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.

SNAREs: a double-edged sword for intravacuolar bacterial pathogens within host cells.

In the tug-of-war between host and pathogen, both evolve to combat each other's defence arsenals. Intracellular phagosomal bacteria have developed strategies to modify the vacuolar niche to suit their requirements best. Conversely, the host tries to target the pathogen-containing vacuoles towards the degradative pathways. The host cells use a robust system through intracellular trafficking to maintain homeostasis inside the cellular milieu. In parallel, intracellular bacterial pathogens have coevolved with the host to harbour strategies to manipulate cellular pathways, organelles, and cargoes, facilitating the conversion of the phagosome into a modified pathogen-containing vacuole (PCV). Key molecular regulators of intracellular traffic, such as changes in the organelle (phospholipid) composition, recruitment of small GTPases and associated effectors, soluble N-ethylmaleimide-sensitive factor-activating protein receptors (SNAREs), etc., are hijacked to evade lysosomal degradation. Legionella, Salmonella, Coxiella, Chlamydia, Mycobacterium, and Brucella are examples of pathogens which diverge from the endocytic pathway by using effector-mediated mechanisms to overcome the challenges and establish their intracellular niches. These pathogens extensively utilise and modulate the end processes of secretory pathways, particularly SNAREs, in repurposing the PCV into specialised compartments resembling the host organelles within the secretory network; at the same time, they avoid being degraded by the host's cellular mechanisms. Here, we discuss the recent research advances on the host-pathogen interaction/crosstalk that involves host SNAREs, conserved cellular processes, and the ongoing host-pathogen defence mechanisms in the molecular arms race against each other. The current knowledge of SNAREs, and intravacuolar bacterial pathogen interactions, enables us to understand host cellular innate immune pathways, maintenance of homeostasis, and potential therapeutic strategies to combat ever-growing antimicrobial resistance.

Real-time heterogeneity of supramolecular assembly of amyloid precursor protein is modulated by an endocytic risk factor PICALM.

Recently, the localization of amyloid precursor protein (APP) into reversible nanoscale supramolecular assembly or "nanodomains" has been highlighted as crucial towards understanding the onset of the molecular pathology of Alzheimer's disease (AD). Surface expression of APP is regulated by proteins interacting with it, controlling its retention and lateral trafficking on the synaptic membrane. Here, we evaluated the involvement of a key risk factor for AD, PICALM, as a critical regulator of nanoscale dynamics of APP. Although it was enriched in the postsynaptic density, PICALM was also localized to the presynaptic active zone and the endocytic zone. PICALM colocalized with APP and formed nanodomains with distinct morphological properties in different subsynaptic regions. Next, we evaluated if this localization to subsynaptic compartments was regulated by the C-terminal sequences of APP, namely, the "Y682ENPTY687" domain. Towards this, we found that deletion of C-terminal regions of APP with partial or complete deletion of Y682ENPTY687, namely, APP-Δ9 and APP-Δ14, affected the lateral diffusion and nanoscale segregation of APP. Lateral diffusion of APP mutant APP-Δ14 sequence mimicked that of a detrimental Swedish mutant of APP, namely, APP-SWE, while APP-Δ9 diffused similar to wild-type APP. Interestingly, elevated expression of PICALM differentially altered the lateral diffusion of the APP C-terminal deletion mutants. These observations confirm that the C-terminal sequence of APP regulates its lateral diffusion and the formation of reversible nanoscale domains. Thus, when combined with autosomal dominant mutations, it generates distinct molecular patterns leading to onset of Alzheimer's disease (AD).

Restoration of ß-GC trafficking improves the lysosome function in Gaucher disease.

