Vesicle-associated membrane protein 2 is a cargo-selective v-SNARE for a subset of GPCRs.

Vesicle fusion at the plasma membrane is critical for releasing hormones and neurotransmitters and for delivering the cognate G protein-coupled receptors (GPCRs) to the cell surface. The SNARE fusion machinery that releases neurotransmitters has been well characterized. In contrast, the fusion machinery that delivers GPCRs is still unknown. Here, using high-speed multichannel imaging to simultaneously visualize receptors and v-SNAREs in real time in individual fusion events, we identify VAMP2 as a selective v-SNARE for GPCR delivery. VAMP2 was preferentially enriched in vesicles that mediate the surface delivery of µ opioid receptor (MOR), but not other cargos, and was required selectively for MOR recycling. Interestingly, VAMP2 did not show preferential localization on MOR-containing endosomes, suggesting that v-SNAREs are copackaged with specific cargo into separate vesicles from the same endosomes. Together, our results identify VAMP2 as a cargo-selective v-SNARE and suggest that surface delivery of specific GPCRs is mediated by distinct fusion events driven by distinct SNARE complexes.

Diversity and specificity in location-based signaling outputs of neuronal GPCRs.

The common mechanisms by which members of the G protein-coupled receptor (GPCR) family respond to neurotransmitters in the brain have been well studied. However, it is becoming increasingly clear that GPCRs show great diversity in their intracellular location, interacting partners and effectors, and signaling consequences. Here we will discuss recent studies on the diversity of location, effectors, and signaling of GPCRs, and how these could interact to generate specific spatiotemporal patterns of GPCR signaling in cells.

Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism.

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol.

Integrity of the Actin Cytoskeleton of Host Macrophages is Necessary for Mycobacterial Entry.

Macrophages are the primary hosts for Mycobacterium tuberculosis (M. tb), an intracellular pathogen, and the causative organism of tuberculosis (TB) in humans. While M. tb has the ability to enter and survive in host macrophages, the precise mechanism of its internalization, and factors that control this essential process are poorly defined. We have previously demonstrated that perturbations in levels of cholesterol and sphingolipids in macrophages lead to significant reduction in the entry of Mycobacterium smegmatis (M. smegmatis), a surrogate model for mycobacterial internalization, signifying a role for these plasma membrane lipids in interactions at the host-pathogen interface. In this work, we investigated the role of the host actin cytoskeleton, a critical protein framework underlying the plasma membrane, in the entry of M. smegmatis into human macrophages. Our results show that cytochalasin D mediated destabilization of the actin cytoskeleton of host macrophages results in a dose-dependent reduction in the entry of mycobacteria. Notably, the internalization of Escherichia coli remained invariant upon actin destabilization of host cells, implying a specific involvement of the actin cytoskeleton in mycobacterial infection. By monitoring the F-actin content of macrophages utilizing a quantitative confocal microscopy-based technique, we observed a close correlation between the entry of mycobacteria into host macrophages with cellular F-actin content. Our results constitute the first quantitative analysis of the role of the actin cytoskeleton of human macrophages in the entry of mycobacteria, and highlight actin-mediated mycobacterial entry as a potential target for future anti-TB therapeutics.

Late endosomal/lysosomal accumulation of a neurotransmitter receptor in a cellular model of Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. It is characterized by accumulation of 7-dehydrocholesterol (the immediate biosynthetic precursor of cholesterol in the Kandutsch-Russell pathway) and an altered cholesterol to total sterol ratio. Because SLOS is associated with neurological malfunction, exploring the function and trafficking of neuronal receptors and their interaction with membrane lipids under these conditions assume significance. In this work, we generated a cellular model of SLOS in HEK-293 cells stably expressing the human serotonin1A receptor (an important neurotransmitter G-protein coupled receptor) using AY 9944, an inhibitor for the enzyme 3ß-hydroxy-steroid-∆7 -reductase (7-DHCR). Using a quantitative flow cytometry based assay, we show that the plasma membrane population of serotonin1A receptors was considerably reduced under these conditions without any change in total cellular expression of the receptor. Interestingly, the receptors were trafficked to sterol-enriched LysoTracker positive compartments, which accumulated under these conditions. To the best of our knowledge, our results constitute one of the first reports demonstrating intracellular accumulation and misregulated traffic of a neurotransmitter GPCR in SLOS-like conditions. We believe these results assume relevance in our overall understanding of the molecular basis underlying the functional relevance of neurotransmitter receptors in SLOS.

