Membrane-bound Secretory Vesicles Research Articles

Dynamic polyhedral actomyosin lattices remodel micron-scale curved membranes during exocytosis in live mice.

Actomyosin networks, the cell's major force production machineries, remodel cellular membranes during myriad dynamic processes1,2 by assembling into various architectures with distinct force generation properties3,4. While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems3, it is not known how actin and myosin networks form and function to remodel membranes in complex three-dimensional mammalian tissues. Here, we use four-dimensional spinning-disc confocal microscopy with image deconvolution to acquire macromolecular-scale detail of dynamic actomyosin networks in exocrine glands of live mice. We address how actin and myosin organize around large membrane-bound secretory vesicles and generate the forces required to complete exocytosis5-7. We find that actin and non-muscle myosin II (NMII) assemble into previously undescribed polyhedral-like lattices around the vesicle membrane. The NMII lattice comprises bipolar minifilaments8-10 as well as non-canonical three-legged configurations. Using photobleaching and pharmacological perturbations in vivo, we show that actomyosin contractility and actin polymerization together push on the underlying vesicle membrane to overcome the energy barrier and complete exocytosis7. Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.

Ultrastructural changes in the sublingual salivary gland of prenatal buffalo (Bubalus bubalis)

Aim:The present study was aimed to elucidate ultrastructural changes in the development of sublingual salivary gland of buffalo during prenatal life.Materials and Methods:The study was carried out on sublingual salivary gland of 36 buffalo fetuses ranging from 13.2 cm curved crown-rump length (CVRL) (88th day) to full term. The fetuses were categorized into three groups based on their CVRL.Results:The cells lining the terminal tubules were undifferentiated with poorly developed cytoplasmic organelles but lacked secretory granules (SGs) at 13.2 cm CVRL (88th day). The SGs appeared first in the form of membrane-bound secretory vesicles with homogeneous electron-dense as well as electron-lucent contents at 21.2 cm CVRL (122nd day); however, mucous acinar cells contained electron-lucent granules, while serous secretory cells as well as serous demilunes showed electron-dense granules at 34 cm CVRL (150th day) of prenatal life. At 53.5 cm CVRL (194th day), both mucous and serous acini were differentiated by the density of SGs.Conclusion:The cytoplasm of acinar cells was filled with mitochondria, rough endoplasmic reticulum, and Golgi profiles in mid and late fetal age groups. The SGs were increased in number during the late fetal age group. The myoepithelial cells (MECs) were located at the base of the acinar cells as well as intercalated and striated ducts and were stellate in shape. The ultrastructure of MEC revealed a parallel stream of myofilaments in the cytoplasm and its processes. The mucous cells were predominantly present in the sublingual salivary gland and were pyramidal in shape.

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.

Neuronal porosome proteome: Molecular dynamics and architecture

Porosomes are the universal secretory portals at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel intravesicular contents to the outside during cell secretion. In the past decade, the neuronal porosome complex, a 10-15nm cup-shaped lipoprotein structure has been isolated, its partial composition and 3D contour map determined, and it has been functionally reconstituted into artificial lipid membrane. Here we further determine the composition of the neuronal porosome proteome using immunoisolation and gel filtration chromatography, followed by tandem mass spectrometry. Results from the study demonstrate nearly 40 proteins to constitute the neuronal porosome proteome. Furthermore, interaction of proteins within the porosome and their resulting arrangement is predicted. The association and dissociation of proteins at the porosome following stimulation of cell secretion demonstrate the dynamic nature of the organelle.

Hypokinetic stress and neuronal porosome complex in the rat brain: The electron microscopic study

Porosomes are the universal secretory machinery in cells, where membrane-bound secretory vesicles transiently dock and fuse to release intravesicular contents to the outside of the cell during cell secretion. Studies using atomic force microscopy, electron microscopy, electron density and 3D contour mapping, provided rich nanoscale information on the structure and assembly of proteins within the neuronal porosome complex in normal brain. However it remains uncertain whether pathological conditions that alter process of neurotransmission, provoke alterations in the porosome structure also. To determine if porosomes are altered in disease states, the current study was undertaken for first time using high resolution electron microscope. One of pathologies that produce subtle alteration at the presynaptic terminals has been demonstrated to be hypokinetic stress. The central nucleus of amygdale is the brain region, where such alterations are mostly expressed. We have examined the width and depth of the neuronal porosome complex and their alterations provoked by chronic hypokinetic stress in above mentioned limbic region. Specifically, we have demonstrated that despite alterations in the presynaptic terminals and synaptic transmission provoked by this pathological condition in this region, the final step/structure in neurosecretion – the porosome – remains unaffected: the morphometric analysis of the depth and diameter of this cup-shaped structure at the presynaptic membrane point out to the heterogeneity of porosome dimensions, but with unchanged fluctuation in norm and pathology.