Lysosomes function as a primary site for catabolism and cellular signaling. These organelles digest a variety of substrates received through endocytosis, secretion and autophagy with the help of resident acid hydrolases. Lysosomal enzymes are folded in the endoplasmic reticulum (ER) and trafficked to lysosomes via Golgi and endocytic routes. The inability of hydrolase trafficking due to mutations or mutations in its receptor or cofactor leads to cargo accumulation (storage) in lysosomes, resulting in lysosome storage disorder (LSD). In Gaucher disease (GD), the lysosomes accumulate glucosylceramide because of low ß-glucocerebrosidase (ß-GC) activity that causes lysosome enlargement/dysfunction. We hypothesize that improving the trafficking of mutant ß-GC to lysosomes may improve the lysosome function in GD. RNAi screen using high throughput based ß-GC activity assay followed by reporter trafficking assay utilizing ß-GC-mCherry led to the identification of nine potential phosphatases. Depletion of these phosphatases in HeLa cells enhanced the ß-GC activity by increasing the folding and trafficking of Gaucher mutants to the lysosomes. Consistently, the lysosomes in primary fibroblasts from GD patients restored their ß-GC activity upon the knockdown of these phosphatases. Thus, these studies provide evidence that altering phosphatome activity is an alternative therapeutic strategy to restore the lysosome function in GD.

Pseudophosphatase STYXL1 depletion enhances glucocerebrosidase trafficking to lysosomes via ER stress.

Pseudophosphatases are catalytically inactive but share sequence and structural similarities with classical phosphatases. STYXL1 is a pseudophosphatase that belongs to the family of dual-specificity phosphatases and is known to regulate stress granule formation, neurite formation and apoptosis in different cell types. However, the role of STYXL1 in regulating cellular trafficking or the lysosome function has not been elucidated. Here, we show that the knockdown of STYXL1 enhances the trafficking of ß-glucocerebrosidase (ß-GC) and its lysosomal activity in HeLa cells. Importantly, the STYXL1-depleted cells display enhanced distribution of endoplasmic reticulum (ER), late endosome and lysosome compartments. Further, knockdown of STYXL1 causes the nuclear translocation of unfolded protein response (UPR) and lysosomal biogenesis transcription factors. However, the upregulated ß-GC activity in the lysosomes is independent of TFEB/TFE3 nuclear localization in STYXL1 knockdown cells. The treatment of STYXL1 knockdown cells with 4-PBA (ER stress attenuator) significantly reduces the ß-GC activity equivalent to control cells but not additive with thapsigargin, an ER stress activator. Additionally, STYXL1-depleted cells show the enhanced contact of lysosomes with ER, possibly via increased UPR. The depletion of STYXL1 in human primary fibroblasts derived from Gaucher patients showed moderately enhanced lysosomal enzyme activity. Overall, these studies illustrated the unique role of pseudophosphatase STYXL1 in modulating the lysosome function both in normal and lysosome-storage disorder cell types. Thus, designing small molecules against STYXL1 possibly can restore the lysosome activity by enhancing ER stress in Gaucher disease.

Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole.

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.

Deceiving the big eaters: Salmonella Typhimurium SopB subverts host cell xenophagy in macrophages via dual mechanisms.

Salmonella, a stealthy facultative intracellular pathogen, utilises an array of host immune evasion strategies. This facilitates successful survival via replicative niche establishment in otherwise hostile environments such as macrophages. Salmonella survives in and utilises macrophages for effective dissemination, ultimately leading to systemic infection. Bacterial xenophagy or macro-autophagy is an important host defense mechanism in macrophages. Here, we report for the first time that the Salmonella pathogenicity island-1 (SPI-1) effector SopB is involved in subverting host autophagy via dual mechanisms. SopB is a phosphoinositide phosphatase capable of altering the phosphoinositide dynamics of the host cell. Here, we demonstrate that SopB mediates escape from autophagy by inhibiting the terminal fusion of Salmonella-containing vacuoles (SCVs) with lysosomes and/or autophagosomes. We also report that SopB downregulates overall lysosomal biogenesis by modulating the Akt-transcription factor EB (TFEB) axis via restricting the latter's nuclear localisation. TFEB is a master regulator of lysosomal biogenesis and autophagy. This reduces the overall lysosome content inside host macrophages, further facilitating the survival of Salmonella in macrophages and systemic dissemination of Salmonella.

KIF13A-A Key Regulator of Recycling Endosome Dynamics.

