Biogenesis of specialized lysosomes in differentiated keratinocytes relies on close apposition with the Golgi apparatus.

Intracellular organelles support cellular physiology in diverse conditions. In the skin, epidermal keratinocytes undergo differentiation with gradual changes in cellular physiology, accompanying remodeling of lysosomes and the Golgi apparatus. However, it was not known whether changes in Golgi and lysosome morphology and their redistribution were linked. Here, we show that disassembled Golgi is distributed in close physical apposition to lysosomes in differentiated keratinocytes. This atypical localization requires the Golgi tethering protein GRASP65, which is associated with both the Golgi and lysosome membranes. Depletion of GRASP65 results in the loss of Golgi-lysosome apposition and the malformation of lysosomes, defined by their aberrant morphology, size, and function. Surprisingly, a trans-Golgi enzyme and secretory Golgi cargoes are extensively localized to the lysosome lumen and secreted to the cell surface, contributing to total protein secretion of differentiated keratinocytes but not in proliferative precursors, indicating that lysosomes acquire specialization during differentiation. We further demonstrate that the secretory function of the Golgi apparatus is critical to maintain keratinocyte lysosomes. Our study uncovers a novel form of Golgi-lysosome cross-talk and its role in maintaining specialized secretory lysosomes in differentiated keratinocytes.

Epidermal Lamellar Body Biogenesis: Insight Into the Roles of Golgi and Lysosomes.

Epidermal lamellar bodies (eLBs) are secretory organelles that carry a wide variety of secretory cargo required for skin homeostasis. eLBs belong to the class of lysosome-related organelles (LROs), which are cell-type-specific organelles that perform diverse functions. The formation of eLBs is thought to be related to that of other LROs, which are formed either through the gradual maturation of Golgi/endosomal precursors or by the conversion of conventional lysosomes. Current evidence suggests that eLB biogenesis presumably initiate from trans-Golgi network and receive cargo from endosomes, and also acquire lysosome characteristics during maturation. These multistep biogenesis processes are frequently disrupted in human skin disorders. However, many gaps remain in our understanding of eLB biogenesis and their relationship to skin diseases. Here, we describe our current understanding on eLB biogenesis with a focus on cargo transport to this LRO and highlight key areas where future research is needed.

Correction: Keratinocyte differentiation promotes ER stress-dependent lysosome biogenesis.

Following publication of this article, the authors realized there was an error in Figure 2b that needed correction. The TFEB panel of Figure 2b (total lysate) appears to be the same as the TFEB panel of Figure 2e (cytosolic fraction); the TFE3 panels of Figure 2b (total lysate) appear to be the same as the TFE3 panels of Figure 2e (cytosolic fraction) which happened during image assembly.This error did not impact the scientific conclusions of the article.

Keratinocyte differentiation promotes ER stress-dependent lysosome biogenesis.

Keratinocytes maintain epidermal integrity through cellular differentiation. This process enhances intraorganelle digestion in keratinocytes to sustain nutritional and calcium-ionic stresses observed in upper skin layers. However, the molecular mechanisms governing keratinocyte differentiation and concomitant increase in lysosomal function is poorly understood. Here, by using primary neonatal human epidermal keratinocytes, we identified the molecular link between signaling pathways and cellular differentiation/lysosome biogenesis. Incubation of keratinocytes with CaCl2 induces differentiation with increased cell size and early differentiation markers. Further, differentiated keratinocytes display enhanced lysosome biogenesis generated through ATF6-dependent ER stress signaling, but independent of mTOR-MiT/TFE pathway. In contrast, chemical inhibition of mTORC1 accelerates calcium-induced keratinocyte differentiation, suggesting that activation of autophagy promotes the differentiation process. Moreover, differentiation of keratinocytes results in lysosome dispersion and Golgi fragmentation, and the peripheral lysosomes showed colocalization with Golgi-tethering proteins, suggesting that these organelles possibly derived from Golgi. In line, inhibition of Golgi function, but not the depletion of Golgi-tethers or altered lysosomal acidity, abolishes keratinocyte differentiation and lysosome biogenesis. Thus, ER stress regulates lysosome biogenesis and keratinocyte differentiation to maintain epidermal homeostasis.

Rab4A organizes endosomal domains for sorting cargo to lysosome-related organelles.

