JCS03023 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Expression of the dominant-negative TbDLP (TbDLP-K39A) inhibits Bax-induced mitochondrial fission. (A) Growth curve of a T. brucei cell line allowing inducible overexpression of TbDLP. (B) ) T. brucei cell line allowing inducible expression of TbDLP-K39A. (C) Cell line allowing inducible coexpression of Bax and TbDLP-K39A. Expression of Bax was verified by immunoblot. The growth curve for the Bax-expressing cell line (as in Fig. 1B), is shown in grey for comparison. Right panels, immunofluorescence as in Fig. 1. Standard errors (n=3-7) are indicated. Bar, 25 μm.

• Supplemental Figure 2 -

  Fig. S2. Expression of the dominant TbDLP-K39A inhibits endocytosis. (A) Overexpression of TbDLP-K39A results in enlarged FPs. FPs in living cells were visualized by fluorescein-conjugated tomato lectin (TLect). Nomarski (Nom) images and the merged images of the tomato lectin and the DAPI-staining of uninduced (0 h) and induced cells (14 h). Bars, 5 μm. (B) Kinetic of appearance of enlarged FPs and loss of endocytic activity after induction of TbDLP-K39A expression. Visualization of the FP in uninduced and induced cells was done by AMCA sulfo-NHS labeling of surface proteins as described (Engstler et al., 2004). Enlarged flagellar pockets in uninduced (−Tet, white diamonds) and induced (+Tet, black diamonds) TbDLP-K39A-expressing 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 the one of the corresponding uninduced cultures. Single-cell analysis showed that 89.8±6.2% of cells having an enlarged FP were defective in endocytosis, whereas 87.4±7.6% of cells with a normal FP showed normal endocytic activity.

• Supplemental Figure 3 -

  Fig. S3. Expression of the dominant TbDLP-K39A leads to a specific arrest of cytokinesis. (A) Analysis of nuclei and kDNA configurations of DAPI-stained cells during induction of TbDLP-K39A expression. 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, is 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 TbDLP-K39A-expressing cell line. (C) NKKN cells have enlarged FPs. Visualization of the FPs was as described in Fig. 2A. Bars, 2.5 μm.