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.

Identification of the porosome complex in the hair cell

Porosomes are proposed to be the universal secretory machinery of the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel their contents to the extracellular space during cell secretion. In neurons, porosomes are manifested as cup-shaped lipoprotein structures in the presynaptic membrane, 12–17 nm in diameter and possessing a central plug. Hair cells of hearing and balance secrete transmitter from synaptic vesicles in sensory signal transduction, but it has not previously been demonstrated that these mechanosensory cells possess porosome structures that could participate in the secretory process. In the present study, we provide evidence obtained using transmission electron microscopy that porosome structures exist in the hair cell, suggesting a mechanism of hair-cell transmitter secretion markedly different from that of the classic view of the exocytotic process.

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.

Two new species of Oregodasys (Gastrotricha: Macrodasyida: Thaumastodermatidae) from Carrie Bow Cay, Belize with ultrastructural observations of the epidermal glandular system

This study represents the first report of Gastrotricha from the coast of Belize. Two new species of Oregodasys (Macrodasyida: Thaumastodermatidae) are described from sublittoral sediments around the island of Carrie Bow Cay in the Belizean barrier reef complex. Oregodasys norenburgi sp. nov. is unique in the possession of paired red ocelli and in the quantity and distribution of lateral and posterior adhesive tubes. Oregodasys katharinae sp. nov. is allied with species that possess ventrolateral cirri in the posterior body region, but is distinguished based on the quantity of cirri and adhesive tubes and the bipartite structure of the caudal organ. Ultrastructural analysis of the bodywall of O. katharinae sp. nov. reveals a highly glandular epithelium covered by a bilayered cuticle consisting of a thin exocuticle and thicker endocuticle containing electron-dense elements. At least three types of papillae are present beneath the cuticle: blunt papillae, triangle-shaped papillae, and sensory papillae. The sensory papillae are two-part structures consisting of a porous epidermal cell and a monociliated epidermal cell. The ultrastructure of blunt papillae was not examined, but triangular papillae are formed from neck-like extensions of underlying glandulocytes and contain a pore to the external environment. Insunk glandulocytes are present between the various papillae and have an elongate neck that extends between epidermal cells. All glandulocytes contain a variety of membrane-bound secretory vesicles with a wide range of staining characteristics, from electron lucent to electron dense.

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.

Cell Secretion: Current Structural and Biochemical Insights

Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent) secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150nm in diameter in acinar cells of the exocrine pancreas to 12nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.

Porosome: The Secretory Portal in Cells

Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles dock and fuse to release intravesicular contents to the outside during cell secretion. The porosome opening to the outside ranges from 150 nm in diameter in acinar cells of the exocrine pancreas to 12 nm in neurons. In the past decade, the composition of the porosome, its structure and dynamics at nanometer resolution in real time, and its functional reconstitution into an artificial lipid membrane have been described. Discovery of the universal secretory machinery in cells, the porosome, came as no surprise since porosome-like "canaliculi" structures for secretion from human platelets, the secretory machinery in single-cell organisms like the secretion apparatus in bacteria and Toxoplasma gondii, and the contractile vacuole in paramecium have been demonstrated. In this review, the discovery of the porosome complex and the molecular mechanism of its function and how this information provides a new understanding of cell secretion are discussed.

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.

