Kenneth T. Lewis

X-ray solution structure of the native neuronal porosome-synaptic vesicle complex: Implication in neurotransmitter release

Nanoportals at the cell plasma membrane called porosomes, mediate secretion from cells. In neurons porosomes are 15 nm cup-shaped lipoprotein structure composed of nearly 40 proteins. The size and complexity of the porosome has precluded determination of its atomic structure. Here we report at nanometer resolution the native 3D structure of the neuronal porosome-synaptic vesicle complex within isolated nerve terminals using small-angle X-ray solution scattering. In addition to furthering our understanding of the porosome structure, results from the study suggests the molecular mechanism involved in neurotransmitter release at the nerve terminal.

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

UNLABELLED 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.

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.

Neuronal porosome lipidome

Cup‐shaped lipoprotein structures called porosomes are the universal secretory portals at the cell plasma membrane, where secretory vesicles transiently dock and fuse to release intravesicular contents. In neurons, porosomes measure ~15 nm and are comprised of nearly 40 proteins, among them SNAREs, ion channels, the Gαo G‐protein and several structural proteins. Earlier studies report the interaction of specific lipids and their influence on SNAREs, ion channels and G‐protein function. Our own studies demonstrate the requirement of cholesterol for the maintenance of neuronal porosome integrity, and the influence of lipids on SNARE complex assembly. In this study, to further understand the role of lipids on porosome structure‐function, the lipid composition of isolated neuronal porosome was determined using mass spectrometry. Using lipid‐binding assays, the affinity of porosome‐associated syntaxin‐1A to various lipids was determined. Our mass spectrometry results demonstrate the presence of phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA) among other lipids, and the enriched presence of ceramide (Cer), lysophosphatidylinositol phosphates (LPIP) and diacylglycerol (DAG). Lipid binding assays demonstrate the binding of neuronal porosome to cardiolipin, and confirm its association with PIPs and PA. The ability of exogenous PA to alter protein–protein interaction and neurotransmitter release is further demonstrated from the study.

Proteome of the insulin-secreting Min6 cell porosome complex: involvement of Hsp90 in its assembly and function.

UNLABELLED Porosomes are secretory portals located at the cell plasma membrane involved in the regulated release of intravesicular contents from cells. Porosomes have been immunoisolated from a number of cells including the exocrine pancreas and neurons, biochemically characterized, and functionally reconstituted into an artificial lipid membrane. In the current study, the proteome of the porosome complex in mouse insulinoma Min6 cells was determined, demonstrating among other proteins, the presence of 30 core proteins including the heat shock protein Hsp90. Half maximal inhibition of Hsp90 using the specific inhibitor 17-demethoxy-17-(2-prophenylamino) geldanamycin, results in the loss of proteins, including the calcium-transporting ATPase type 2C and the potassium channel subfamily K member 2 from the Min6 porosome. This loss of porosome proteins is reflected in the observed inhibition of glucose stimulated insulin release from Min6 cells exposed to the Hsp90 specific inhibitor. Results from the study implicate Hsp90 in the assembly and function of the porosome complex. BIOLOGICAL SIGNIFICANCE In the present study, the porosome proteome in the insulin-secreting mouse β-cell line Min6 has been determined. Nearly 30 core proteins including the heat shock protein Hsp90 are found to compose the Min6 porosome complex. Results from the study implicate Hsp90 in the assembly of the Min6 porosome. These new findings will facilitate understanding of the porosome assembly and its function in insulin secretion.

Unique Lipid Chemistry of Synaptic Vesicle and Synaptosome Membrane Revealed Using Mass Spectrometry

Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal.

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 β-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 β-cells.

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.

The neuronal porosome complex in health and disease

Cup-shaped secretory portals at the cell plasma membrane called porosomes mediate the precision release of intravesicular material from cells. Membrane-bound secretory vesicles transiently dock and fuse at the base of porosomes facing the cytosol to expel pressurized intravesicular contents from the cell during secretion. The structure, isolation, composition, and functional reconstitution of the neuronal porosome complex have greatly progressed, providing a molecular understanding of its function in health and disease. Neuronal porosomes are 15 nm cup-shaped lipoprotein structures composed of nearly 40 proteins, compared to the 120 nm nuclear pore complex composed of >500 protein molecules. Membrane proteins compose the porosome complex, making it practically impossible to solve its atomic structure. However, atomic force microscopy and small-angle X-ray solution scattering studies have provided three-dimensional structural details of the native neuronal porosome at sub-nanometer resolution, providing insights into the molecular mechanism of its function. The participation of several porosome proteins previously implicated in neurotransmission and neurological disorders, further attest to the crosstalk between porosome proteins and their coordinated involvement in release of neurotransmitter at the synapse.

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.