Mol. Cells OS, 517-529, December 31, 2008 Molecules Minireview and Cells ©2008 KSMCB Porosome: the Universal Molecular Machinery for Cell Secretion Bhanu P. Jena* Porosomes are supramolecular, lipoprotein structures at vesicles following secretion. The journey leading to the discov- the cell plasma membrane, where membrane-bound secre- ery of the ‘éçêçëçãÉ’, a nanometer-size structure at the cell tory vesicles transiently dock and fuse to release inrave- plasma membrane -the universal secretory machinery, and its sicular contents to the outside during cell secretion. The structure, function, isolation, chemistry, and reconstitution into mouth of the porosome opening to the outside, range in lipid membrane, the molecular mechanism of secretory vesicle size from 150 nm in diameter in acinar cells of the exocrine swelling and fusion at the base of porosomes, is discussed. pancreas, to 12 nm in neurons, which dilates during cell The isolation of the porosome, and the determination of its secretion, returning to its resting size following completion biochemical composition, its structure and dynamics at nm of the process. In the past decade, the composition of the resolution and in real time, and its functional reconstitution into porosome, its structure and dynamics at nm resolution architecture lipid membrane (Cho et al., 2002a; 2002b; 2004; and in real time, and its functional reconstitution into artifi- 2007; Jena, 2005; 2007; Jena et al., 2003; Jeremic et al., 2003; cial lipid membrane, have all been elucidated. In this mini Schneider et al., 1997), has greatly advanced our understand- review, the discovery of the porosome, its structure, func- ing of the secretory process in cells. tion, isolation, chemistry, and reconstitution into lipid The establishment of continuity between the secretory vesi- membrane, the molecular mechanism of secretory vesicle cle membrane and the membrane at the porosome base, re- swelling and fusion at the base of porosomes, and how quires the participation of specific membrane proteins called this new information provides a paradigm shift in our un- SNAREs. At the nerve terminal for example, target membrane derstanding of cell secretion, is discussed. proteins SNAP-25 and syntaxin, collectively called t-SNAREs present at the base of the neuronal porosome complex, and synaptic vesicle-associated protein v-SNARE, are involved in INTRODUCTION fusion of synaptic vesicles at the porosome base. To under- stand SNARE-induced membrane fusion, required an under- The story of cell secretion, a fundamental process as old as life standing of the interaction and assembly of membrane- itself, occurs in all organisms- from the simple yeast to cells in associated v-SNARE and t-SNAREs. The structure and ar- humans. Secretion is responsible for numerous physiological rangement of membrane-associated t-/v-SNARE complex, was activities in living organisms, such as neurotransmission and first determined using AFM. Results from the study demon- the release of hormones and digestive enzymes. Secretory strate that t-SNAREs and v-SNARE, when present in opposing defects in cells are responsible for a host of debilitating dis- bilayers, interact in a circular array to form ring complexes or eases, and hence this field has been the subject of intense channels, each measuring a few nanometers (Cho et al., study for over half a century. In the past 15 years, primarily 2002c). The size of the ring complex is directly proportional to using the atomic force microscope -a force spectroscope, a the curvature of the opposing bilayers. In the presence of cal- detailed understanding of the molecular machinery and mecha- cium, the ring-complex enables the establishment of continuity nism of cell secretion has come to light. As opposed to the between compartments across the opposing bilayers. In con- commonly held belief that during cell secretion secretory trast however, in the absence of membrane, soluble v- and t- vesicles completely merge at the cell plasma membrane (to be SNAREs fail to assemble in such specific and organized pat- endocytosed later), it has become clear that secretory vesicles tern, nor form such conducting channels (Cho et al., 2002c; transiently dock, fuse, partially expel their contents, and disso- Cook et al., 2008). Once v-SNARE and t-SNAREs residing in ciate, allowing multiple such rounds. It has been further deter- opposing bilayers meet, the resulting SNARE complex over- mined that swelling of secretory vesicles is required for the come the repulsive forces between opposing bilayers, bringing expulsion if intravesicular contents during cell secretion. These them closer to within a distance of 2.8-3 Å, allowing calcium findings have led to a paradigm shift in our understanding of bridging of the opposing phospholipids headgroups, leading to cell secretion, and explains the generation of partially empty local dehydration and membrane fusion (Jeremic et al., 2004a; Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA *Correspondence: [email protected] Received