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Porosome - The Secretory NanoMachine in Cells

Porosome - The Secretory NanoMachine in Cells

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06/16/2014

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345
Douglas J. Taatjes and Jürgen Roth (eds.),
Cell Imaging Techniques: Methods and Protocols
, Methods in Molecular Biology,vol. 931, DOI 10.1007/978-1-62703-056-4_17, © Springer Science+Business Media, LLC 2012
 Chapter 17
Porosome: The Secretory NanoMachine in Cells
Bhanu P. Jena
Abstract
Cells synthesize and store within membranous sacs products such as hormones, growth factors,neurotransmitters, or digestive enzymes, for release on demand. As recently as just 15 years ago, it wasbelieved that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasmamembrane resulting in the diffusion of intravesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation, however, failed to explain the genera-tion of partially empty vesicles observed in electron micrographs following secretion. Logically therefore,in a 1993 News and Views article in the journal
Nature 
 , Prof. Erwin Neher wrote “It seems terribly waste-ful that, during the release of hormones and neurotransmitters from a cell, the membrane of a vesicleshould merge with the plasma membrane to be retrieved for recycling only seconds or minutes later.” Thediscovery of permanent secretory portals or nanomachines at the cell plasma membrane calledPOROSOMES, where membrane-bound secretory vesicles transiently dock and fuse to release intravesicu-lar contents to the cell exterior, has finally resolved this conundrum. Following this discovery, the composi-tion of the porosome, its structure and dynamics visualized with high-resolution imaging techniquesatomic force and electron microscopy, and its functional reconstitution into articial lipid membrane haveprovided a molecular understanding of cell secretion. In agreement, it has been demonstrated that “secre-tory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells” (ProcNatl Acad Sci U S A 100:2070–2075, 2003); that “single synaptic vesicles fuse transiently and successively  without loss of identity” (Nature 423:643–647, 2003); and that “zymogen granule exocytosis is character-ized by long fusion pore openings and preservation of vesicle lipid identity” (Proc Natl Acad Sci U S A 101:6774–6779, 2004). It made no sense all these years to argue that mammalian cells possess an “all ornone” mechanism of cell secretion resulting from complete vesicle merger at the cell plasma membrane, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such asthe secretion apparatus of 
Toxoplasma gondii 
 , contractile vacuoles in paramecium, and different types of secretory structures in bacteria. The discovery of the porosome and its functional reconstitution in articiallipid membrane, and an understanding of its morphology, composition, and dynamics, has resulted in aparadigm shift in our understanding of the secretory process in cells.
Key words:
Porosome, Fusion pore, Secretion, Membrane fusion
 
346B.P. Jena
 
For nearly half a century, the prevailing notion was that during cellsecretion, membrane-bound secretory vesicles completely mergeat the cell plasma membrane resulting in the diffusion of intrave-sicular contents to the outside and the compensatory retrieval of the excess membrane by endocytosis. This explanation of cellularsecretion, however, contradicted the observation of partially empty  vesicles in electron micrographs following secretion. Further, suchan “all or none” mechanism of cell secretion by complete mergerof secretory vesicle membrane at the cell plasma membrane leaveslittle regulation and control by the cell on the amount of contentrelease. Furthermore, it made no sense for mammalian cells to uti-lize such “all or none” process of cell secretion, when even single-cell organisms have developed specialized and sophisticatedsecretory machinery, such as the secretion apparatus of 
Toxoplasma  gondii 
 , the contractile vacuoles in paramecium, or the various typesof secretory structures in bacteria. Therefore in the 1960s, experi-mental data concerning neurotransmitter release mechanisms by Katz and Folkow ( 1, 2 ) advanced that limitation of the quantal packet may be set by the nerve membrane, in which case the sizeof the packet may actually correspond to just a fraction of the ves-icle content ( 3, 4 ). Again, in 1993 in a
News and Views 
article inthe journal
Nature 
( 5 ), E. Neher noted “It seems terribly wasteful that, during the release of hormones and neurotransmitters from acell, the membrane of a vesicle should merge with the plasma mem-brane to be retrieved for recycling only seconds or minutes later.”This conundrum on the molecular mechanism of cell secretion wasnally resolved in 1996 (( 6 ), published on-line ahead of print) fol- lowing discovery of the “porosome,” a nanomachine at the cellplasma membrane and the universal secretory portal in cells.Porosomes are supramolecular lipoprotein structures at the cellplasma membrane, where membrane-bound secretory vesiclestransiently dock and fuse to release intravesicular contents to theoutside. In the past 15 years, the composition of the porosome, itsstructure and dynamics at nanometer resolution and in real time,and its functional reconstitution into articial lipid membrane havebeen determined ( 6– 42 ). Isolated live pancreatic acinar cells in near physiological buffer when imaged using atomic force micros-copy (AFM) demonstrate the size and shape of the secretory vesi-cles called zymogen granules (ZG) lying immediately below theapical plasma membrane of the cell ( 43 ). Following stimulation of  secretion, secretory products are released with no loss in vesicles( 43 ). Furthermore, since porosomes in exocrine and neuroendo- crine cells measure 100–180 nm, and only a 20–45% increase inporosome diameter is demonstrated following the docking andfusion of 0.2–1.2
μ
 m in diameter secretory vesicles, it is concluded
1. Introduction
 
