Conformation states of the neuronal porosome complex

Porosomes are the universal secretory machinery of the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel intravesicular contents to the environment during cell secretion. In neurons, 12- to 17-nm cup-shaped lipoprotein structures possessing a central plug are present at the presynaptic membrane, where 40-50 nm in diameter synaptic vesicles transiently dock and fuse to release neurotransmitters. The neuronal porosome complex has been isolated, its composition determined and it has been both structurally and functionally reconstituted in artificial lipid membranes. Earlier studies using AFM (atomic force microscopy), EM (electron microscopy), electron density and 3D contour mapping provide the structure and assembly of proteins within the neuronal porosome complex at the nanoscale level. A set of eight protein units lining the neuronal porosome cup is present, each connected via spoke-like elements to a central plug, hypothesized for the rapid opening and closing of the structure to the outside. In the present study, ultrahigh-resolution imaging of the presynaptic membrane of isolated synaptosome preparations demonstrate, for the first time, the presence of neuronal porosomes in both their open and close conformations. The results suggests that the central plug is retracted into the porosome cup in its open conformation and pushed outward to seal the porosome opening, supporting the hypothesis that it operates as the opening-closing device of the complex.     

Neuronal porosome in the rat and cat brain

It is well established that during cell secretion, membrane-bound secretory vesicles dock and fuse at the base of supramolecular cup-shaped structures at the cell plasma membrane called "porosomes", to expel intra-vesicular contents to the outside. In neurons, it has been demonstrated that 12-17 nm cup-shaped lipoprotein structure possessing a central plug are present at the presynaptic membrane, where 50 nm in diameter synaptic vesicles transiently dock and fuse to release neurotransmitter. In the past decade, the neuronal porosome has been isolated and its major chemical composition determined. Additionally, the porosome has been both structurally and functionally reconstituted into artificial lipid membrane, establishing its role as the secretory portal in neurons. Studies utilizing atomic force and electron microscopy, combined with electron density and 3D contour mapping, provide at the nanoscale, the structure and assembly of proteins within the neuronal porosome. In the current study, ultrahigh resolution imaging of the presynaptic membrane of isolated brains from both rats and cats, demonstrate for the first time, the presence of neuronal porosomes in cat brain, and further confirms the presence of porosomes at the presynaptic membrane in rat brain synaptosomes. Results from the present study further confirm the cup-shaped morphology of porosomes in the rat brain, and demonstrates their similar shape and size in the cat nerve terminal. The study also demonstrates for the first time, the universal presence of similar porosomes in different species of mammals.     

Neuronal porosome in the rat and cat brain

It is well established that during cell secretion, membrane-bound secretory vesicles dock and fuse at the base of supramolecular cup-shaped structures at the cell plasma membrane called “porosomes”, to expel intra-vesicular contents to the outside. In neurons, it has been demonstrated that 12–17 nm cupshaped lipoprotein structure possessing a central plug are present at the presynaptic membrane, where 50 nm in diameter synaptic vesicles transiently dock and fuse to release neurotransmitters. In the past decade, the neuronal porosome has been isolated and its major chemical composition determined. Additionally, the porosome has been both structurally and functionally reconstituted into artificial lipid membrane, establishing its role as the secretory portal in neurons. Studies utilizing atomic force and electron microscopy, combined with electron density and 3D contour mapping, provide at the nanoscale, the structure and assembly of proteins within the neuronal porosome. In the current study, ultrahigh resolution imaging of the presynaptic membrane of isolated brains from both rats and cats, demonstrate for the first time, the presence of neuronal porosomes in cat brain, and further confirms the presence of porosomes at the presynaptic membrane in rat brain synaptosomes. Results from the present study further confirm the cup-shaped morphology of porosomes in the rat brain, and demonstrates their similar shape and size in the cat nerve terminal. The study also demonstrates for the first time, the universal presence of similar porosomes in different species of mammals.     

The story of cell secretion: events leading to the discovery of the 'porosome' - the universal secretory machinery in cells

Cell secretion has come of age, and a century old quest has been elegantly solved. We have come a long way since earlier observations of what appeared to be 'fibrillar regions' at the cell plasma membrane, and electrophysiological studies suggesting the presence of 'fusion pores' at the cell plasma membrane where secretion occurs. Finally, the fusion pore or 'porosome' has been discovered, and its morphology and dynamics determined at nm resolution and in real time in live secretory cells. The porosome has been isolated, its composition determined and it has been both structurally and functionally reconstituted in artificial lipid membrane. The discovery of the porosome as the universal secretory machinery in cells and the discovery of the molecular mechanism of vesicular content expulsion during cell secretion have finally enabled a clear understanding of this important cellular process. This review outlines the fascinating and exciting journey leading to the discovery of the porosome, ultimately solving one of the most difficult, significant, and fundamental cellular process -cell secretion.     

