IP3R2 mediated inter-organelle Ca2+ signaling orchestrates melanophagy

Abstract

Organelle dynamics and crosstalk play a critical role in cellular functions thereby regulating physiological processes and pathological conditions. A variety of cellular processes are outcome of a balance between organelle biogenesis and degradation. Pigmentation is one such homeostatic state that is a result of melanosome biogenesis and melanosome degradation. Although melanosome biogenesis is partially understood, the melanosome degradation i.e. melanophagy remains largely unappreciated. Here, we reveal that Inositol 1,4,5-trisphosphate receptor 2 (IP3R2) is a negative regulator of melanophagy. In this study, we developed two de novo ratio metric imaging probes to study melanophagy in live-cells. Using these probes, biochemical assays, ultrastructural studies, confocal microscopy, molecular analyses and calcium imaging; we demonstrate that IP3R2, but not IP3R1 or IP3R3, keeps melanophagy in check. In vivo studies in zebrafish model system further substantiate IP3R2s functional relevance in pigmentation. Mechanistically, IP3R2 silencing decreases mitochondrial Ca2+ uptake, augments ADP/ATP ratio and thereby activates melanophagy. Simultaneously, IP3R2 knockdown increases ER-lysosome proximity, enhances lysosomal Ca2+ levels and decreases lysosomal pH. This in turn activates lysosomal TRPML1 channel and stimulates nuclear translocation of TFEB transcription factor, which facilitates transcription of key autophagy and two known melanophagy drivers. Taken together, we uncover that IP3R2-mediated Ca2+ signaling across organelles is a critical determinant of melanophagy and thereby skin pigmentation. Hence, this signaling cascade offers potential therapeutic prospects for the management of pigmentary disorders and skin malignancies. Highlights[tpltrtarr] IP3R2, but not IP3R1 and IP3R3, is a critical positive regulator of melanogenesis in vitro and in vivo. [tpltrtarr]Generation and validation of two de novo ratio-metric live cells imaging probes reveal crucial role of IP3R2 in melanophagy. [tpltrtarr]IP3R2 knockdown decreases mitochondrial Ca2+ uptake, augments ADP/ATP ratio and thereby activates melanophagy via AMPK-ULK1 pathway. [tpltrtarr]IP3R2 silencing enhances ER-lysosomal proximity, elevates lysosomal Ca2+ levels and reduces lysosomal pH. [tpltrtarr]IP3R2 knockdown stimulates lysosomal TRPML1 channel activity thereby facilitating nuclear translocation of TFEB transcription factor. [tpltrtarr]TFEB transcriptionally upregulates genes involved in the melanophagy process, leading to enhanced degradation of melanosomes and decreased pigmentation.