Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biology

First Advisor

Michael Marks

Abstract

Melanosomes are pigment cell-specific lysosome-related organelles (LROs) in which melanin pigments are synthesized and stored. Melanosome maturation requires biosynthetic delivery of melanogenic enzymes (e.g. TYRP1), transporters (e.g. OCA2), and SNAREs (e.g. VAMP7) from early endosomes (EEs). One essential pathway requires the multisubunit complex, BLOC-1, on EEs to generate tubular transport carriers to deliver cargos to melanosomes. The molecular mechanisms by which BLOC-1 facilitates tubule formation are not entirely known, but several studies suggest that phosphatidylinositol-4-phosphate (PtdIns4P) and the type II PtdIns-4-kinases, PI4KIIα and PI4KIIβ, may play a role. Moreover, PI4KIIα and PI4KIIβ interact respectively with AP-3 and AP-1, both of which control BLOC-1-dependent cargo sorting. We hypothesized that type II PtdIns-4-kinases support BLOC-1-dependent tubule formation via PtdIns4P to regulate melanosome biogenesis. We found that depletion of either PI4KIIα or PI4KIIβ in melanocytes reduced melanin content and impaired the melanosomal localization of TYRP1, OCA2, and VAMP7 by immunofluorescence microscopy (IFM) and bright-field microscopy. TYRP1 was largely mislocalized to late endosomes/lysosomes in PI4KIIα- or β-depleted cells, and not exclusively to EEs as observed in BLOC-1-/- melanocytes. This suggested that type II PtdIns-4kinases are not absolutely required for BLOC-1-dependent cargo exit from EEs but function downstream of BLOC-1. Consistently, depletion of PI4KIIα or PI4KIIβ in BLOC-1-deficient cells did not affect TYRP1 accumulation in EEs by IFM, and depletion of PI4KIIα or PI4KIIβ in wild-type melanocytes did not affect BLOC-1 association with membranes by subcellular fractionation. Rescue studies revealed that enzymatic activity and AP-binding abilities of both PI4KIIα and PI4KIIβ are necessary for TYRP1 localization to melanosomes. Live cell imaging showed that PI4KIIα associates with tubules from the onset of tubule formation, whereas PI4KIIβ accumulates after the tubule has formed, suggesting that PI4KIIα acts earlier than PI4KIIβ. Depletion of either PI4KIIα or PI4KIIβ reduced PtdIns4P on endosomal tubules and impaired tubule elongation. These data together suggest that PI4KIIα and PI4KIIβ function sequentially and non-redundantly downstream of BLOC-1 by generating PtdIns4P on endosomal tubules during tubule elongation and that both isoforms are necessary for efficient melanosome contact and content delivery to melanosome. Our study demonstrates the important roles of PtdIns4P and both type II PtdIns-4-kinases in BLOC-1-dependent tubular cargo transport for melanosome biogenesis and extends the cohort of effectors required for LRO biogenesis.

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