UC Irvine

Epidermal melanocytes synthesize melanin within specialized lysosome-related organelles known as melanosomes. These melanosomes are transferred to neighboring keratinocytes where they surround nuclei to protect against ultraviolet-induced UV damage. The synthesis of melanin, a process known as melanogenesis, is subject to complex and intricate regulation at multiple levels. While extensive studies have elucidated many of the transcriptional and regulatory pathways, melanosomal trafficking comparatively far less understood. To further complicate matters, several lysosomal and autophagic components are utilized during these processes, but it is unclear what delineates the specificity of their regulation. To help elucidate these mechanisms, our laboratory conducted a genome-wide siRNA screen, which identified several novel regulators of melanogenesis. Several autophagy components, including the phosphoinositol binding protein WIPI1, were among those implicated in the screen. Through further investigation, it was determined that WIPI1 regulates both melanosomal transcription and trafficking, but the mechanism by which it coordinated both processes was not thoroughly elucidated. WIPI1 binds to three different phosphoinositides, PI(3)P, PI(3,5)P2, and PI(5)P, the latter two of which are synthesized by the PIKfyve kinase complex. PI(3,5)P2 and PI(5)P have been priorly implicated in regulating membrane trafficking and signaling pathways. Furthermore, mutation in select PI(3,5)P2 effectors or in the PIKfyve complex itself has been shown to affect pigment accumulation, though the mechanisms have not been priorly elucidated. In our studies, we have demonstrated that loss of PIKfyve in vivo results in the progressive loss of pigmentation. Disruption of the PIKfyve leads to defects in vesicle trafficking and severe vacuolization. Our in vitro and in vivo studies have identified similar disruption in melanocytes resulting in the inhibition of melanosome maturation and melanosomal acidification. These mechanisms would suggest an important role of PI(3,5)P2 in melanogenesis. In contrast, partial depletion of PIKfyve depleted WIPI1 and resulted in downregulation of these signaling pathways and expression of MITF-M and TYR, while the addition of PI(5)P had the opposite effect. Taken together these studies demonstrate novel regulatory roles of the PIKfyve complex, elucidating distinct roles of PI(3,5)P2 and PI(5)P in melanogenesis. These studies expand upon the fields of knowledge in both melanocyte and phosphoinositide biology and provide significant insight into the regulatory mechanisms of melanocytes.