A non-canonical lysosome biogenesis pathway generates Golgi-associated lysosomes during epidermal differentiation

Keratinocytes maintain epidermis integrity and function including physical and antimicrobial barrier through cellular differentiation. This process is predicted to be controlled by calcium ion gradient and nutritional stress. Keratinocytes undergo proteome changes during differentiation, which enhances the intracellular organelle digestion to sustain the stress conditions. However, the molecular mechanism between epidermal differentiation and organelle homeostasis is poorly understood. Here, we used primary neonatal human epidermal keratinocytes to study the link between cellular differentiation, signaling pathways and organelle turnover. Upon addition of calcium chloride (2 mM) to the culture medium, keratinocytes increased their cell size and the expression of differentiation markers. Moreover, differentiated keratinocytes showed enhanced lysosome biogenesis that was dependent on ATF6-arm of UPR signaling but independent of mTOR-MiT/TFE transcription factors. Furthermore, chemical inhibition of mTOR has increased keratinocyte differentiation and relocalized the MiT/TFE TFs to the lysosome membranes, indicating that autophagy activation promotes the epidermal differentiation. Interestingly, differentiation of keratinocytes resulted in dispersal of fragmented Golgi and lysosomes, and the later organelles showed colocalization with Golgi-tethering proteins, suggesting that these lysosomes possibly originated from Golgi, hence named as Golgi-associated lysosomes (GALs). Consistent to this prediction, inhibition of Golgi function using brefeldin A completely abolished the formation of GALs and the keratinocyte differentiation. Thus, ER stress regulates the biogenesis of GALs, which maintains keratinocyte differentiation and epidermal homeostasis.