The optimized quantum dot mediated thermometry reveals isoform specific differences in efficiency of myosin extracted from muscle mini bundles.

The biophysical function of myosin in vitro has been extensively investigated in different motility assays, but the study of myosin ATPase properties at the fiber level is insufficiently investigated. In this study, quantum dot (QD) mediated thermometry measurements were optimized to measure the efficiency of myosin extracted from muscle mini bundles. A reduction in fluorescent intensity of QD reflects an increase in temperature caused by the heat released during ATP hydrolysis and denotes the efficiency of the motor protein myosin. The procedure for extracting myosin was similar to the single fiber in vitro motility assay with some small modifications, and the concentration of myosin was represented by the extracted total protein since the ratio of extracted myosin to total protein was constant. Moreover, the efficiencies of myosin extracted from preparations containing different myosin heavy chain isoforms reveal lower efficiency of slow compared to fast myosin isoforms. Specifically, more heat was released in slow myosin enzymatic reaction, resulting in faster decay of QD fluorescence intensity. Hence, the optimized QD mediated thermometry provides a novel and sensitive approach to evaluate efficiency of myosin ATPase obtained from small muscle samples, representing a significant advantage in the clinical evaluation of neuromuscular disorders.

Nanoscale imaging using differential expansion microscopy.

Expensive and time-consuming approaches of immunoelectron microscopy of biopsy tissues continues to serve as the gold-standard for diagnostic pathology. The recent development of the new approach of expansion microscopy (ExM) capable of fourfold lateral expansion of biological specimens for their morphological examination at approximately 70 nm lateral resolution using ordinary diffraction limited optical microscopy, is a major advancement in cellular imaging. Here we report (1) an optimized fixation protocol for retention of cellular morphology while obtaining optimal expansion, (2) an ExM procedure for up to eightfold lateral and over 500-fold volumetric expansion, (3) demonstrate that ExM is anisotropic or differential between tissues, cellular organelles and domains within organelles themselves, and (4) apply image analysis and machine learning (ML) approaches to precisely assess differentially expanded cellular structures. We refer to this enhanced ExM approach combined with ML as differential expansion microscopy (DiExM), applicable to profiling biological specimens at the nanometer scale. DiExM holds great promise for the precise, rapid and inexpensive diagnosis of disease from pathological specimen slides.

vH+-ATPase-induced intracellular acidification is critical to glucose-stimulated insulin secretion in beta cells.

Swelling of secretory vesicles is critical for the regulated release of intra-vesicular contents from cells during secretion. At the secretory vesicle membrane of the exocrine pancreas and neurons, GTP-binding G proteins, vH+-ATPase, potassium channels and AQP water channels, are among the players implicated in vesicle volume regulation. Here we report in the endocrine insulin-secreting MIN6 cells, the similar requirement of vH+-ATPase-mediated intracellular acidification on glucose-stimulated insulin release. MIN6 cells exposed to the vH+-ATPase inhibitor Bafilomycin A show decreased acidification of the cytosolic compartment that include insulin-carrying granules. Additionally, a loss of insulin granules near the cell plasma membrane following Bafilomycin A treatment, suggests impaired transport of insulin granules and consequent decrease in glucose-stimulated insulin secretion and accumulation of intracellular insulin. These results suggest that vH+-ATPase-mediated intracellular acidification is required for insulin secretion in beta cells.

Self-Assembly and Biogenesis of the Cellular Membrane are Dictated by Membrane Stretch and Composition.

The cell plasma membrane is a highly dynamic organelle governing a wide range of cellular activities including ion transport, secretion, cell division, growth, and development. The fundamental process involved in the addition of new membranes to pre-existing plasma membranes, however, is unclear. Here, we report, using biophysical, morphological, biochemical, and molecular dynamic simulations, the selective incorporation of proteins and lipids from the cytosol into the cell plasma membrane dictated by membrane stretch and composition. Stretching of the cell membrane as a consequence of volume increase following incubation in a hypotonic solution and results in the incorporation of cytosolic proteins and lipids into the existing plasma membrane. Molecular dynamic simulations further confirm that increased membrane stretch results in the rapid insertion of lipids into the existing plasma membrane. Similarly, depletion of cholesterol from the cell plasma membrane selectively alters the incorporation of lipids into the membrane.

Nanothermometry Reveals Calcium-Induced Remodeling of Myosin.