A molecular sensor for cholesterol in the human serotonin1A receptor.

The function of several G protein-coupled receptors (GPCRs) exhibits cholesterol sensitivity. Cholesterol sensitivity of GPCRs could be attributed to specific sequence and structural features, such as the cholesterol recognition/interaction amino acid consensus (CRAC) motif, that facilitate their cholesterol-receptor interaction. In this work, we explored the molecular basis of cholesterol sensitivity exhibited by the serotonin1A receptor, the most studied GPCR in the context of cholesterol sensitivity, by generating mutants of key residues in CRAC motifs in transmembrane helix 2 (TM2) and TM5 of the receptor. Our results show that a lysine residue (K101) in one of the CRAC motifs is crucial for sensing altered membrane cholesterol levels. Insights from all-atom molecular dynamics simulations showed that cholesterol-sensitive functional states of the serotonin1A receptor are associated with reduced conformational dynamics of extracellular loops of the receptor. These results constitute one of the first reports on the molecular mechanism underlying cholesterol sensitivity of GPCRs.

Membrane cholesterol regulates endocytosis and trafficking of the serotonin1A receptor: Insights from acute cholesterol depletion.

Endocytosis and intracellular trafficking constitute important regulatory features associated with G protein-coupled receptor (GPCR) function. GPCR endocytosis involves several remodeling events at the plasma membrane orchestrated by a concerted interplay of a large number of proteins and membrane lipids. Although considerable literature exists on the protein framework underlying GPCR endocytosis, the role of membrane lipids in this process remains largely unexplored. In order to explore the role of membrane cholesterol (an essential and important lipid in higher eukaryotes) in GPCR endocytosis, we monitored the effect of acute cholesterol depletion using methyl-ß-cyclodextrin (MßCD) on endocytosis and intracellular trafficking of the serotonin1A receptor, an important neurotransmitter GPCR. Our results show that the serotonin1A receptor exhibits agonist-induced clathrin-mediated endocytosis with a concentration-dependent inhibition in internalization with increasing concentrations of MßCD, which was restored upon cholesterol replenishment. Interestingly, subsequent to internalization under these conditions, serotonin1A receptors were re-routed toward lysosomal degradation, instead of endosomal recycling observed under normal conditions, thereby implicating membrane cholesterol in modulation of intracellular trafficking of the receptor. This raises the possibility of a novel cholesterol-dependent role of intracellular sorting proteins in GPCR trafficking. These results differ from our previous observations on the endocytosis of the serotonin1A receptor upon statin-induced chronic cholesterol depletion, in terms of endocytic pathway. We conclude that analysis of complex cellular trafficking events such as GPCR endocytosis under acute and chronic cholesterol depletion conditions should be carried out with caution due to fundamental differences underlying these processes.

Cholesterol-dependent endocytosis of GPCRs: implications in pathophysiology and therapeutics.

G protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins that relay extracellular signals across the plasma membrane and elicit an intricate cascade of cellular signaling events. A significantly large fraction of available drugs target GPCRs in order to exert fine control over functional outcomes from these receptors in pathological conditions. In this context, endocytosis and intracellular trafficking of GPCRs stringently regulate signaling outcomes from GPCRs within physiologically relevant spatiotemporal regimes. The membrane microenvironment around GPCRs has recently emerged as a key player in receptor function. Cholesterol is the single most abundant lipid in the eukaryotic plasma membrane and plays a central role in membrane organization and dynamics, with far-reaching functional implications in cellular physiology. In this review, we discuss current excitements in GPCR endocytosis and trafficking, with an emphasis on the role of membrane cholesterol. We envision that a detailed understanding of the contribution of membrane lipids such as cholesterol in spatiotemporal regulation of GPCR signaling would enable the development of therapeutic interventions fine-tuned to receptors residing in specific membrane microenvironments.

Statin-Induced Chronic Cholesterol Depletion Switches GPCR Endocytosis and Trafficking: Insights from the Serotonin1A Receptor.