34717 Porosome: The Secretory NanoMachine in Cells
that secretory vesicles “transiently” dock and fuse, as opposed tocomplete merger at the porosome base, to release intravesicularcontents to the outside. In agreement, it has also been demon-strated that “secretory granules are recaptured largely intact afterstimulated exocytosis in cultured endocrine cells” ( 44 ); that “single synaptic vesicles fuse transiently and successively withoutloss of identity” ( 45 ); and that in the “zymogen granule (the secretory vesicle in exocrine pancreas) exocytosis is characterizedby long fusion pore openings and preservation of vesicle lipididentity” ( 46 ). Microtubules have been recognized as the railroad for move-ment of organelles over long distances within the cell (>1 mm), whereas the actin system is responsible for transport over shorterdistances, typically from tens to a few hundred nanometers. Thus,microtubule-dependent motors such as kinesin and kinesin-relatedproteins, and the superfamily of actin-dependent myosin motors,have all been implicated in intracellular organelle transport ( 47,  48 ). Myosin motors include the conventional myosin (myosin II) and a large group of unconventional myosins (myosin I, III, V, and VI). In recent years, the prime candidate for secretory vesicle trans-port in cells has been reported to be the class V of myosin motors( 49– 51 ). Myosin V is composed of two heavy chains that dimerise  via a coiled-coil motif, located in the stalk region of the heavy chain( 52 ). The heavy chain contains an amino-terminal actin-binding motor domain ( 52 ), followed by a neck region where up to six regulatory light chains can bind. The carboxy-terminus globulardomain of the heavy chain is thought to mediate organelle-bindingspecicity ( 53 ). Interaction between the actin and the microtubule transport system seems to be a requirement for the correct delivery of intracellular cargo such as secretory vesicles ( 54– 56 ). Studies have been undertaken to determine whether secretory vesicles inlive cells remain free-oating, only to associate with the transportsystems following a secretory stimulus, or whether they are alwaystethered. Studies using isolated live pancreatic acinar cells demon-strate that all secretory vesicles within the cell are tethered and notfree-oating ( 57 ). Nocodazole and cytochalasin B disrupt much of  this tether. Immunoblot analysis of isolated secretory vesicles furtherdetermines the association of actin, myosin V, and kinesin to them( 57 ). These studies demonstrate for the first time that secretory   vesicles in live pancreatic acinar cells are tethered and not free- oating, suggesting that following vesicle biogenesis they areplaced on their own specic railroad track, ready to be transportedto their final destination when required ( 57 ). Intuitively, this makes sense, since precision and regulation are the hallmarks of all cellularprocesses, and therefore would also hold true for the transport andlocalization of subcellular organelles within the cell.Using the cellular railroad system, once secretory vesicles dock at the porosome base following a secretory stimulus, the fusion of 

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