Ions greatly influence protein structure-function and are critical to health and disease. A 10, 000-fold higher calcium in the sarcoplasmic reticulum (SR) of muscle suggests elevated calcium levels near active calcium channels at the SR membrane and the impact of localized high calcium on the structure-function of the motor protein myosin. In the current study, combined quantum dot (QD)-based nanothermometry and circular dichroism (CD) spectroscopy enabled detection of previously unknown enthalpy changes and associated structural remodeling of myosin, impacting its function following exposure to elevated calcium. Cadmium telluride QDs adhere to myosin, function as thermal sensors, and reveal that exposure of myosin to calcium is exothermic, resulting in lowering of enthalpy, a decrease in alpha helical content measured using CD spectroscopy, and the consequent increase in motor efficiency. Isolated muscle fibers subjected to elevated levels of calcium further demonstrate fiber lengthening and decreased motility of actin filaments on myosin-functionalized substrates. Our results, in addition to providing new insights into our understanding of muscle structure-function, establish a novel approach to understand the enthalpy of protein-ion interactions and the accompanying structural changes that may occur within the protein molecule.

Valproate inhibits glucose-stimulated insulin secretion in beta cells.

Valproate (VPA), an FDA approved anti-epileptic drug with a half-life of 12-18 h in humans, has been shown to perturb the vacuolar proton pump (vH+-ATPase) function in yeasts by inhibiting myo-inositol phosphate synthase, the first and rate-limiting enzyme in inositol biosynthesis, thereby resulting in inositol depletion. vH+-ATPase transfers protons (H+) across cell membranes, which help maintain pH gradients within cells necessary for various cellular functions including secretion. This proton pump has a membrane (V0) and a soluble cytosolic (V1) domain, with C-subunit associated with V1. In secretory cells such as neurons and insulin-secreting beta cells, vH+-ATPase acidifies vesicles essential for secretion. In this study, we demonstrate that exposure of insulin-secreting Min6 cells to a clinical dose of VPA results in inositol depletion and loss of co-localization of subunit C of vH+-ATPase with insulin-secreting granules. Consequently, a reduction of glucose-stimulated insulin secretion is observed following VPA exposure. These results merit caution and the reassessment of the clinical use of VPA.

Secretion induces cell pH dynamics impacting assembly-disassembly of the fusion protein complex: A combined fluorescence and atomic force microscopy study.

A wide range of cellular activities including protein folding and cell secretion, such as neurotransmission or insulin release, are all governed by intracellular pH homeostasis, underscoring the importance of pH on critical life processes. Nano- scale pH measurements of cells and biomolecules therefore hold great promise in understanding a plethora of cellular functions, in addition to disease detection and therapy. In the current study, a novel approach using cadmium telluride quantum dots (CdTeQDs) as pH sensors, combined with fluorescent imaging, spectrofluorimetry, atomic force microscopy (AFM), and Western blot analysis, enabled the study of intracellular pH dynamics at 1 milli-pH sensitivity and 80nm pixel resolution, during insulin secretion. Additionally, the pH-dependent interaction between membrane fusion proteins, also called the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE), was determined. Glucose stimulation of CdTeQD-loaded insulin secreting Min-6 mouse insulinoma cell line demonstrated the initial (5-6min) intracellular acidification reflected as a loss in QD fluorescence, followed by alkalization and a return to resting pH in 10min. Analysis of the SNARE complex in insulin secreting Min-6 cells demonstrated an initial gain followed by loss of complexed SNAREs in 10min. Stabilization of the SNARE complex at low intracellular pH is further supported by results from studies utilizing both native and AFM measurements of liposome-reconstituted recombinant neuronal SNAREs, providing a molecular understanding of the role of pH during cell secretion.

Human Platelet Vesicles Exhibit Distinct Size and Proteome.

In the past 50 years, isolated blood platelets have had restricted use in wound healing, cancer therapy, and organ and tissue transplant, to name a few. The major obstacle for its unrestricted use has been, among others, the presence of ultrahigh concentrations of growth factors and the presence of both pro-angiogenic and anti-angiogenic proteins. To overcome this problem requires the isolation and separation of the membrane bound secretory vesicles containing the different factors. In the current study, high-resolution imaging of isolated secretory vesicles from human platelets using atomic force microscopy (AFM) and mass spectrometry enabled characterization of the remaining vesicles size and composition following their immunoseparation. The remaining vesicles obtained following osmotic lysis, when subjected to immunoseparation employing antibody to different vesicle-associated membrane proteins (VAMPs), demonstrate for the first time that VAMP-3-, VAMP-7-, and VAMP-8-specific vesicles each possesses distinct size range and composition. These results provide a window into our understanding of the heterogeneous population of vesicles in human platelets and their stability following both physical manipulation using AFM and osmotic lysis of the platelet. This study further provides a platform for isolation and the detailed characterization of platelet granules, with promise for their future use in therapy. Additionally, results from the study demonstrate that secretory vesicles of different size found in cells reflect their unique and specialized composition and function.

