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Fig. 4. CED-9-altered mitochondria can be partially suppressed by co-expression of DRP-1. (A) Co-expression of DRP-1 and CED-9 from the same transgene resulted in three classes of mitochondrial morphology: tubular, fragmented and interconnected (percentages represent the mean value of three independently generated transgenic lines; raw data is presented in C and Table S1 in supplementary material). These three phenotypes suggest that increased DRP-1 expression is able to partially suppress the effects of CED-9 on mitochondria, or vice-versa. n, nucleus. Bar, 10 µm. (B) Structure of CED-9 (yellow surface) bound to a portion of the BH3 containing protein EGL-1 (blue ribbon) (1TY4.pdb). In the gain-of-function ced-9(n1950sd) allele, glycine 169, which resides in the CED-9 BH3 binding pocket, is mutated to glutamate (G169E). This mutation has been reported to decrease EGL-1 binding to CED-9 (del Peso et al., 2000). (C) Co-expression of DRP-1 and CED-9(G169E) from the same transgene also resulted in three classes of mitochondrial morphology. (D) Each point represents the proportion of cells with interconnected mitochondria from an independently generated transgenic line; the mean value is indicated by a horizontal bar, and error bars represent the standard error. No mean was calculated for the CED-9(G169E) construct because of the apparent bimodal distribution. This variation among lines expressing CED-9(G169E) was examined by western blot analysis (Fig. S4 in supplementary material). The difference in proportions between co-expression of DRP-1 with CED-9 and co-expression of DRP-1 with CED-9(G169E) is significant, z=2.59, P<0.01. (E) The GTPase activity of 2.5 µM human DRP1 was assayed using a continuous assay coupled to a regenerative system. (F) The amount of GTP hydrolyzed by 2.5 µM human DRP1 with 12.5 µM CED-9 after 40 minutes (296±23 µM) was significantly higher than the amount of GTP hydrolyzed by 2.5 µM human DRP1 alone after 40 minutes (221±17 µM), t(4)=4.56, P<0.025.
