Porosome: the universal secretory portal in cells

In the past 50 years it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the 'all or none' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. Moreover, it makes no sense for mammalian cells to possess such 'all or none' mechanism of cell secretion, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such as the secretion apparatus of Toxoplasma gondii, the contractile vacuoles in paramecium, or the various types of secretory structures in bacteria. Therefore, in the 1960's, experimental data concerning neurotransmitter release mechanisms by B. Katz and B. Folkow brilliantly hypothesized that limitation of the quantal packet may be set by the nerve membrane, in which case the size of the packet may actually correspond to just a fraction of the vesicle content. This conundrum in the molecular mechanism of cell secretion was finally resolved in 1997 following discovery of the porosome, the universal secretory machinery in the cell. Porosomes are supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release inravesicular contents to the outside during cell secretion. In the past decade, the composition of the porosome, its structure and dynamics at nanometer (nm) resolution and in real time, and its functional reconstitution into artificial lipid membrane, have all been elucidated. Since porosomes in exocrine and neuroendocrine cells measure 100-180 nm, and only 20-45% increase in porosome diameter is demonstrated following the docking and fusion of secretory vesicles (0.2-1.2 μm in diameter), it is concluded that secretory vesicles "transiently" dock and fuse, rather than completely merge at the base of the porosome complex to release their contents to the outside. In agreement, it has been demonstrated that "secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells"; that "single synaptic vesicles fuse transiently and successively without loss of identity"; and that "zymogen granule (the secretory vesicle in exocrine pancreas) exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity". In this review, the discovery of the porosome, resulting in a paradigm shift in our understanding of cell secretion, is briefly presented.Biomedical Reviews 2010; 21: 1-15

Self-Assembly and Disassembly of the SNARE Complex: Examined Using Circular Dichroism and Atomic Force Microscopy

In this study, we report for the first time that both t-SNAREs and v-SNARE and their complexes in buffered suspension, exhibit defined peaks at CD signals of 208 and 222 nm wavelengths, consistent with a higher degree of helical secondary structure. Surprisingly, when incorporated in lipid membrane, both SNAREs and their complexes exhibit reduced folding. In presence of NSF-ATP, the SNARE complex disassembles, as reflected from the CD signals demonstrating elimination of α-helices within the structure

Gd-Doped Superparamagnetic Magnetite Nanoparticles for Potential Cancer Theranostics

Nanotechnology has facilitated the applications of a class of nanomaterials called superparamagnetic iron oxide nanoparticles (SPIONs) in cancer theranostics. This is a new discipline in biomedicine that combines therapy and diagnosis in one platform. The multifunctional SPIONs, which are capable of detecting, visualizing, and destroying the neoplastic cells with fewer side effects than the conventional therapies, are reviewed in this chapter for theranostic applications. The chapter summarizes the design parameters such as size, shape, coating, and target ligand functionalization of SPIONs, which enhance their ability to diagnose and treat cancer. The review discusses the methods of synthesizing SPIONs, their structural, morphological, and magnetic properties that are important for theranostics. The applications of SPIONs for drug delivery, magnetic resonance imaging, and magnetic hyperthermia therapy (MHT) are included. The results of our recent MHT study on Gd-doped SPION as a possible theranostic agent are highlighted. We have also discussed the challenges and outlook on the future research for theranostics in clinical settings

Structure of membrane-associated neuronal SNARE complex: implication in neurotransmitter release.

To enable fusion between biological membranes, t-SNAREs and v-SNARE present in opposing bilayers, interact and assemble in a circular configuration forming ring-complexes, which establish continuity between the opposing membranes, in presence of calcium ions. The size of a t-/v-SNARE ring complex is dictated by the curvature of the opposing membrane. Hence smaller vesicles form small SNARE-ring complexes, as opposed to large vesicles. Neuronal communication depends on the fusion of 40-50 nm in diameter membrane-bound synaptic vesicles containing neurotransmitters at the nerve terminal. At the presynaptic membrane, 12-17 nm in diameter cup-shaped neuronal porosomes are present where synaptic vesicles transiently dock and fuse. Studies demonstrate the presence of SNAREs at the porosome base. Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t-SNAREs, are found assembled in a ring conformation. To further determine the structure and arrangement of the neuronal t-/v-SNARE complex, 50 nm t-and v-SNARE proteoliposomes were mixed, allowing t-SNARE-vesicles to interact with v-SNARE vesicles, followed by detergent solubilization and imaging of the resultant t-/v-SNARE complexes formed using both AFM and EM. Our results demonstrate formation of 6-7 nm membrane-directed self-assembled t-/v-SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes. The smaller SNARE ring at the porosome base may reflect the 3-4 nm base diameter, where 40-50 nm in diameter v-SNARE-associated synaptic vesicle transiently dock and fuse to release neurotransmitters

Structure of membrane-associated neuronal SNARE complex: implication in neurotransmitter release.

To enable fusion between biological membranes, t-SNAREs and v-SNARE present in opposing bilayers, interact and assemble in a circular configuration forming ring-complexes, which establish continuity between the opposing membranes, in presence of calcium ions. The size of a t-/v-SNARE ring complex is dictated by the curvature of the opposing membrane. Hence smaller vesicles form small SNARE-ring complexes, as opposed to large vesicles. Neuronal communication depends on the fusion of 40-50 nm in diameter membrane-bound synaptic vesicles containing neurotransmitters at the nerve terminal. At the presynaptic membrane, 12-17 nm in diameter cup-shaped neuronal porosomes are present where synaptic vesicles transiently dock and fuse. Studies demonstrate the presence of SNAREs at the porosome base. Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t-SNAREs, are found assembled in a ring conformation. To further determine the structure and arrangement of the neuronal t-/v-SNARE complex, 50 nm t-and v-SNARE proteoliposomes were mixed, allowing t-SNARE-vesicles to interact with v-SNARE vesicles, followed by detergent solubilization and imaging of the resultant t-/v-SNARE complexes formed using both AFM and EM. Our results demonstrate formation of 6-7 nm membrane-directed self-assembled t-/v-SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes. The smaller SNARE ring at the porosome base may reflect the 3-4 nm base diameter, where 40-50 nm in diameter v-SNARE-associated synaptic vesicle transiently dock and fuse to release neurotransmitters

Reconstituted Fusion Pore

Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the pores. Transmission electron microscopy (TEM) further confirmed the presence of fusion pores, and immunoAFM, and immunochemical studies demonstrated t-SNAREs to localize at the base of the fusion pore. In the present study, the morphology, function, and composition of the immunoisolated fusion pore was investigated. TEM studies reveal in further detail the structure of the fusion pore. Immunoblot analysis of the immunoisolated fusion pore reveals the presence of several isoforms of the proteins, identified earlier in addition to the association of chloride channels. TEM and AFM micrographs of the immunoisolated fusion pore complex were superimposable, revealing its detail structure. Fusion pore reconstituted into liposomes and examined by TEM, revealed a cup-shaped basket-like morphology, and were functional, as demonstrated by their ability to fuse with isolated secretory vesicles