Endocytosis is a key regulatory mechanism adopted by G protein-coupled receptors (GPCRs) to modulate downstream signaling responses within a stringent spatiotemporal regime. Although the role of membrane lipids has been extensively studied in the context of the function, organization, and dynamics of GPCRs, their role in receptor endocytosis remains largely unexplored. Cholesterol, the predominant sterol in higher eukaryotes, plays a crucial role in maintaining the structure and organization of cell membranes and is involved in essential cellular processes in health and disease. The serotonin1A receptor is a representative GPCR involved in neuronal development and in neuropsychiatric disorders such as anxiety and depression. We recently combined quantitative flow cytometric and confocal microscopic approaches to demonstrate that the serotonin1A receptor undergoes clathrin-mediated endocytosis upon agonist stimulation and subsequently traffics along the endosomal recycling pathway. In this work, we show that statin-induced chronic cholesterol depletion switches the endocytic pathway of the serotonin1A receptor from clathrin- to caveolin-mediated endocytosis. Interestingly, under these conditions, a significant proportion of endocytosed receptors is rerouted toward lysosomal degradation. To the best of our knowledge, these results constitute one of the first comprehensive reports on the role of membrane cholesterol in GPCR endocytosis and trafficking. These results are significant in our overall understanding of the modulatory effects of membrane lipids on GPCR endocytosis and trafficking and could provide novel insight in developing therapeutic interventions against neuropsychiatric disorders such as depression.

Leishmania donovani Internalizes into Host Cells via Caveolin-mediated Endocytosis.

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis, a major cause of mortality and morbidity worldwide. The host plasma membrane serves as the portal of entry for Leishmania to gain access to the cellular interior. Although several host cell membrane receptors have been shown to be involved in the entry of Leishmania donovani into host cells, the endocytic pathway involved in the internalization of the parasite is not known. In this work, we explored the endocytic pathway involved in the entry of Leishmania donovani into host macrophages, utilizing specific inhibitors against two major pathways of internalization, i.e., clathrin- and caveolin-mediated endocytosis. We show that pitstop 2, an inhibitor for clathrin-mediated endocytosis, does not affect the entry of Leishmania donovani promastigotes into host macrophages. Interestingly, a significant reduction in internalization was observed upon treatment with genistein, an inhibitor for caveolin-mediated endocytosis. These results are supported by a similar trend in intracellular amastigote load within host macrophages. These results suggest that Leishmania donovani utilizes caveolin-mediated endocytosis to internalize into host cells. Our results provide novel insight into the mechanism of phagocytosis of Leishmania donovani into host cells and assume relevance in the development of novel therapeutics against leishmanial infection.

Exploring Endocytosis and Intracellular Trafficking of the Human Serotonin1A Receptor.

G protein-coupled receptors (GPCRs) represent the largest class of receptors involved in signal transduction across cell membranes and are major drug targets in all clinical areas. Endocytosis of GPCRs offers a regulatory mechanism for sustaining their signaling within a stringent spatiotemporal regime. In this work, we explored agonist-induced endocytosis of the human serotonin1A receptor stably expressed in HEK-293 cells and the cellular machinery involved in receptor internalization and intracellular trafficking. The serotonin1A receptor is a popular GPCR implicated in neuropsychiatric disorders such as anxiety and depression and serves as an important drug target. In spite of its pharmacological relevance, its mechanism of endocytosis and intracellular trafficking is less understood. In this context, we have utilized a combination of robust population-based flow cytometric analysis and confocal microscopic imaging to address the path and fate of the serotonin1A receptor during endocytosis. Our results, utilizing inhibitors of specific endocytosis pathways and intracellular markers, show that the serotonin1A receptor undergoes endocytosis predominantly via the clathrin-mediated pathway and subsequently recycles to the plasma membrane via recycling endosomes. These results would enhance our understanding of molecular mechanisms of GPCR endocytosis and could offer novel insight into the underlying mechanism of antidepressants that act via the serotonergic pathway. In addition, our results could be relevant in understanding cell (or tissue)-specific GPCR endocytosis.

Macrophage sphingolipids are essential for the entry of mycobacteria.

Mycobacteria are intracellular pathogens that can invade and survive within host macrophages. Mycobacterial infections remain a major cause of mortality and morbidity worldwide, with serious concerns of emergence of multi and extensively drug-resistant tuberculosis. While significant advances have been made in identifying mycobacterial virulence determinants, the detailed molecular mechanism of internalization of mycobacteria into host cells remains poorly understood. Although several studies have highlighted the crucial role of sphingolipids in mycobacterial growth, persistence and establishment of infection, the role of sphingolipids in the entry of mycobacteria into host cells is not known. In this work, we explored the role of host membrane sphingolipids in the entry of Mycobacterium smegmatis into J774A.1 macrophages. Our results show that metabolic depletion of sphingolipids in host macrophages results in a significant reduction in the entry of M. smegmatis. Importantly, the entry of Escherichia coli into host macrophages under similar conditions remained invariant, implying the specificity of the requirement of sphingolipids in mycobacterial entry. To the best of our knowledge, our results constitute the first report demonstrating the role of host macrophage sphingolipids in the entry of mycobacteria. Our results could help in the development of novel therapeutic strategies targeting sphingolipid-mediated entry of mycobacteria into host cells.

