THE DEVELOPMENT OF NOVEL FLUORESCENT PROBES FOR THE INVESTIGATION OF MELANOSOME TRAFFICKING

Melanosomes are pigment cell-specific organelles in which melanin is produced and trafficked within the cell, before being transferred to neighbouring keratinocytes. Their production is essential for the protection of skin cells from UV radiation. Melanosomes are an ideal model for the study of vesicular transport, due to the ease with which they can be detected in darkly pigmented cells. Extensive research has identified the key proteins involved in melanogenesis, including Pmel17, TRP2 and Rab27A. However, melanosomal research thus far has been mostly static, using techniques such as electron microscopy. As a result, the dynamic movement of melanosomes both within melanocytes and during transfer to keratinocytes has not been observed. Here, the three melanosome-related proteins Rab27A, TRP2 and Pmel17 are fluorescently tagged and examined in SKMEL28 cells for their use in the observation of melanosome dynamics. Rab27A is implicated in the transport of mature pigmented melanosomes to the cell periphery. TRP2 is associated with early stage melanosomes and mediates pigmentation, while Pmel17 initiates melanosome maturation, forming fibrils onto which melanin is deposited. Novel fluorescent probes tdTomato-TRP2 and mNG-Pmel17 were designed, and the latter was determined to be the most effective for the visualisation of melanosomes. The mNG-Pmel17 fluorescent probe produced punctate structures in live cells, of a size consistent with melansomes. The probe co-localised with the actin cytoskeleton and was shown to be distinct from lysosomal compartments. Finally, mNG-Pmel17 is reported as a valuable tool for the evaluation of the effects of bioactive compounds of interest. The novel mNG-Pmel17 probe was shown to be a useful tool for dynamic melanosome observation, and may be used in further studies for the elucidation of the mechanism(s) of melanosome transfer to keratinocytes