Nanoscale 3D contour map of protein assembly within the astrocyte porosome complex

The astrocyte porosome complex, the secretory machinery at the plasma membrane of astrocytes, is a 10-15 nm cup-shaped lipoprotein structure possessing a central plug. Since the porosome is a membrane-associated, multi-protein complex, it has precluded the generation of 3D crystals for X-ray diffraction studies, nor structural analysis at the atomic level using the solution NMR. These limitations were partially overcome in the current studies, furthering our understanding of the porosome structure in astrocytes. Using atomic force microscopy, electron microscopy, and electron density and 3D contour mapping, finally provides at the nanoscale, the structure and assembly of proteins within the astrocyte porosome complex. Results from this study demonstrate a set of protein units lining the porosome cup, each connected via spoke-like elements to a central plug region within the structure. In contrast to the neuronal porosome, which possess eight globular proteins at the outer rim of the complex, the porosome complex appear to possess 12 such globular structures. Nature has designed the porosome as the universal secretory machinery, but has fine-tuned its use to suite secretion from different cell types. The isolation of intact astrocyte porosomes for near-atomic resolution using cryo-electron diffraction measurements, is finally possible.     

‘Porosome’ discovered nearly 20 years ago provides molecular insights into the kiss‐and‐run mechanism of cell secretion

Secretion is a fundamental cellular process in living organisms, from yeast to cells in humans. Since the 1950s, it was believed that secretory vesicles completely merged with the cell plasma membrane during secretion. While this may occur, the observation of partially empty vesicles in cells following secretion suggests the presence of an additional mechanism that allows partial discharge of intra‐vesicular contents during secretion. This proposed mechanism requires the involvement of a plasma membrane structure called ‘porosome’, which serves to prevent the collapse of secretory vesicles, and to transiently fuse with the plasma membrane (Kiss‐and‐run), expel a portion of its contents and disengage. Porosomes are cup‐shaped supramolecular lipoprotein structures at the cell plasma membrane ranging in size from 15 nm in neurons and astrocytes to 100–180 nm in endocrine and exocrine cells. Neuronal porosomes are composed of nearly 40 proteins. In comparison, the 120 nm nuclear pore complex is composed of >500 protein molecules. Elucidation of the porosome structure, its chemical composition and functional reconstitution into artificial lipid membrane, and the molecular assembly of membrane‐associated t‐SNARE and v‐SNARE proteins in a ring or rosette complex resulting in the establishment of membrane continuity to form a fusion pore at the porosome base, has been demonstrated. Additionally, the molecular mechanism of secretory vesicle swelling, and its requirement for intra‐vesicular content release during cell secretion has also been elucidated. Collectively, these observations provide a molecular understanding of cell secretion, resulting in a paradigm shift in our understanding of the secretory process.     

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 G_αo 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 (PIP's) 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 PIP's and PA. The ability of exogenous PA to alter protein–protein interaction and neurotransmitter release is further demonstrated from the study.     

Direct interaction between SNAP-23 and L-type Ca2+ channel

During secretion, membrane-bound secretory vesicles dock and fuse at the base of porosomes in the cell plasma membrane. Among other proteins, the porosome is composed of SNAREs and Ca2+-channels. Ca2+-channels and SNAREs have been implicated in cell secretion. Several immunoprecipitation and binding studies suggest the physical interaction of the t-SNARE proteins, Syntaxin-1 and SNAP-25 with various Ca2+-channels. In this study, using yeast two-hybrid and immunoanalysis, we demonstrate for the first time, direct interaction of SNAP-23 and a L-type Ca2+-channel at the plasma membrane in pancreas.     

Molecular machinery and mechanism of cell secretion

Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them dock and fuse at plasma membrane-associated supramolecular structures, called porosomes, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release use a highly regulated secretory process. Similar to other fundamental cellular processes, cell secretion is precisely regulated. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the porosome as the universal secretory machinery, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The molecular mechanism of secretory vesicle swelling and the fusion of opposing bilayers, that is, the fusion of secretory vesicle membrane at the base of the porosome membrane, have also been resolved. These findings reveal, for the first time, the universal molecular machinery and mechanism of secretion in cells.     