Statin-induced chronic cholesterol depletion inhibits Leishmania donovani infection: Relevance of optimum host membrane cholesterol.

Leishmania are obligate intracellular protozoan parasites that invade and survive within host macrophages leading to leishmaniasis, a major cause of mortality and morbidity worldwide, particularly among economically weaker sections in tropical and subtropical regions. Visceral leishmaniasis is a potent disease caused by Leishmania donovani. The detailed mechanism of internalization of Leishmania is poorly understood. A basic step in the entry of Leishmania involves interaction of the parasite with the host plasma membrane. In this work, we have explored the effect of chronic metabolic cholesterol depletion using lovastatin on the entry and survival of Leishmania donovani in host macrophages. We show here that chronic cholesterol depletion of host macrophages results in reduction in the attachment of Leishmania promastigotes, along with a concomitant reduction in the intracellular amastigote load. These results assume further relevance since chronic cholesterol depletion is believed to mimic physiological cholesterol modulation. Interestingly, the reduction in the ability of Leishmania to enter host macrophages could be reversed upon metabolic replenishment of cholesterol. Importantly, enrichment of host membrane cholesterol resulted in reduction in the entry and survival of Leishmania in host macrophages. As a control, the binding of Escherichia coli to host macrophages remained invariant under these conditions, thereby implying specificity of cholesterol requirement for effective leishmanial infection. To the best of our knowledge, these results constitute the first comprehensive demonstration that an optimum content of host membrane cholesterol is necessary for leishmanial infection. Our results assume relevance in the context of developing novel therapeutic strategies targeting cholesterol-mediated leishmanial infection.

The membrane as the gatekeeper of infection: Cholesterol in host-pathogen interaction.

The cellular plasma membrane serves as a portal for the entry of intracellular pathogens. An essential step for an intracellular pathogen to gain entry into a host cell therefore is to be able to cross the cell membrane. In this review, we highlight the role of host membrane cholesterol in regulating the entry of intracellular pathogens using insights obtained from work on the interaction of Leishmania and Mycobacterium with host cells. The entry of these pathogens is known to be dependent on host membrane cholesterol. Importantly, pathogen entry is inhibited either upon depletion (or complexation), or enrichment of membrane cholesterol. In other words, an optimum level of host membrane cholesterol is necessary for efficient infection by pathogens. In this overall context, we propose a general mechanism, based on cholesterol-induced conformational changes, involving cholesterol binding sites in host cell surface receptors that are implicated in this process. A therapeutic strategy targeting modulation of membrane cholesterol would have the advantage of avoiding the commonly encountered problem of drug resistance in tackling infection by intracellular pathogens. Insights into the role of host membrane cholesterol in pathogen entry would be instrumental in the development of novel therapeutic strategies to effectively tackle intracellular pathogenesis.

Dissecting the membrane cholesterol requirement for mycobacterial entry into host cells.

Mycobacteria are intracellular pathogens that can invade and survive within host macrophages, and are a major cause of mortality and morbidity worldwide. The molecular mechanism involved in the internalization of mycobacteria is poorly understood. In this work, we have explored the role of host membrane cholesterol in the entry of the avirulent surrogate mycobacterial strain Mycobacterium smegmatis into THP-1 macrophages. Our results show that depletion of host membrane cholesterol using methyl-ß-cyclodextrin results in a significant reduction in the entry of M. smegmatis into host cells. More importantly, we show that the inhibition in the ability of M. smegmatis to enter host macrophages could be reversed upon replenishment of membrane cholesterol. To the best of our knowledge, these results constitute the first report showing that membrane cholesterol replenishment can reverse the inhibition in the entry of mycobacteria into host cells. In addition, we demonstrate that cholesterol complexation using amphotericin B (without physical depletion) is sufficient to inhibit mycobacterial entry. Importantly, we observed a significant reduction in mycobacterial entry upon enrichment of host membrane cholesterol. Taken together, our results demonstrate, for the first time, that an optimum host plasma membrane cholesterol is necessary for the entry of mycobacteria. These results assume relevance in the context of developing novel therapeutic strategies targeting cholesterol-mediated mycobacterial host cell entry.

Integrity of the actin cytoskeleton of host macrophages is essential for Leishmania donovani infection.

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.