Molecular motors of the kinesin superfamily (KIF) are a class of ATP-dependent motor proteins that transport cargo, including vesicles, along the tracks of the microtubule network. Around 45 KIF proteins have been described and are grouped into 14 subfamilies based on the sequence homology and domain organization. These motors facilitate a plethora of cellular functions such as vesicle transport, cell division and reorganization of the microtubule cytoskeleton. Current studies suggest that KIF13A, a kinesin-3 family member, associates with recycling endosomes and regulates their membrane dynamics (length and number). KIF13A has been implicated in several processes in many cell types, including cargo transport, recycling endosomal tubule biogenesis, cell polarity, migration and cytokinesis. Here we describe the recent advances in understanding the regulatory aspects of KIF13A motor in controlling the endosomal dynamics in addition to its structure, mechanism of its association to the membranes, regulators of motor activity, cell type-specific cargo/membrane transport, methods to measure its activity and its association with disease. Thus, this review article will provide our current understanding of the cell biological roles of KIF13A in regulating endosomal membrane remodeling.

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking.

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

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.

KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell.

Endocytic recycling is a complex itinerary, critical for many cellular processes. Membrane tubulation is a hallmark of recycling endosomes (REs), mediated by KIF13A, a kinesin-3 family motor. Understanding the regulatory mechanism of KIF13A in RE tubulation and cargo recycling is of fundamental importance but is overlooked. Here, we report a unique mechanism of KIF13A dimerization modulated by Rab22A, a small guanosine triphosphatase, during RE tubulation. A conserved proline between neck coil-coiled-coil (NC-CC1) domains of KIF13A creates steric hindrance, rendering the motors as inactive monomers. Rab22A plays an unusual role by binding to NC-CC1 domains of KIF13A, relieving proline-mediated inhibition and facilitating motor dimerization. As a result, KIF13A motors produce balanced motility and force against multiple dyneins in a molecular tug-of-war to regulate RE tubulation and homeostasis. Together, our findings demonstrate that KIF13A motors are tuned at a single-molecule level to function as weak dimers on the cellular cargo.

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.

Rab22A recruits BLOC-1 and BLOC-2 to promote the biogenesis of recycling endosomes.

Recycling endosomes (REs) are transient endosomal tubular intermediates of early/sorting endosomes (E/SEs) that function in cargo recycling to the cell surface and deliver the cell type-specific cargo to lysosome-related organelles such as melanosomes in melanocytes. However, the mechanism of RE biogenesis is largely unknown. In this study, by using an endosomal Rab-specific RNAi screen, we identified Rab22A as a critical player during RE biogenesis. Rab22A-knockdown results in reduced RE dynamics and concurrent cargo accumulation in the E/SEs or lysosomes. Rab22A forms a complex with BLOC-1, BLOC-2 and the kinesin-3 family motor KIF13A on endosomes. Consistently, the RE-dependent transport defects observed in Rab22A-depleted cells phenocopy those in BLOC-1-/BLOC-2-deficient cells. Further, Rab22A depletion reduced the membrane association of BLOC-1/BLOC-2. Taken together, these findings suggest that Rab22A promotes the assembly of a BLOC-1-BLOC-2-KIF13A complex on E/SEs to generate REs that maintain cellular and organelle 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.

Opsin3-A Link to Visible Light-Induced Skin Pigmentation.

Skin pigmentation is primarily dependent on melanogenesis, a physiological process that occurs in melanosomes of melanocytes. Solar radiation modulates pigmentation through variety of signaling pathways, but the mechanism of visible light-induced hyperpigmentation remains uncharacterized. Passeron's group recently reported that visible light stimulates opsin3-regulated calcium-dependent microphthalmia-associated transcription factor activation that increases pigment gene expression and that it also causes the clustering of melanogenic enzymes. Together, these processes possibly contribute to long-lasting hyperpigmentation in the melanocompetent skins.

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.

Visualization of Intracellular Tyrosinase Activity in vitro.

Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels results in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we described a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes.

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.