|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. Two TPR motifs are important for interaction with DLP1 or DLP1-containing complex. (A) Helix-breaking point mutations in 
1 (L14P), 2 (L42P), and 3 (L58P), 4 (L77P), 5 (L91P), and 6 (L110P) were made in full-length hFis1 and overexpressed in Clone 9 cells to test their effects on mitochondrial fragmentation. The L42P, L58P, L77P, and L91P point mutations in helices forming TPR abolished the mitochondrial fragmentation effectively, showing that less than 15% of the point mutant-expressing cells contained fragmented mitochondria. L14P and L110P point mutations in non-TPR helices were less effective in abolishing mitochondrial fragmentation. (B) Gallery of cells expressing TPR mutations showing the dominant-negative mitochondrial phenotype. These mitochondrial morphologies were found throughout cells overexpressing four TPR mutants. Mitochondria are elongated and often severely entangled around the nucleus indicative of a fission-defective mitochondrial phenotype, suggesting that these point mutants exert a dominant-negative effect. N, nucleus; scale bar, 10 µm. (C,D) Morphometric analyses of mitochondrial elongation in cells expressing TPR mutants. Cell counting revealed that 25-60% of cells expressing TPR mutants displayed the dominant-negative mitochondrial phenotype whereas non-TPR mutants showed no mitochondrial elongation (C). For morphometric analysis, five mutant cells with less severely entangled mitochondria and five untransfected normal cells were selected, and pixel lengths were measured only in clearly discernible mitochondria. Pixel values representing the length of mitochondria were sorted from the longest to the shortest, and the five longest mitochondrial tubules from each cell are presented (D). Note that overall lengths of mitochondrial tubules are longer in mutant cells, showing approximately two-fold increase. (E) 4 and 5 participate in binding DLP1 or DLP1-containing complex. Three helix-breaking mutants L77P, L91P, and L110P in Myc-hFis1[32-152] were tested for DLP1 binding by co-immunoprecipitations. Both L77P and L91P mutations greatly reduced the binding to DLP1, whereas the L110P mutant showed the DLP1 interaction to the level similar to Myc-hFis1[32-152]. 1% of starting materials and 50% of immunoprecipitated materials were loaded onto the gel for immunoblot analyses.
|
