Reilein AR , Tint IS , Peunova NI , Enikolopov GN , Gelfand VI .

GM 52111 NIGMS NIH HHS , R01 GM052111 NIGMS NIH HHS

	Figure 1. Inhibition of PP2A by the small t antigen blocks melanosome aggregation. (A) Melanophores transfected with plasmids encoding the small t antigen or GFP were treated with 10 nM melatonin for 90 min and fixed with formaldehyde. Cells expressing the small t antigen were identified by immunofluorescent staining with an antibody against the SV-40 small t antigen. The vertical axis shows the percentage of cells that were scored as aggregated (white), partially dispersed (grey), or dispersed (black). Scoring was done as described in Materials and Methods. Note the almost complete inhibition of pigment aggregation in the small t-expressing cells. The data are from over 400 small t-expressing and over 500 GFP-expressing cells from three separate experiments. (B) A cell expressing small t maintained its pigment in a dispersed state after melatonin treatment (left), whereas a GFP control cell aggregated pigment normally (right). The cells are shown by overlaying bright-field images onto fluorescent images. Bar, 20 μm.
	Figure 2. Overexpression of PP2A inhibits melanosome dispersion at low levels of cAMP. Cells transfected with plasmids encoding the HA-tagged PP2A catalytic subunit or GFP were treated with 25 μM dexamethasone for 24 h to induce PP2A expression. Cells were then treated with 10 nM melatonin for 1 h to aggregate pigment, followed by 1 nM or 10 nM MSH for 30 min. PP2A-expressing cells were identified by immunofluorescent staining with an antibody to HA, and 100 cells were scored per treatment. The data is from a typical experiment; similar results were obtained in three independent experiments.
	Figure 3. Phase-contrast images of nontransfected cells (A), and cells transfected with the PKA inhibitor plasmid (B), or with the plasmid encoding the inactive analogue of the inhibitor (C). After 72 h, a large percentage of cells in the culture expressing the active inhibitor had fully aggregated pigment. Control cells maintained their melanosomes in the dispersed state. Bar, 50 μm.
	Figure 4. Quantitative analysis of the effects of the active and inactive PKA and PKC inhibitor peptides on melanosome aggregation and dispersion. Cells expressing the PKA inhibitor aggregated their pigment and could not disperse it when treated with MSH. These cells partially dispersed melanosomes in response to PMA. Cells expressing the inactive PKA inhibitor dispersed melanosomes normally in response to MSH and PMA. Cells expressing the PKC inhibitor dispersed pigment when treated with MSH, but were inhibited from dispersing pigment after aggregation with melatonin and treatment with PMA. Percentages are from scoring 200 cells per treatment. Similar results were obtained in at least three other independent experiments with each inhibitor.
	Figure 7. Immunoprecipitations of kinesin II and cytoplasmic dynein from 32Pi- labeled melanophore extract. Left lane, cell extract. Immunoprecipitates of dynein from melatonin-treated and MSH-treated cells show strong phosphorylation at the molecular weights of the dynein intermediate chain (83 kD) and the dynein heavy chain, and at an unidentified band at 200 kD (arrowheads). Immunoprecipitates of kinesin II from melatonin-treated and MSH-treated cells have phosphorylation in a broad band at 95 kD (bracket). No consistent phosphorylation differences have been observed in immunoprecipitations of dynein or kinesin II.
	Figure 6. Phosphorylation of melanosome proteins in cells treated with 10 nM melatonin, 100 nM MSH, 0.1 μM PMA, or 1 mM IBMX. Phosphorylation differences were reproduced in three separate experiments. (A) Cells were labeled with 32Pi for 21 h before melanosome purification. The differences in phosphorylation are found in the 85–95 kD area. Melanosomal proteins from melatonin-treated cells show phosphorylation at ∼86, 92, and 95 kD (arrows), whereas proteins from MSH- and IBMX-treated cells are phosphorylated at 87 and 94 kD. Proteins from untreated and PMA-treated cells show phosphorylation in a broad band from 88–95 kD. (B) Western blot of melanosomal proteins with the anti-phosphothreonine antibody. The pattern of threonine phosphorylation looks very similar in the mol wt range 85–95 kD to the pattern of total phosphorylation in A. There is less phosphorylation on melanosomes purified from cells treated with IMBX and MSH than on melanosomes purified from cells treated with melatonin, whereas it appears that untreated and PMA-treated cells are in a phosphorylation state intermediate between that of aggregating and dispersing cells. In addition, phosphorylation of the band at 50 kD is reduced with melatonin treatment.

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