Nanothermometry Measure of Muscle Efficiency.

Despite recent advances in thermometry, determination of temperature at the nanometer scale in single molecules to live cells remains a challenge that holds great promise in disease detection among others. In the present study, we use a new approach to nanometer scale thermometry with a spatial and thermal resolution of 80 nm and 1 mK respectively, by directly associating 2 nm cadmium telluride quantum dots (CdTe QDs) to the subject under study. The 2 nm CdTe QDs physically adhered to bovine cardiac and rabbit skeletal muscle myosin, enabling the determination of heat released when ATP is hydrolyzed by both myosin motors. Greater heat loss reflects less work performed by the motor, hence decreased efficiency. Surprisingly, we found rabbit skeletal myosin to be more efficient than bovine cardiac. We have further extended this approach to demonstrate the gain in efficiency of Drosophila melanogaster skeletal muscle overexpressing the PGC-1α homologue spargel, a known mediator of improved exercise performance in humans. Our results establish a novel approach to determine muscle efficiency with promise for early diagnosis and treatment of various metabolic disorders including cancer.

Functionalized nanoparticles enable tracking the rapid entry and release of doxorubicin in human pancreatic cancer cells.

Efficient drug delivery is critical to therapy. Using electron microscopy, X-ray, and light microscopy, we have characterized functionalized superparamagnetic iron oxide (SPIO) nanoparticles, and determined their ability for rapid entry and release of the cancer drug doxorubicin in human pancreatic cancer cells. Dextran-coated SPIO nanoparticle ferrofluid, functionalized with the red-autofluorescing doxorubicin and the green-fluorescent dye fluorescein isothiocyanate as a reporter, enables tracking the intracellular nanoparticle transport and drug release. This engineered nanoparticle enables a >20 fold rapid entry and release of the drug in human pancreatic cancer cells, holding therapeutic potential as an advanced drug delivery and imaging platform. The low extracellular pH of most tumors precluding the entry of a number of weakly basic drugs such as doxorubicin, conferring drug resistance, can now be overcome.

Functional Reconstitution of the Insulin-Secreting Porosome Complex in Live Cells.

Supramolecular cup-shaped lipoprotein structures called porosomes embedded in the cell plasma membrane mediate fractional release of intravesicular contents from cells during secretion. The presence of porosomes, have been documented in many cell types including neurons, acinar cells of the exocrine pancreas, GH-secreting cells of the pituitary, and insulin-secreting pancreatic ß-cells. Functional reconstitution of porosomes into artificial lipid membranes, have also been accomplished. Earlier studies on mouse insulin-secreting Min6 cells report 100-nm porosome complexes composed of nearly 30 proteins. In the current study, porosomes have been functionally reconstituted for the first time in live cells. Isolated Min6 porosomes reconstituted into live Min6 cells demonstrate augmented levels of porosome proteins and a consequent increase in the potency and efficacy of glucose-stimulated insulin release. Elevated glucose-stimulated insulin secretion 48 hours after reconstitution, reflects on the remarkable stability and viability of reconstituted porosomes, documenting the functional reconstitution of native porosomes in live cells. These results, establish a new paradigm in porosome-mediated insulin secretion in ß-cells.

Matriptase activation and shedding through PDGF-D-mediated extracellular acidosis.

