FIGURE 3:

smFRAP analysis of the turnover of melanosome-associated Mlph in melanocytes. Melanocytes were cultured in glass-bottomed dishes, transfected with a vector expressing GFP-Mlph, and the dynamics of the association of GFP-Mlph with melanosomes were investigated using confocal FRAP analysis (see Materials and Methods). (A–C) Top panels are example images showing the distribution of GFP-Mlph and melanosomes in living wild-type (melan-a) (A), Mlph –/– (melan-ln) (B), and myosin-Va –/– (melan-d) (C) cells, respectively. Bottom panels are high-magnification images taken from example FRAP series captured at the indicated time relative to photobleaching (t = 0). Red arrows highlight the position over time of melanosomes that were selectively photobleached. White boxes in the top panels indicate the parts of cells that were subjected to FRAP analysis and are shown in the high-magnification images below. Scale bars = 20 μm. (D) Line plot showing the fluorescence intensity associated with photobleached melanosomes over time; n = 10 (A), 5 (B), and 5 (C) melanosomes analyzed, respectively.