JCS010207 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1A
• Supplemental Figure S1B -

  Fig. S1. Depletion of EB1 protein by siRNA oligonucleotide transfection of wild-type melan-ink melanocytes. Wild-type melanocytes were transfected with the indicated siRNA oligonucleotides and depletion of protein analysed 72 hours later (as described in Materials and methods). Panel A upper part shows the result of siRNA depletion of EB1 and Mlph upon a population of cells as determined by immunoblotting of cell lysates using the indicated antibodies as well as the expression level of the control protein actin. The lower part is a quantification of the level of depletion by siRNA transfection. For this each transfection was carried out in triplicate and then the average ratio of band intensities for EB1 and Mlph versus actin was determined and expressed as a percentage of the control siRNA transfected cells. For B, cells transfected with the indicated siRNAs were stained with antibodies specific for EB1 and Mlph, merged fluorescence images show the EB1 (green) and Mlph (red) and transmission images showing the distribution of melanosomes. Apparent localisation of Mlph to the nucleus is an artefact resulting from MeOH fixation as the same staining pattern is observed in melan-ln cells that lack Mlph. Bar, 20 μm.

• Supplemental Figure S2 -

  Fig. S2. Immunofluorescence microscopy of primary cultured melanocytes reveals Mlph requires Rab27a but not MyoVa for stability and melanosome targeting. Primary cultures of melanocytes derived from Rab27a null (ashen) and MyoVa null (dilute) mutant mice were fixed and stained using Mlph-specific antibodies and the distribution of melanosomes (left-hand images) together with Mlph was observed (right-hand). The bottom panels are high magnification images that show the colocalisation of Mlph on melanosomes in dilute primary melanocytes. Bar, 20 μm.

• Movie 1 -

  Movie 1. Overlaid movie of the movements of EB1-EGFP comets and melanosomes in wild-type melanoctyes. Wild-type melanocytes were transiently transfected with plasmid allowing expression of EB1-EGFP. 18 hours later the movements of the fluorescent protein and melanosomes were observed using sequential TIRFM and phase contrast time lapse microscopy. The series of phase contrast images were inverted and false coloured and then individual frames were merged with corresponding TIRFM frames allowing simultaneous visualisation of the movements of EB1-EGFP comets (green) and melanosomes (red).

• Movie 2 -

  Movie 2. Overlaid movie of the movements of EB1-EGFP comets and melanosomes in melan-ln melanoctyes. As described for Movie 1, melan-ln melanocytes were transiently transfected with plasmid allowing expression of EB1-EGFP and then the movements of EB1-EGFP comets (green) and melanosomes (red) were recorded.