Activation of ß-platelet-derived growth factor receptor (ß-PDGFR) is associated with prostate cancer (PCa) progression and recurrence after prostatectomy. Analysis of the ß-PDGFR ligands in PCa revealed association between PDGF-D expression and Gleason score as well as tumor stage. During the course of studying the functional consequences of PDGF ligand-specific ß-PDGFR signaling in PCa, we discovered a novel function of PDGF-D for activation/shedding of the serine protease matriptase leading to cell invasion, migration, and tumorigenesis. The present study showed that PDGF-D, not PDGF-B, induces extracellular acidification, which correlates with increased matriptase activation. A cDNA microarray analysis revealed that PDGF-D/ß-PDGFR signaling upregulates expression of the acidosis regulator carbonic anhydrase IX (CAIX), a classic target of the transcriptional factor hypoxia-inducible factor-1α (HIF-1α). Cellular fractionation displayed a strong HIF-1α nuclear localization in PDGF-D-expressing cells. Treatment of vector control or PDGF-B-expressing cells with the HIF-1α activator CoCl2 led to increased CAIX expression accompanied by extracellular acidosis and matriptase activation. Furthermore, the analysis of the CAFTD cell lines, variants of the BPH-1 transformation model, showed that increased PDGF-D expression is associated with enhanced HIF-1α activity, CAIX induction, cellular acidosis, and matriptase shedding. Importantly, shRNA-mediated knockdown of CAIX expression effectively reversed extracellular acidosis and matriptase activation in PDGF-D-transfected BPH-1 cells and in CAFTD variants that express endogenous PDGF-D at a high level. Taken together, these novel findings reveal a new paradigm in matriptase activation involving PDGF-D-specific signal transduction leading to extracellular acidosis.

Nanometric features of myosin filaments extracted from a single muscle fiber to uncover the mechanisms underlying organized motility.

The single muscle fiber in vitro motility assay (SF-IVMA) is characterized by organized linear motility of actin filaments, i.e., actin filaments motility showing a parallel or anti-parallel direction with similar speed independent of direction in the central part of the flow-cell where density of myosin is high. In contrast, the low myosin density region in the flow-cell exhibits random filament movements, but the mechanisms underlying the organized motility remain unknown. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) imaging techniques have been combined to investigate the morphological features of myosin extracted from single muscle fiber segments in the flow cell. Nanometric scale imaging of myosin filaments in the SF-IVMA showed intact spatial distances between myosin heads being essential for myosin filament function. However, angular spectrum analyses of myosin filaments in the high myosin density region showed organized myosin filament orientation only in small areas, while unorganized filament orientation were dominantly presented when larger areas were analyzed. Thus, parallel myosin filament organization is a less likely mechanism underlying the organized motility of actin filaments and the high myosin density per se is therefore forwarded as the primary "driver" that promotes organized linear motility.

COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and ß-Cell ER Homeostasis.

Pancreatic ß-cells possess a highly active protein synthetic and export machinery in the endoplasmic reticulum (ER) to accommodate the massive production of proinsulin. ER homeostasis is vital for ß-cell functions and is maintained by the delicate balance between protein synthesis, folding, export, and degradation. Disruption of ER homeostasis by diabetes-causing factors leads to ß-cell death. Among the 4 components to maintain ER homeostasis in ß-cells, the role of ER export in insulin biogenesis is the least understood. To address this knowledge gap, the present study investigated the molecular mechanism of proinsulin ER export in MIN6 cells and primary islets. Two inhibitory mutants of the secretion-associated RAS-related protein (Sar)1 small GTPase, known to specifically block coat protein complex II (COPII)-dependent ER export, were overexpressed in ß-cells using recombinant adenoviruses. Results from this approach, as well as small interfering RNA-mediated Sar1 knockdown, demonstrated that defective Sar1 function blocked proinsulin ER export and abolished its conversion to mature insulin in MIN6 cells, isolated mouse, and human islets. It is further revealed, using an in vitro vesicle formation assay, that proinsulin was packaged into COPII vesicles in a GTP- and Sar1-dependent manner. Blockage of COPII-dependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and ß-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (p-eIF2α) and inositol-requiring protein 1 (IRE1)/x-box binding protein 1 (Xbp1) pathways but not via activating transcription factor 6 (ATF6). Collectively, results from the study demonstrate that COPII-dependent ER export plays a vital role in insulin biogenesis, ER homeostasis, and ß-cell survival.

Porosome in Cystic Fibrosis.

