|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 2. Deletion of 
1-helix of hFis1 abolishes mitochondrial fragmentation and increases the DLP1-hFis1 interaction. (A) Deletions in the N-terminal cytosolic domain of hFis1 abolished mitochondrial fragmentation. Myc-tagged hFis1 full length (WT) and deletion constructs, hFis1[11-152], hFis1[21-152], hFis1[32-152], hFis1[61-152] and hFis1[92-152] were transfected into Clone 9 cells harboring GFP-labeled mitochondria and cells overexpressing the Myc-tagged proteins were detected by immunofluorescence using the anti-Myc antibody. Two-hundred to 300 untransfected cells and cells transfected with each construct were counted to score mitochondrial morphology. The upper three panels represent the three mitochondrial morphologies used for scoring the different mitochondrial phenotypes. Mitochondria were detected by GFP fluorescence and anti-Myc staining completely overlapped with the GFP signal. While untransfected cells contained normal tubular mitochondria (top left, bottom UnTr), mitochondria in cells overexpressing hFis1-WT were finely fragmented, showing a punctate mitochondrial phenotype (top middle, bottom WT). The number of cells containing fragmented mitochondria decreased as N-terminal amino acids were further deleted. The third phenotype that shows swollen, ball-shape mitochondria (top right) were prevalent in cells overexpressing Myc-hFis1[32-152]. Scale bar, 10 µm. (B) Deletion of the N-terminal region of hFis1 enhances the hFis1-DLP1 interaction. BHK-21 cells were transfected with full length (FL) or N-terminally truncated hFis1 constructs and whole cell extract (WCE) was subjected to immunoprecipitation by anti-DLP1 antibodies. Immunoprecipitated proteins were analyzed by immunoblotting with anti-DLP1 and anti-Myc antibodies. All Myc-tagged hFis1 constructs, FL and deletion mutants, were expressed in cells as detected in the WCE. Myc-hFis1[21-152], Myc-hFis1[32-152], and Myc-hFis1[61-152] were readily detected in immune complexes isolated by the anti-DLP1 antibody while little binding was obtained with cells expressing FL and Myc-hFis1[11-152]. No binding was detected with Myc-hFis1[92-152]. 1% of starting materials and 50% of immunoprecipitated materials were loaded onto the gel for immunoblot analyses.
|
