Fig. 3: MFF modulates melanosome function and morphology.

A Representative SR images of MFF, DRP1, and melanosomes (LAMP1). Scale bar 2 µm, and 1 µm in the magnified ROIs (A, B). Orange arrows point to MFF and DRP1 colocalization in mitochondria, while white (MFF) and yellow (DRP1) arrows indicate the presence of these proteins in melanosomal structures (LAMP1 positive). Representative images of two independent experiments. B Representative Western Blotting image showing the expression level of MFF and DRP1  after silencing in melan-a cells, and the expression levels of melanogenesis-related proteins (five biological replicates). C Representative SR images of HMB45 immunostaining in melan-a cells under the indicated silencing conditions. Scale bar 2 μm. Red arrows highlight melanosomes with different morphology. D, E Quantification of melanosome number and perimeter (n = 4083 cells for control siRNA; n = 42597 cells for Mff siRNA; n = 3846 cells for Drp1 siRNA; n = 4 independent experiments; px = pixel). Mean ± SEM. Kruskal–Wallis test followed by Dunn’s multiple comparisons test, *p = 0.0194. F Representative TEM images of stage I and stage II melanosomes with dysfunctional morphology and lumen buildup in Mff -silenced melan-a compared to control (three biological replicates). Scale bar 1 μm. G Live-cell imaging sequence showing mitochondria (Mitotracker, green) and an elongated melanosomal structure (LAMP1-RFP, magenta) that does not successfully undergo fission (white arrows) in Mff-silenced cells. Scale bar 1 μm. H The fission of melanosomes was observed using time-lapse imaging in both control and Mff -silenced melan-a cells. The number of fission events per cell is shown. n = 20 cells from three independent experiments. Mean ± SEM; two-tailed unpaired Student’s t test, *p = 0.0457. I Representative images of Mff -silenced melanocytes with HMB45 immunostaining: the insets show an increased colocalization of HMB45-positive organelles with melanosomes that are enriched in melanin, as seen in brightfield (BF) images. Scale bar 2 μm. J HMB45 colocalization with melanin was quantified from confocal images of the respective conditions. Mean ± SEM; n = 40 cells from four independent experiments. Two-tailed Mann–Whitney test, *p = 0.0411.