Macromolecular structures embedded in the cell plasma membrane called 'porosomes', are involved in the regulated fractional release of intravesicular contents from cells during secretion. Porosomes range in size from 15 nm in neurons and astrocytes to 100-180 nm in the exocrine pancreas and neuroendocrine cells. Porosomes have been isolated from a number of cells, and their morphology, composition, and functional reconstitution well documented. The 3D contour map of the assembly of proteins within the porosome complex, and its native X-ray solution structure at sub-nm resolution has also advanced. This understanding now provides a platform to address diseases that may result from secretory defects. Water and ion binding to mucin impart hydration, critical for regulating viscosity of the mucus in the airways epithelia. Appropriate viscosity is required for the movement of mucus by the underlying cilia. Hence secretion of more viscous mucus prevents its proper transport, resulting in chronic and fatal airways disease such as cystic fibrosis (CF). CF is caused by the malfunction of CF transmembrane conductance regulator (CFTR), a chloride channel transporter, resulting in viscous mucus in the airways. Studies in mice lacking functional CFTR secrete highly viscous mucous that adhered to the epithelium. Since CFTR is known to interact with the t-SNARE protein syntaxin-1A, and with the chloride channel CLC-3, which are also components of the porosome complex, the interactions between CFTR and the porosome complex in the mucin-secreting human airway epithelial cell line Calu-3 was hypothesized and tested. Results from the study demonstrate the presence of approximately 100 nm in size porosome complex composed of 34 proteins at the cell plasma membrane in Calu-3 cells, and the association of CFTR with the complex. In comparison, the nuclear pore complex measures 120 nm and is comprised of over 500 protein molecules. The involvement of CFTR in porosome-mediated mucin secretion is hypothesized, and is currently being tested.

Proteome of the porosome complex in human airway epithelia: interaction with the cystic fibrosis transmembrane conductance regulator (CFTR).

The surface of the airways is coated with a thin film of mucus composed primarily of mucin, which is under continuous motion via ciliary action. Mucin not only serves to lubricate the airways epithelia, but also functions as a trap for foreign particles and pathogens, thereby assisting in keeping the airways clean and free of particulate matter and infections. Altered mucin secretion especially increased mucin viscosity, results in mucin stagnation due to the inability of the cilia to propel them, leading to infections and diseases such as cystic fibrosis (CF). Since porosomes have been demonstrated to be the secretory portals at the cell plasma membrane in cells, their presence, structure, and composition in the mucin-secreting human airway epithelial cell line Calu-3 expressing CF transmembrane receptor (CFTR), were investigated. Atomic force microscopy (AFM) of Calu-3 cells demonstrates the presence of approximately 100nm in diameter porosome openings at the plasma membrane surface. Electron microscopy confirms the AFM results, and tandem mass spectrometry and immunoanalysis performed on isolated Calu-3 porosomes, reveal the association of CFTR with the porosome complex. These new findings will facilitate understanding of CFTR-porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease. BIOLOGICAL SIGNIFICANCE: In the present study, the porosome proteome in human airway epithelia has been determined. The interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the porosome complex in the human airway epithelia is further demonstrated. The possible regulation by CFTR on the quality of mucus secretion via the porosome complex at the cell plasma membrane is hypothesized. These new findings will facilitate understanding of CFTR-porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease.

CXCR2 macromolecular complex in pancreatic cancer: a potential therapeutic target in tumor growth.

The signaling mediated by the chemokine receptor CXC chemokine receptor 2 (CXCR2) plays an important role in promoting the progression of many cancers, including pancreatic cancer, one of the most lethal human malignancies. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif at its carboxyl termini, which might interact with potential PDZ scaffold/adaptor proteins. We have previously reported that CXCR2 PDZ motif-mediated protein interaction is an important regulator for neutrophil functions. Here, using a series of biochemical assays, we demonstrate that CXCR2 is physically coupled to its downstream effector phospholipase C-ß3 (PLC-ß3) that is mediated by PDZ scaffold protein Na(+)/H(+) exchange regulatory factor 1 (NHERF1) into a macromolecular signaling complex both in vitro and in pancreatic cancer cells. We also observe that disrupting the CXCR2 complex, by gene delivery or peptide delivery of exogenous CXCR2 C-tail, significantly inhibits the biologic functions of pancreatic cancer cells (i.e., proliferation and invasion) in a PDZ motif-dependent manner. In addition, using a human pancreatic tumor xenograft model, we show that gene delivery of CXCR2 C-tail sequence (containing the PDZ motif) by adeno-associated virus type 2 viral vector potently suppresses human pancreatic tumor growth in immunodeficient mice. In summary, our results suggest the existence of a physical and functional coupling of CXCR2 and PLC-ß3 mediated through NHERF1, forming a macromolecular complex that is critical for efficient and specific CXCR2 signaling in pancreatic cancer progression. Disrupting this CXCR2 complex could represent a novel and effective treatment strategy against pancreatic cancer.

