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. 2012 Jan 17;109(5):1661–1666. doi: 10.1073/pnas.1113166109

Fig. 2.

Fig. 2.

Sacsin localizes to mitochondria and regulates mitochondrial morphology. (A) Immunofluorescence panels of cultured hippocampal neurons double-labeled with rabbit antibody against sacsin and mouse monoclonal antibody against cytochrome C (cyto C). Images in Ai and Aii are 10× and 4× zooms, respectively, of the indicated areas from A Top. Images in Aiii are 2.5× zooms from the indicated area in Aii. N, nucleus. Double arrows, arrows, and arrowheads indicate sacsin/cytochrome C colocalization in the soma, dendrites, and axons, respectively. (B) SH-SY5Y cells treated with scrambled (scrm) siRNA or siRNA targeting sacsin were transfected with pAcGFP1-Mito, and defined 2 × 2 μm regions of interest were bleached by using a 488-nm laser line. Recovery was monitored over 20 cycles of imaging with a 1-s interval. Points represent mean ± SEM of FRAP measurements from 32 cells from two independent experiments. (C and D) Control fibroblasts and fibroblasts from two ARSACS patients were prepared for Western blot with polyclonal antibody against sacsin (C), or cells were fixed and processed for immunofluorescence with antibody against TOM20 (green) to reveal mitochondria and with phalloidin Alex647 to stain actin filaments, outlining the cells (D). The areas boxed in D Upper are indicated in a 4× magnification in D Lower. Arrows indicated balloon-like or bulbed mitochondria, characteristic of a hyperfused phenotype (30, 31, 33). (E) Minimally 15 fields (containing from one to five cells) from each cell type from each of three independent experiments were coded and counted blind and independently by two individuals. The combined assessment of the hyperfused status is presented as a mean and SD. (F) SH-SY5Y cells were mock-transfected (control) or were transfected with a FLAG-tagged construct encoding residues 1–1368 of sacsin. Immunoprecipitation (IP) was performed with anti-FLAG antibody, and immunoblots (IB) were performed with anti-FLAG or anti-Drp1 antibodies as indicated. The inputs of FLAG-sacsin and endogenous Drp1 are indicated. (Scale bars: A Top, 20 μm; Ai, 2 μm; Aii, 5 μm; Aiii, 2 μm; D Upper, 10 μm; D Lower, 2.5 μm.)