are shown in the lower panel. Whilst most cells had diminished staining 24 hours post induction or later, some TbCentrin4 staining remained in a few cells having normal DNA content. The cell variation is indicated below each cell.

• Supplemental Figure S6 -

  Fig. S6. Flagella remained attached to cell bodies until late stages of TbCentrin4-depletion. TbCentrin4-RNAi cells induced with 10 µg/ml tetracycline were fixed and stained with DAPI for DNA (blue) and anti-PFRA for paraflagellar rod (a protein complex structure running in parallel to the flagellar axoneme; green) to monitor flagellar morphology. Most flagella remained associated with the cell bodies, in both normal (1K1N, 2K1N and 2K2N) and abnormal cells zoids, 1K2N and multinucleated (MN). Cell variations are indicated below each cell. Only at late stage of the induction, (48 and 72 hours) when cells became increasingly multi-nucleated and more zoids appeared, were more detachments observed (arrows).