Porosome in astrocytes

Secretion is a universal cellular process occurring in bakers yeast, to the complex multicellular organisms, to humans beings. Neurotransmission, digestion, immune response or the release of hormones occur as a result of cell secretion. Secretory defects result in numerous diseases and hence a molecular understanding of the process is critical. Cell secretion involves the transport of vesicular products from within cells to the outside. Porosomes are permanent cup-shaped supramolecular structures at the cell plasma membrane, where secretory vesicles transiently dock and transiently fuse to release intravesicular contents to the outside. In the past decade, porosomes have been determined to be the universal secretory machinery in cells, present in the exocrine pancreas, endocrine and neuroendocrine cells, and in neurons. In this study, we report for the first time the presence of porosomes in rat brain astrocytes. Using atomic force microscopy on live astrocytes, cup-shaped porosomes measuring 10–15 nm are observed at the cell plasma membrane. Further studies using electron microscopy confirm the presence of porosomes in astrocytes. Analogous to neuronal porosomes, there is a central plug in the astrocyte porosome complex. Immunoisolation and reconstitution of the astrocyte porosome in lipid membrane, demonstrates a structure similar to what is observed in live cells. These studies demonstrate that in astrocytes, the secretory apparatus at the cell plasma membrane is similar to what is found in neurons.     

Cell secretion: an update

This past decade has witnessed the publication of a flurry of scientific papers and reports on the subject of cell secretion, following discovery of a permanent plasma membrane structure termed 'porosome' and its determination as the universal secretory machinery in cells. This discovery has led to a paradigm shift in our understanding of the secretory process, demonstrating that membrane-bound secretory vesicles transiently dock and fuse at the porosome base to release their contents to the cell exterior. The regulated release of intravesicular contents during cell secretion is governed by dilation of the porosome opening to the outside, and the extent of vesicle swelling. In agreement, a great number of articles have been written and studies performed, which are briefly discussed in this article.     

Discovery of the Porosome: revealing the molecular mechanism of secretion and membrane fusion in cells

Secretion and membrane fusion are fundamental cellular processes involved in the physiology of health and disease. Studies within the past decade reveal the molecular mechanism of secretion and membrane fusion in cells. Studies reveal that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized plasma membrane structures. Swelling of secretory vesicles result in a build-up of intravesicular pressure, which allows expulsion of vesicular contents. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution, are discussed. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, have been resolved. With these findings a new understanding of cell secretion has emerged and confirmed by a number of laboratories.     

Discovery of the 'porosome'; the universal secretory machinery in cells

The release of neurotransmitters at the nerve terminal for neurotransmission, release of insulin from beta-cells of the endocrine pancreas for regulating blood glucose levels, the release of growth hormone from GH cells of the pituitary gland to regulate body growth, or the expulsion of zymogen from exocrine pancreas to digest food, are only a few examples of key physiological processes made possible by cell secretion. It comes as no surprise that defects in cell secretion are the cause for numerous diseases, and have been under intense investigation for over half century. Only in the last decade, the molecular machinery and mechanism of cell secretion has become clear. Cell secretion involves the docking and transient fusion of membrane-bound secretory vesicles at the base of plasma membrane structures called porosomes, and the regulated expulsion of intravesicular contents to the outside, by vesicle swelling. The discovery of the porosome in live cells, its morphology and dynamics at nanometer resolution and in real time, its isolation, its composition, and its structural and functional reconstitution in lipid membrane, are complete. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and the regulated expulsion of intravesicular contents during cell secretion, are also resolved. In this minireview, the monumental discovery of the porosome, a new cellular structure at the cell plasma membrane, is briefly discussed.     

Neuronal fusion pore assembly requires membrane cholesterol

Cholesterol has been proposed to play a critical role in regulating neurotransmitter release and synaptic plasticity. The neuronal porosome/fusion pore, the secretory machinery at the nerve terminal, is a 12-17 nm cup-shaped lipoprotein structure composed of cholesterol and a number of proteins, among them calcium channels, and the t-SNARE proteins Syntaxin-1 and SNAP-25. During neurotransmission, synaptic vesicles dock and fuse at the porosome via interaction of their v-SNARE protein with t-SNAREs at the porosome base. Membrane-associated neuronal t-SNAREs interact in a circular array with liposome-associated neuronal v-SNARE to form the t-/v-SNARE ring complex. The SNARE complex along with calcium is required for the establishment of continuity between opposing bilayers. Here we show that although cholesterol is an integral component of the neuronal porosome and is required for maintaining its physical integrity and function, it has no influence on the conformation of the SNARE ring complex.     