Lysophosphatidylcholine inhibits membrane-associated SNARE complex disassembly.

In cells, N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors called SNAREs are involved in membrane fusion. In neurons, for example, target membrane proteins SNAP-25 and syntaxin called t-SNAREs present at the pre-synaptic membrane, and a synaptic vesicle-associated membrane protein (VAMP) or v-SNARE, is part of the conserved protein complex involved in neurotransmission. Cholesterol and LPC (L-α-lysophosphatidylcholine) are known to contribute to the negative and positive curvature respectively of membranes. In this study, using purified recombinant neuronal membrane-associated SNAREs, we demonstrate for the first time that membrane-curvature-influencing lipids profoundly influence SNARE complex disassembly. Exposure of cholesterol-associated t-SNARE and v-SNARE liposome mixtures to NSF-ATP results in dissociated vesicles. In contrast, exposure of LPC-associated t-SNARE and v-SNARE liposome mixtures to NSF-ATP, results in inhibition of t-/v-SNARE disassembly and the consequent accumulation of clustered vesicles. Similarly, exposure of isolated rat brain slices and pancreas to cholesterol or LPC, also demonstrates LPC-induced inhibition of SNARE complex disassembly. Earlier studies demonstrate a strong correlation between altered plasma LPC levels and cancer. The altered plasma LPC levels observed in various cancers may in part contribute to defects in SNARE assembly-disassembly and membrane fusion, consequently affecting protein maturation and secretion in cancer cells.

Involvement of ß-adrenergic receptor in synaptic vesicle swelling and implication in neurotransmitter release.

Secretory vesicle swelling is required for vesicular discharge during cell secretion. The G(αo) -mediated water channel aquaporin-6 (AQP-6) involvement in synaptic vesicle (SV) swelling in neurons has previously been reported. Studies demonstrate that in the presence of guanosine triphosphate (GTP), mastoparan, an amphiphilic tetradecapeptide from wasp venom, activates G(o) protein GTPase, and stimulates SV swelling. Stimulation of G proteins is believed to occur via insertion of mastoparan into the phospholipid membrane to form a highly structured α-helix that resembles the intracellular loops of G protein-coupled adrenergic receptors. Consequently, the presence of adrenoceptors and the presence of an endogenous ß-adrenergic agonist at the SV membrane is suggested. Immunoblot analysis of SV using ß-adrenergic receptor antibody, and vesicle swelling experiments using ß-adrenergic agonists and antagonists, demonstrate the presence of functional ß-adrenergic receptors at the SV membrane. Since a recent study shows vH(+) -ATPase to be upstream of AQP-6 in the pathway leading from G(αo) -mediated swelling of SV, participation of an endogenous ß-adrenergic agonist, in the binding and stimulation of its receptor to initiate the swelling cascade is demonstrated.

Involvement of cholesterol in synaptic vesicle swelling.

Studies demonstrate that cholesterol plays a critical role in the regulation of neurotransmitter release and that secretory vesicle swelling is a requirement for the regulated expulsion of intravesicular contents during cell secretion. In view of this, the involvement of cholesterol in synaptic vesicle swelling was hypothesized and tested in the present study, using isolated synaptic vesicles from rat brain and the determination of their swelling competency in the presence and absence of cholesterol. The involvement of the water channel aquaporin-6 (AQP-6) and proton pump vH(+)-ATPase in GTP-G(alpha o)-mediated synaptic vesicle swelling has been reported previously. Mastoparan, the amphiphilic tetradecapeptide from wasp venom, known to activate the GTPase activity of G(alpha o/i) proteins, stimulates synaptic vesicle swelling in the presence of GTP. In the current study, using nanometer-scale precision measurements of isolated synaptic vesicles, we report for the first time that depletion of cholesterol from synaptic vesicle membrane results in a significant loss of GTP-mastoparan-stimulable synaptic vesicle swelling. In contrast, incorporation of cholesterol into the synaptic vesicle membrane potentiates GTP-mastoparan-stimulable vesicle swelling. Our study further demonstrates that this effect of cholesterol is due, in part, to its involvement in the interactions between AQP-6, vH(+)-ATPase and the GTP-binding G(alpha o) protein at the synaptic vesicle membrane.