November 6, 2008; accepted November 10, 2008; published online November 17, 2008 Keywords: cell secretion, membrane fusion, porosome 518 The Molecular Machinery and Mechanism of Cell Secretion 2004b; Potoff et al., 2008). A B In this review, the discovery of the porosome as the universal secretory machinery in cells, its structure, dynamics, its isola- tion, composition, its functional reconstitution in artificial lipid membrane, and the molecular mechanism of SNARE-induced fusion of secretory vesicle at the porosome base, is discussed. The article is primarily focused on porosome in a slow secretory cell, i.e., the acinar cell of the exocrine pancreas, and in a fast secretory cell, the neuron. Similarly, results from studies on neuronal SNAREs, have been used to explain the molecular mechanism of SNARE-induced membrane fusion, i.e., the fusion of synaptic vesicle at the porosome base. C D Discovery of the ‘porosome’ -the universal secretory machinery in cells Porosomes were first discovered in acinar cells of the exocrine pancreas (Schneider et al., 1997). Exocrine pancreatic acinar cells are polarized secretory cells possessing an apical and a basolateral end. This well characterized cell of the exocrine pancreas, synthesize digestive enzymes, which is stored within 0.2-1.2 μm in diameter apically located membranous sacs or secretory vesicles, called zymogen granules (ZG). Following a E F secretory stimulus, ZG’s dock and fuse with the apical plasma membrane to release their contents to the outside. Contrary to neurons, where secretion of neurotransmitters occurs in the millisecond time regime, the pancreatic acinar cells secrete digestive enzymes over minutes following a secretory stimulus. Being a slow secretory cell, pancreatic acinar cells were ideal for investigation of the molecular steps involved in cell secretion. In the mid 1990’s, AFM studies were undertaken on live pan- creatic acinar cells to evaluate at high resolution, the structure and dynamics of the apical plasma membrane in both resting and following stimulation of cell secretion. To our surprise, iso- Fig. 1. Porosomes or previously referred to as ‘depression’ at the lated live pancreatic acinar cells in physiological buffer, when plasma membrane in pancreatic acinar cell and at the nerve termi- imaged using the AFM (Schneider et al., 1997), reveal new nal. (A) AFM micrograph depicting ‘pits’ (yellow arrow) and ‘poro- cellular structures. At the apical plasma membrane, a group of somes’ within (blue arrow), at the apical plasma membrane in a live circular ‘pits’ measuring 0.4-1.2 μm in diameter, contain smaller pancreatic acinar cell. (B) To the right is a schematic drawing de- ‘depressions’ were observed. Each depression measure be- picting porosomes at the cell plasma membrane (PM), where tween 100-180 nm in diameter, and typically 3-4 depressions membrane-bound secretory vesicles called zymogen granules (ZG), are found within a pit. The basolateral membrane in acinar cells, dock and fuse to release intravesicular contents. (C) A high resolu- are devoid of such structures. High-resolution AFM images of tion AFM micrograph shows a single pit with four 100-180 nm poro- depressions in live acinar cells further reveal a cone-shaped somes within. (D) An electron micrograph depicting a porosome morphology, and the depth of each cone measure 15-35 nm. (red arrowhead) close to a microvilli (MV) at the apical plasma Subsequent studies over the years, demonstrate the presence membrane (PM) of a pancreatic acinar cell. Note association of the of depressions in all secretory cells examined. Analogous to porosome membrane (yellow arrowhead), and the zymogen gran- pancreatic acinar cells, examination of resting GH secreting ule membrane (ZGM) (red arrow head) of a docked ZG (inset). cells of the pituitary (Cho et al., 2002b) and chromaffin cells of Cross section of a circular complex at the mouth of the porosome is the adrenal medulla (Cho et al., 2002d) also reveal the pres- seen (blue arrow head). (E) The bottom left panel shows an elec- ence of pits and depressions at the cell plasma membrane. The tron micrograph of a porosome (red arrowhead) at the nerve termi- presence of depressions or porosomes in neurons, astrocytes, nal, in association with a synaptic vesicle (SV) at the presynaptic β-cells of the endocrine pancreas, and in mast cells have also membrane (Pre-SM). Notice a central plug at the neuronal poro- been elucidated (Cho et al., 2004; 2007; Jena, 2004), demon- some opening. (F) The bottom right panel is an AFM micrograph of strating their universal presence (Figs. 1-3). a neuronal porosome in physiological buffer, also showing the cen- Exposure of pancreatic acinar cells to a secretagogue (mas- tral plug (red arrowhead) at its opening. It is believed that the central toparan) results in a time-dependent increase (25-45%) in both plug in neuronal porosomes may regulate its rapid close-open con- the diameter and relative depth of depressions. Studies dem- formation during neurotransmitter release.