Stimulation inhibits the mobility of recycling peptidergic vesicles in astrocytes

Astrocytes are increasingly viewed as playing many roles in the integration of brain function. These cells store among other gliotransmitters also neuroactive peptides in membrane bound vesicles, the trafficking and release of which, may be changed in altered conditions, therefore affecting the physiological status of neurons. In general, peptidergic membrane-bound secretory vesicles fuse with the plasma membrane in the process of exocytosis. Some of them are retrieved from the plasma membrane to be recycled back into the cytosol. The mobility of retrieving vesicles in astrocytes was not studied yet, however, understanding the mechanisms of such trafficking would highlight the communication paths between astrocytes and neurons. We labeled vesicles with antibodies against the vesicle atrial natriuretic peptide (ANP), which is stored inside secretory vesicles. ANP-vesicles in astrocytes have been proposed to enter Ca2+-dependent secretion and here we show that they are associated with synaptotagmin IV (SytIV), a regulator of exocytosis in astrocytes. Moreover, the results show that recycling ANP-vesicles are to a significant extent acidic. Their velocity (0.06+/-0.001 microm/s) is one order of magnitude lower than the velocity of vesicles trafficking to the plasma membrane (Potokar et al. (2005) Biochem Biophys Res Commun 329:678-683; Potokar et al. (2007) Traffic 8:12-20). Interestingly, ionomycin or ATP application further attenuated ANP-vesicle mobility to 0.02+/-0.002 and to 0.03+/-0.001 microm/s, respectively. In summary, the mobility of recycling peptidergic vesicles appears to be slower than the vesicle traffic to the plasma membrane and it requires an intact cytoskeleton. Physiological implications of attenuated traffic of ANP-vesicles are considered in the discussion.

Prostasomes, Angiogenesis, and Tissue Factor

Prostasomes are membrane-bound secretory vesicles produced by prostatic epithelial cells. They are known to carry many proteins, including tissue factor, and have membranes unusually rich in cholesterol and sphingomyelin. Prostasomes have well-documented effects on fertility, promoting sperm motility, stabilizing the acrosome reaction, and facilitating immunosuppression. This article reviews the evidence of the effects of prostasomes on in vitro angiogenesis assays, and the mechanism by which these effects occur. Seminal prostasomes seem to inhibit angiogenesis, whereas the equivalent particles released by malignant prostate cells promote angiogenesis. In both cases, the effects seem preserved after heat treatment to denature the protein content, suggesting an important role for lipid transfer, in particular, transfer of sphingomyelin.

Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2–1.2 μm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.

Discovery of the ‘porosome’ the universal secretory machinery in cells

The release of neurotransmitters at the nerve terminal for neurotransmission, release of insulin from β-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.

Patch Clamped Single Pancreatic Zymogen Granules: Direct Measurements of Ion Channel Activities at the Granule Membrane

Background/Aim: Pancreatic acinar cells are involved in the secretion of digestive enzymes. Digestive enzymes in pancreatic acinar cells are stored in membrane-bound secretory vesicles called zymogen granules (ZGs). The swelling of ZGs is implicated in the regulation of the expulsion of intravesicular contents during secretion. The molecular mechanism of ZG swelling has been previously elucidated. It has been further demonstrated that the water channel aquaporin-1, the potassium channel IRK-8, and the chloride channel CLC-2, are present in the ZG membrane and involved in ZG swelling. However, a direct measurement of these ion channels at the ZG membrane in intact ZGs had not been performed. The aim of this study was to investigate the electrical activity of single ZGs and verify the types of channels found within their membrane. Methods: ZGs from pancreatic acinar cells were isolated from the pancreas of Sprague-Dawley rats. Direct measurements of whole vesicle currents, in the presence and absence of ion channel blockers (quinine, glyburide and DIDS), were recorded following successful patching of single ZGs. Conclusion: In this study, we were able, for the first time, to patch single ZGs and study ion channels in their membrane. We were able to record currents across the ZG membrane and, utilizing ion channel blockers, confirm the presence of the chloride channels CLC-2 and the potassium channel IRK-8 (Kir6.1), and additionally demonstrate the presence of a second chloride channel CLC-3.

Cell secretion and membrane fusion

Secretion occurs in all cells of multicellular organisms and involves the delivery of secretory products packaged in membrane-bound vesicles to the cell exterior. Specialized cells for neurotransmission, enzyme secretion or hormone release utilize a highly regulated secretory process. Secretory vesicles are transported to specific sites at the plasma membrane, where they dock and fuse to release their contents. Similar to other cellular processes, cell secretion is found to be highly regulated and a precisely orchestrated event. It has been demonstrated that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized supramolecular structures at the cell plasma membrane. Swelling of secretory vesicles results in a build-up of pressure, allowing expulsion of intravesicular contents. The extent of secretory vesicle swelling dictates the amount of intravesicular contents expelled during secretion. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution into artificial lipid membrane, have been determined. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, has also been resolved. These findings reveal the molecular mechanism of cell secretion.

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.