Sorting endosomes (SEs) are the regulatory hubs for sorting cargo to multiple organelles, including lysosome-related organelles, such as melanosomes in melanocytes. In parallel, melanosome biogenesis is initiated from SEs with the processing and sequential transport of melanocyte-specific proteins toward maturing melanosomes. However, the mechanism of cargo segregation on SEs is largely unknown. Here, RNAi screening in melanocytes revealed that knockdown of Rab4A results in defective melanosome maturation. Rab4A-depletion increases the number of vacuolar endosomes and disturbs the cargo sorting, which in turn lead to the mislocalization of melanosomal proteins to lysosomes, cell surface and exosomes. Rab4A localizes to the SEs and forms an endosomal complex with the adaptor AP-3, the effector rabenosyn-5 and the motor KIF3, which possibly coordinates cargo segregation on SEs. Consistent with this, inactivation of rabenosyn-5, KIF3A or KIF3B phenocopied the defects observed in Rab4A-knockdown melanocytes. Further, rabenosyn-5 was found to associate with rabaptin-5 or Rabip4/4' (isoforms encoded by Rufy1) and differentially regulate cargo sorting from SEs. Thus, Rab4A acts a key regulator of cargo segregation on SEs.This article has an associated First Person interview with the first author of the paper.

Aqueous humor tyrosinase activity is indicative of iris melanocyte toxicity.

Antibiotics such as fluoroquinolones (FQLs) are commonly used to treat ocular infections but are also known to cause dermal melanocyte toxicity. The release of dispersed pigments from the iris into the aqueous humor has been considered a possible ocular side effect of the systemic administration of FQLs such as Moxifloxacin, and this condition is known as bilateral acute iris transillumination (BAIT). Bilateral acute depigmentation of iris (BADI) is a similar condition, with iris pigment released into the aqueous, but it has not been reported as a side effect of FQL. Iris pigments are synthesized by the melanogenic enzyme tyrosinase (TYR) and can be detected but not quantified by using slit-lamp biomicroscopy. The correlation between dispersed pigments in the aqueous and the extent of melanocyte toxicity due to topical antibiotics in vivo is not well studied. Here, we aimed to study the effect of topical FQLs on iris tissue, the pigment release in the aqueous humor and the development of clinically evident iris atrophic changes. We evaluated this process by measuring the activity of TYR in the aqueous humor of 82 healthy eyes undergoing cataract surgery following topical application of FQLs such as Moxifloxacin (27 eyes, preservative-free) or Ciprofloxacin (29 eyes, with preservative) or the application of non-FQL Tobramycin (26 eyes, with preservative) as a control. In addition, the patients were questioned and examined for ocular side effects in pre- and post-operative periods. Our data showed a significantly higher mean TYR activity in the aqueous humor of Ciprofloxacin-treated eyes compared to Moxifloxacin- (preservative free, p < 0.0001) or Tobramycin-treated eyes (p < 0.0001), which indicated that few quinolones under certain conditions are toxic to the iris melanocytes. However, the reduced TYR activity in the aqueous of Moxifloxacin-treated eyes was possibly due to the presence of a higher drug concentration, which inhibits TYR activity. Consistently, immunoblotting analysis of the aqueous humor from both Ciprofloxacin- and Moxifloxacin-treated eyes showed the presence of soluble TYR enzyme, thus reflecting its toxicity to iris melanocytes and corresponding to its activity in the aqueous humor. Intriguingly, none of these patients developed any clinically appreciable ocular side effects characteristic of BAIT or BADI. Overall, our results suggest that topical antibiotics cause different levels of iris melanocyte toxicity, releasing dispersed pigments into the aqueous humor, which can be measured through TYR enzyme activity. Hence, we conclude that topical FQLs may cause subclinical toxicity to the iris melanocytes but may not be the sole cause of the development of BAIT or BADI.

SNAREs in the maturation and function of LROs.

The early/recycling endosomes of an eukaryotic cell perform diverse cellular functions. In addition, the endosomal system generates multiple organelles, including certain cell type-specific organelles called lysosome-related organelles (LROs). The biosynthesis of these organelles possibly occurs through a sequential maturation process in which the cargo-containing endosomal vesicular/tubular structures are fused with the maturing organelle. The molecular machinery that regulates the cargo delivery or the membrane fusion during LRO biogenesis is poorly understood. Here, we describe the known key molecules, such as SNAREs, that regulate both the biogenesis and secretion of multiple LROs. Moreover, we also describe other regulatory molecules, such as Rab GTPases and their effectors that modulate the SNARE activity for cargo delivery to one such LRO, the melanosome. Overall, this review will increase our current understanding of LRO biogenesis and function.

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes.

Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky-Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes.