Metabolic depletion of sphingolipids inhibits agonist-induced endocytosis of the serotonin1A receptor.

G protein-coupled receptors (GPCRs) are vital cellular signaling machinery and currently represent ~40% drug targets. Endocytosis of GPCRs is an important process that allows stringent spatiotemporal control over receptor population on the cell surface. Although the role of proteins in GPCR endocytosis is well addressed, the contribution of membrane lipids in this process is rather unexplored. Sphingolipids are essential functional lipids in higher eukaryotes and are implicated in several neurological functions. To understand the role of sphingolipids in GPCR endocytosis, we subjected cells expressing human serotonin1A receptors (an important neurotransmitter GPCR involved in cognitive and behavioral functions) to metabolic sphingolipid depletion using fumonisin B1 , an inhibitor of sphingolipid biosynthetic pathway. Our results, using flow cytometric analysis and confocal microscopic imaging, show that sphingolipid depletion inhibits agonist-induced endocytosis of the serotonin1A receptor in a concentration-dependent manner, which was restored when sphingolipid levels were replenished. We further show that there was no change in the internalization of transferrin, a marker for clathrin-mediated endocytosis, under sphingolipid-depleted condition, highlighting the specific requirement of sphingolipids for endocytosis of serotonin1A receptors. Our results reveal the regulatory role of sphingolipids in GPCR endocytosis and highlight the importance of neurotransmitter receptor trafficking in health and disease.

Quantitation of F-actin in cytoskeletal reorganization: Context, methodology and implications.

The actin cytoskeleton is involved in a large number of cellular signaling events in addition to providing structural integrity to the cell. Actin polymerization is a key event during cellular signaling. Although the role of actin cytoskeleton in cellular processes such as trafficking and motility has been extensively studied, the reorganization of the actin cytoskeleton upon signaling has been rarely explored due to lack of suitable assays. Keeping in mind this lacuna, we developed a confocal microscopy based approach that relies on high magnification imaging of cellular F-actin, followed by image reconstruction using commercially available software. In this review, we discuss the context and relevance of actin quantitation, followed by a detailed hands-on approach of the methodology involved with specific points on troubleshooting and useful precautions. In the latter part of the review, we elucidate the method by discussing applications of actin quantitation from our work in several important problems in contemporary membrane biology ranging from pathogen entry into host cells, to GPCR signaling and membrane-cytoskeleton interaction. We envision that future discovery of cell-permeable novel fluorescent probes, in combination with genetically encoded actin-binding reporters, would allow real-time visualization of actin cytoskeleton dynamics to gain deeper insights into active cellular processes in health and disease.

Statin-induced increase in actin polymerization modulates GPCR dynamics and compartmentalization.

The function of the actin cytoskeleton in cellular motility and trafficking has been widely studied. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol and its consequences on membrane dynamics are addressed only rarely. In a recent work, we reported that chronic cholesterol depletion using statins leads to significant polymerization of the actin cytoskeleton. In this work, we explore the effect of reorganization of the actin cytoskeleton on membrane dynamics under cholesterol-depleted condition. Specifically, we explore the role of actin cytoskeleton in regulating the dynamics of the serotonin1A receptor, a crucial neurotransmitter G protein-coupled receptor (GPCR) that plays a major role in the generation and modulation of cognitive and behavioral functions. For this, we analyzed the lateral dynamics of the serotonin1A receptor in cholesterol-depleted cells (using statins) by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) measurements. Our results indicate that lateral diffusion parameters of serotonin1A receptors in normal cells are consistent with models describing the diffusion of molecules in a homogeneous membrane. Interestingly, these parameters are altered in cholesterol-depleted cells and the receptor exhibits dynamic confinement. Notably, our results show that statin-induced dynamic confinement could be reversed by destabilization of the actin cytoskeleton. On a broader perspective, these results assume significance in understanding the modulatory role of the membrane environment on the organization and dynamics of GPCRs in diseases caused by altered cholesterol biosynthesis.

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.

Role of Cholesterol and its Biosynthetic Precursors on Membrane Organization and Dynamics: A Fluorescence Approach.

Cholesterol is the most representative sterol present in membranes of higher eukaryotes, and is the end product of a long and multistep biosynthetic pathway. Lathosterol and zymosterol are biosynthetic precursors of cholesterol in Kandutsch-Russell and Bloch pathways, respectively. Lathosterol differs with cholesterol merely in the position of the double bond in the sterol ring, whereas zymosterol differs with cholesterol in position and number of double bonds. In this work, we have monitored the effect of cholesterol and its biosynthetic precursors (lathosterol and zymosterol) on membrane organization and dynamics in fluid and gel phase membranes. Toward this goal, we have utilized two fluorescent membrane probes, DPH and its cationic derivative TMA-DPH. Our results using these probes show that cholesterol and its biosynthetic precursors (lathosterol and zymosterol) exhibit similar trend in maintaining membrane organization and dynamics (as reported by fluorescence anisotropy and apparent rotational correlation time), in fluid phase POPC membranes. Notably, although lathosterol and zymosterol show similar trend in maintaining membrane organization and dynamics, the corresponding change for cholesterol is different in gel phase DPPC membranes. These results demonstrate that the position and number of double bonds in sterols is an important determinant in maintaining membrane physical properties. Our results assume significance since accumulation of precursors of cholesterol have been reported to be associated with severe pathological conditions.

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.

Cholesterol in GPCR Structures: Prevalence and Relevance.

Bound cholesterol molecules are emerging as important hallmarks of GPCR structures. In this commentary, we analyze their statistical prevalence and biological relevance.

Lysine 101 in the CRAC Motif in Transmembrane Helix 2 Confers Cholesterol-Induced Thermal Stability to the Serotonin1A Receptor.

G protein-coupled receptors (GPCRs) constitute the largest class of membrane proteins that transduce signals across the plasma membrane and orchestrate a multitude of physiological processes within cells. The serotonin1A receptor is a crucial neurotransmitter receptor in the GPCR family involved in a multitude of neurological, behavioral and cognitive functions. We have previously shown, using a combination of experimental and simulation approaches, that membrane cholesterol acts as a key regulator of organization, dynamics, signaling and endocytosis of the serotonin1A receptor. In addition, we showed that membrane cholesterol stabilizes the serotonin1A receptor against thermal deactivation. In the present work, we explored the molecular basis of cholesterol-induced thermal stability of the serotonin1A receptor. For this, we explored the possible role of the K101 residue in a cholesterol recognition/interaction amino acid consensus (CRAC) motif in transmembrane helix 2 in conferring the thermal stability of the serotonin1A receptor. Our results show that a mutation in the K101 residue leads to loss in thermal stability of the serotonin1A receptor imparted by cholesterol, independent of membrane cholesterol content. We envision that our results could have potential implications in structural biological advancements of GPCRs and design of thermally stabilized receptors for drug development.

Effect of Local Anesthetics on Dipole Potential of Different Phase Membranes: A Fluorescence Study.

The molecular mechanism behind the action of local anesthetics is not well understood. Phenylethanol (PEtOH) is an ingredient of essential oils with a rose-like odor, and it has previously been used as a local anesthetic. In this work, we explored the effect of PEtOH on dipole potential in membranes representing biologically relevant phases, employing the dual-wavelength ratiometric method utilizing the potential-sensitive probe di-8-ANEPPS. Our results show that PEtOH reduces membrane dipole potential in membranes of all biologically relevant phases (gel, liquid-ordered, and fluid) in a concentration-dependent manner. To the best of our knowledge, these results constitute one of the early reports describing reduction of membrane dipole potential induced by local anesthetics, irrespective of membrane phase.

Metabolic Depletion of Sphingolipids Does Not Alter Cell Cycle Progression in Chinese Hamster Ovary Cells.

The cell cycle is a sequential multi-step process essential for growth and proliferation of cells comprising multicellular organisms. Although a number of proteins are known to modulate the cell cycle, the role of lipids in regulation of cell cycle is still emerging. In our previous work, we monitored the role of cholesterol in cell cycle progression in CHO-K1 cells. Since sphingolipids enjoy a functionally synergistic relationship with membrane cholesterol, in this work, we explored whether sphingolipids could modulate the eukaryotic cell cycle using CHO-K1 cells. Sphingolipids are essential components of eukaryotic cell membranes and are involved in a number of important cellular functions. To comprehensively monitor the role of sphingolipids on cell cycle progression, we carried out metabolic depletion of sphingolipids in CHO-K1 cells using inhibitors (fumonisin B1, myriocin, and PDMP) that block specific steps of the sphingolipid biosynthetic pathway and examined their effect on individual cell cycle phases. Our results show that metabolic inhibitors led to significant reduction in specific sphingolipids, yet such inhibition in sphingolipid biosynthesis did not show any effect on cell cycle progression in CHO-K1 cells. We speculate that any role of sphingolipids on cell cycle progression could be context and cell-type dependent, and cancer cells could be a better choice for monitoring such regulation, since sphingolipids are differentially modulated in these cells.

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.

Conformational plasticity and dynamic interactions of the N-terminal domain of the chemokine receptor CXCR1.

The dynamic interactions between G protein-coupled receptors (GPCRs) and their cognate protein partners are central to several cell signaling pathways. For example, the association of CXC chemokine receptor 1 (CXCR1) with its cognate chemokine, interleukin-8 (IL8 or CXCL8) initiates pathways leading to neutrophil-mediated immune responses. The N-terminal domain of chemokine receptors confers ligand selectivity, but unfortunately the conformational dynamics of this intrinsically disordered region remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations, complemented by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo-receptor N-terminal domain is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we corroborated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies and show that the trends are similar. Our results indicate that chemical shift perturbation is often not a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces.

Insights into cellular signaling from membrane dynamics.

Biological membranes allow morphological compartmentalization of cells and represent complex micro-heterogeneous fluids exhibiting a range of dynamics. The plasma membrane occupies a central place in cellular signaling which allows the cell to perform a variety of functions. In this review, we analyze cellular signaling in a dynamic biophysical framework guided by the "mobile receptor hypothesis". We describe a variety of examples from literature in which lateral diffusion of signaling membrane proteins acts as an important determinant in the efficiency of signaling. A major focus in our review is on membrane-embedded G protein-coupled receptors (GPCRs) which act as cellular signaling hubs for diverse cellular functions. Taken together, we describe a dynamics-based signaling paradigm with chosen examples from literature to elucidate how such a paradigm helps us understand signaling by GPCRs, maintenance of cellular polarity in yeast and infection by pathogens. We envision that with further technological advancement, it would be possible to explore cellular signaling more holistically as cells undergo development, differentiation and aging, thereby providing us a robust window into the dynamics of the cellular interior and its functional correlates.

Membrane electrostatics sensed by tryptophan anchors in hydrophobic model peptides depends on non-aromatic interfacial amino acids: implications in hydrophobic mismatch.

WALPs are synthetic α-helical membrane-spanning peptides that constitute a well-studied system for exploring hydrophobic mismatch. These peptides represent a simplified consensus motif for transmembrane domains of intrinsic membrane proteins due to their hydrophobic core of alternating leucine and alanine flanked by membrane-anchoring aromatic tryptophan residues. Although the modulation of mismatch responses in WALPs by tryptophan anchors has been reported earlier, there have been limited attempts to utilize the intrinsic tryptophan fluorescence of this class of peptides in mismatch sensors. We have previously shown, utilizing the red edge excitation shift (REES) approach, that interfacial WALP tryptophan residues in fluid phase bilayers experience a dynamically constrained membrane microenvironment. Interestingly, emerging reports suggest the involvement of non-aromatic interfacially localized residues in modulating local structure and dynamics in WALP analogs. In this backdrop, we have explored the effect of interfacial amino acids, such as lysine (in KWALPs) and glycine (in GWALPs), on the tryptophan microenvironment of WALP analogs in zwitterionic and negatively charged membranes. We show that interfacial tryptophans in KWALP and GWALP experience a more restricted microenvironment, as reflected in the substantial increase in magnitude of REES and apparent rotational correlation time, relative to those in WALP in zwitterionic membranes. Interestingly, in contrast to WALP, the tryptophan anchors in KWALP and GWALP appear insensitive to the presence of negatively charged lipids in the membrane. These results reveal a subtle interplay between non-aromatic flanking residues in transmembrane helices and negatively charged lipids at the membrane interface, which could modulate the membrane microenvironment experienced by interfacially localized tryptophan residues. Since interfacial tryptophans are known to influence mismatch responses in WALPs, our results highlight the possibility of utilizing the fluorescence signatures of tryptophans in membrane proteins or model peptides such as WALP as markers for assessing protein responses to hydrophobic mismatch. More importantly, these results constitute one of the first reports on the influence of lipid headgroup charge in fine-tuning hydrophobic mismatch in membrane bilayers, thereby enriching the existing framework of hydrophobic mismatch.

Lack of Environmental Sensitivity of a Naturally Occurring Fluorescent Analog of Cholesterol.

Dehydroergosterol (DHE, Δ5,7,9(11),22-ergostatetraen-3ß-ol) is a naturally occurring fluorescent analog of cholesterol found in yeast. Since DHE has been shown to faithfully mimic cholesterol in a large number of biophysical, biochemical, and cell biological studies, it is widely used to explore cholesterol organization, dynamics and trafficking in model and biological membranes. In this work, we show that DHE, in spite of its localization at the membrane interface, does not exhibit red edge excitation shift (REES) in model membranes, irrespective of the membrane phase. These results are reinforced by semi-empirical quantum chemical calculations of dipole moment changes of DHE in ground and excited states, which show a very small change in the dipole moment of DHE upon excitation. We conclude that DHE fluorescence exhibits lack of environmental sensitivity, despite its usefulness in monitoring cholesterol organization, dynamics and traffic in model and biological membranes.

Environment-Sensitive Fluorescence of 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-Labeled Ligands for Serotonin Receptors.

Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin1A receptor stably expressed in CHO-K1 cells could be specifically labeled with one of the fluorescent ligands with minimal nonspecific labeling. Interestingly, we show by spectral imaging that the NBD-labeled ligand exhibits a red edge excitation shift (REES) of 29 nm when bound to the receptor, implying that it is localized in a restricted microenvironment. Taken together, our results show that NBD-labeled serotonin analogs offer an attractive fluorescent approach for elucidating the molecular environment of the serotonin binding site in serotonin receptors. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these fluorescent ligands would be useful in future studies involving serotonin receptors.

Role of Actin Cytoskeleton in Dynamics and Function of the Serotonin1A Receptor.

G protein-coupled receptors (GPCRs) are important membrane proteins in higher eukaryotes that carry out a vast array of cellular signaling and act as major drug targets. The serotonin1A receptor is a prototypical member of the GPCR family and is implicated in neuropsychiatric disorders such as anxiety and depression, besides serving as an important drug target. With an overall goal of exploring the functional consequence of altered receptor dynamics, in this work, we probed the role of the actin cytoskeleton in the dynamics, ligand binding, and signaling of the serotonin1A receptor. We monitored receptor dynamics utilizing single particle tracking, which provides information on relative distribution of receptors in various diffusion modes in addition to diffusion coefficient. We show here that the short-term diffusion coefficient of the receptor increases upon actin destabilization by cytochalasin D. In addition, analysis of individual trajectories shows that there are changes in relative populations of receptors undergoing various types of diffusion upon actin destabilization. The release of dynamic constraint was evident by an increase in the radius of confinement of the receptor upon actin destabilization. The functional implication of such actin destabilization was manifested as an increase in specific agonist binding and downstream signaling, monitored by measuring reduction in cellular cAMP levels. These results bring out the interdependence of GPCR dynamics with cellular signaling.

Structural Stringency and Optimal Nature of Cholesterol Requirement in the Function of the Serotonin1A Receptor.

The role of membrane cholesterol in modulating G protein-coupled receptor (GPCR) structure and function has emerged as a powerful theme in contemporary biology. In this paper, we report the subtlety and stringency involved in the interaction of sterols with the serotonin1A receptor. For this, we utilized two immediate biosynthetic precursors of cholesterol, 7-dehydrocholesterol (7-DHC) and desmosterol, which differ with cholesterol merely in a double bond in their chemical structures in a position-dependent manner. We show that whereas 7-DHC could not support the ligand binding function of the serotonin1A receptor in live cells, desmosterol could partially support it. Importantly, depletion and enrichment of membrane cholesterol over basal level resulted in an increase and reduction of the basal receptor activity, respectively. These results demonstrate the relevance of optimal membrane cholesterol in maintaining the activity of the serotonin1A receptor, thereby elucidating the relevance of cellular cholesterol homeostasis.

Percutaneous rescue therapy in a child with blocked cavo-pulmonary shunt.

A 7-month-old infant presented with bilateral blocked cavo-pulmonary anastomosis within 2 months of surgery. Due to extreme haemodynamic instability, surgical options were abandoned and rescue intervention from left jugular line was planned. Acute thrombosis of the left-sided Glenn was noted with significant anastomotic narrowing. Successful rescue thrombolysis was done using recombinant tissue plasminogen activator (Alteplase) along with balloon dilatation of the attenuated segments.

Novel use of Konar-multifunctional occluder.

INTRODUCTION: Lifetech Konar-multifunctional occluder is a novel device which is primarily used for the closure of ventricular septal defects. Being "multifunctional", the occluder has the potential to be useful in various structural cardiac defects. MATERIALS AND METHODS: We share our retrospective review from two centres regarding non-conventional usage of multifunctional occluders in CHD. Eight patients who underwent interventions using multifunctional occluders for lesions other than ventricular septal defects between March 2019 to September 2019 were included in the study. The patients were analysed based on demography, the size and type of lesion, procedural success, and development of complications. All patients were followed up in the outpatient department for a minimum period of 6 months. RESULTS: The median age and weight of the cohort were 3.2 years and 9 kg, respectively. Six patients had patent ductus arteriosus, while one patient had aorto-pulmonary window and one had a coronary arterio-venous fistula. The sizing of the occluders and the procedural approach were based on the underlying pathology. The most commonly used occluder was 6 × 4 mm variant. One patient had successful implantation but had significant intra-device residual flow and was thus replaced by a different occluder. There were no major complications, nor any incidences of device embolisation or malposition. On follow-up, all patients had uneventful course. CONCLUSION: Konar-multifunctional occluder can be safely used in lesions other than ventricular septal defects, when needed under specific circumstances. Its unique characteristics make it a versatile choice in a variety of cardiac lesions.