Cellular secretion studied by force microscopy

Using the optical microscope, real adventures in cellular research began in earnest in the latter half of the nineteenth century. With the development of the electron microscope, ultramicroscopy, and improved cell staining techniques, significant advances were made in defining intracellular structures at the nanometer level. The invention of force microscopy, the atomic force microscope (AFM) in the mid 1980s, and the photonic force microscope (PFM) in the mid 1990s, finally provided the opportunity to study live cellular structure-function at the nanometer level. Working with the AFM, dynamic cellular and subcellular events at the molecular level were captured in the mid 1990s, and a new cellular structure 'the porosome' in the plasma membrane of all secretory cells has been defined, where specific docking and fusion of secretory vesicles occur. The molecular mechanism of fusion of the secretory vesicle membrane at the base of the porosome membrane in cells, and the regulated release of intravesicular contents through the porosome opening to the extracellular space, has been determined. These seminal discoveries provide for the first time a molecular mechanism of cell secretion, and the possibility to ameliorate secretory defects in disease states.     

Discovery of a new cellular structure--porosome

A new cell structure--"porosome", discovered by the American scientist Bhanu Jena and co-wokers, is described. Mechanisms of budding and fusion of transport vesicle are elucidated in addition to those of fusion of secretory vesicles at the cell plasma membrane, and of release of intravesicular contents. The morphology of porosomes, their contents and functional reconstruction in lipid bilayer membranes were examined at a near nanometer resolution. Using atomic force microscopy, the presence of circular "pits", measuring 400-1200 nm in diameter with small 100-150 nm wide "depressions" inside and 3-4 deep pores, called porosomes, was demonstrated. A porosome is cup-shaped and 15-30 nm wide. Porosomes are the places where secretory vesicles fuse with the plasma cell membrane, and where the intravesicular content is released.     

Defining the role of the Escherichia coli chaperone SecB using comparative proteomics

To improve understanding and identify novel substrates of the cytoplasmic chaperone SecB in Escherichia coli, we analyzed a secB null mutant using comparative proteomics. The secB null mutation did not affect cell growth but caused significant differences at the proteome level. In the absence of SecB, dynamic protein aggregates containing predominantly secretory proteins accumulated in the cytoplasm. Unprocessed secretory proteins were detected in radiolabeled whole cell lysates. Furthermore, the assembly of a large fraction of the outer membrane proteome was slowed down, whereas its steady state composition was hardly affected. In response to aggregation and delayed sorting of secretory proteins, cytoplasmic chaperones DnaK, GroEL/ES, ClpB, IbpA/B, and HslU were up-regulated severalfold, most likely to stabilize secretory proteins during their delayed translocation and/or rescue aggregated secretory proteins. The SecB/A dependence of 12 secretory proteins affected by the secB null mutation (DegP, FhuA, FkpA, OmpT, OmpX, OppA, TolB, TolC, YbgF, YcgK, YgiW, and YncE) was confirmed by "classical" pulse-labeling experiments. Our study more than triples the number of known SecB-dependent secretory proteins and shows that the primary role of SecB is to facilitate the targeting of secretory proteins to the Sec-translocase.     

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.     

Vesicle swelling regulates content expulsion during secretion

The involvement of secretory vesicle swelling has been proposed in secretion; however, little is known about its role. Using both the pancreatic acinar cell and neuronal model, we show secretory vesicle swelling in live cells. Our study reveals that vesicle swelling potentiates its fusion at the cell plasma membrane, and is required for expulsion of intravesicular contents. Since the extent of swelling is directly proportional to the amount of vesicular contents expelled, this provides cells with the ability to regulate release of secretory products. These direct observations of the requirement of secretory vesicle swelling in secretion, provides an understanding of the appearance of partially empty vesicles following the process.     

Small GTP-binding proteins in parietal cells: candidate modulators of parietal cell membrane dynamics.

The stimulated fusion of intracellular H/K-ATPase-containing tubulovesicles with a target canalicular membrane surface is central to the process of acid secretion. A super-family of small GTP-binding proteins (smGTPBPs) has been implicated in many aspects of intracellular dynamics and vesicle membrane trafficking. We have investigated the presence of smGTPBPs in isolated rabbit parietal cells. Parietal cells possess a number of smGTPBP species with molecular masses of 18-28 kDa. One 23 kDa smGTPBP has been localized to tubulovesicles and identified immunochemically as rab2. Rab2 redistributes during stimulation in concert with the movement of the H/K-ATPase. The results demonstrate that specific smGTPBPs are associated with the parietal cell secretory apparatus. Small GTP-binding proteins are important candidate regulators of parietal secretory membrane dynamics.