Studies on the mechanism of melanosome secretion

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Membrane traffic describes the flow of both lipids and proteins between intracellular compartments. This process is crucial for the establishment and maintenance of the distinct nature of these organelles and is mediated by small vesicles. In addition, proteins synthesized in the cell can be extruded via the secretory pathway where vesicles fuse with the plasma membrane. Such secreted proteins can be taken up by neighboring cells as is the case with the pigment melanin. This pigment is synthesized in specialized cells called melanocytes and stored in a unique organelle called the melanosome. These organelles are transported to the cell periphery and by as as yet unidentified mechanism transfer their melanin to neighboring keratinocytes. The melanin serves to protect the keratinocyte DNA from the harmful effects of ultraviolet radiation. The exact mechanism whereby melanin is transferred from melanocytes to keratinocytes is unknown and it remains unclear if melanin or melanosomes are actually transferred. This application proposes to use a two-pronged approach to address this problem by (i) examining factors known to be involved in this process as well as by (ii) taking an unbiased approach by setting up an RNA interference-based screen. Both approaches rely on the observation that failure of the B16 melanocyte cell line to release melanin results in the accumulation of the pigment within the cells and such "darkened" cells can be selectively isolated from a mixed population of cells by flow cytometry. The screen will involve expression of random interfering RNAs, sorting of the melanin-accumulating cells by flow cytometry and then using a bioinformatics approach to identify the protein that was knocked-down. It is anticipated that this approach will identify components involved in many different phases of melanosome biology as any defect that prevents their movement or release will lead to darkened cells. Proteins identified by the screen or by the more directed approach will be validated by a variety of biochemical and cell biological methods. In addition, this screen can be adapted to the study of other secretory pathway processes making it widely applicable to the study of many aspects of protein secretion.