|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Secretory vesicles transiently dock and fuse at the porosome to discharge contents during cell secretion
Bhanu P. Jena1
Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A.
In contrast with the observation in electron micrographs of partially empty vesicles in cells following secretion, it has been believed since the 1950s that during cell secretion, secretory vesicles completely merge at the cell plasma membrane, resulting in the diffusion of intravesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. In the interim, a large body of work has been published arguing both for and against the complete merger of secretory vesicle membrane at the cell plasma membrane during secretion. The only definitive determination of the mechanism of cell secretion remained in its direct observation at nanometre resolution in live cells. In the past decade, this finally became a reality through the power and scope of the atomic force microscope, which has made it possible to resolve a major conundrum in cell biology. This paradigm shift in our understanding of cell secretion is briefly outlined here.
Key words: atomic force microscopy, membrane fusion, pancreatic acinar cell, porosome, secretoy vesicle, SNARE
Abbreviations: AFM, atomic force microscopy, GH, growth hormones, NSF, N-ethylmaleimide-sensitive factor, VAMP, vesicle-associated membrane protein, ZG, zymogen granules
Secretion is a fundamental cellular process as old as life, and occurs in all living organisms from the simple yeast to cells in humans. Secretion is responsible for a variety of physiological activities in living organisms, such as neurotransmission, and the release of hormones and digestive enzymes. Secretory defects in cells are responsible for a host of debilitating diseases. Since the mid-1950s, it has been believed that during cell secretion, secretory vesicles completely merge at the cell plasma membrane resulting in diffusing out of intravesicular contents, and the compensatory retrieval of the excess membrane by endocytosis. In contrast, the observation in electron micrographs of partially empty vesicles in cells following secretion could not be justified according to the above mechanism. In the 1960s, experimental data concerning neurotransmitter release mechanisms by Katz (1962) and Folkow et al. (1967) 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 (Folkow and Häggendal, 1970; Folkow et al., 1967, 1997). In the interim, a large body of work was published both for and against the complete merger of secretory vesicles at the cell plasma membrane during secretion, further deepening the controversy. The only definitive determination of the mechanism of cell secretion was a direct observation of the process at nanometre resolution in live cells. The conundrum was finally resolved using AFM (atomic force microscopy) (Kelly et al., 2004). Isolated live pancreatic acinar cells in near physiological buffer when imaged using AFM at high force (200–300 pN) demonstrate the size and shape of the secretory vesicles called ZG (zymogen granules) lying immediately below the apical plasma membrane of the cell (Figure 1). Within 2.5 min of exposure to a physiological secretory stimulus (1 μM carbamylcholine), the majority of ZG within cells swell (Figure 1), followed by a decrease in ZG size (Figure 1), by which time secretion is complete (Figure 1). These studies revealed for the first time in live cells the intracellular swelling of secretory vesicles following stimulation of secretion and their deflation after partial discharge of vesicular contents (Kelly et al., 2004). No loss of secretory vesicles was observed throughout the experiment, demonstrating transient fusion and partial discharge of vesicular contents during cell secretion.
The other major breakthrough in our understanding of cell secretion came with the discovery of a new cellular structure—the ‘porosome'—by using AFM (Schneider et al., 1997; Cho et al., 2002b, 2002e, 2004, 2007, 2008; Jena et al., 2003; Jeremic et al., 2003). In the past 12 years, the porosome has been determined as the universal secretory machinery in cells. Porosomes are supramolecular, lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release intravesicular contents to the outside during cell secretion. The mouth of the porosome opening to the outside, ranges in size from 150 nm in diameter (in acinar cells of the exocrine pancreas) to 12 nm (in neurons), and dilates during cell secretion, returning to its resting size following completion of the process. In the past decade, the composition of the porosome, its structure and dynamics at nanometre 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 a 20–35% increase in porosome diameter is demonstrated following the docking and fusion of 0.2–1.2 μm in diameter secretory vesicles, it can be concluded that secretory vesicles ‘transiently' dock and fuse at the base of the porosome complex to release their contents to the outside (Figure 2). In agreement, studies demonstrate that “secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells” (Taraska et al., 2003), “single synaptic vesicles fusing transiently and successively without loss of identity” (Aravanis et al., 2003) and “zymogen granule exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity” (Thorn et al., 2004).
2. Discovery of the ‘porosome'
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, which synthesizes digestive enzymes, is stored within 0.2–1.2 μm in diameter, apically located membranous sacs or secretory vesicles called ZG. Following a secretory stimulus, ZG dock and fuse with the apical plasma membrane to release their contents to the outside. In contrast with neurons, where the secretion of neurotransmitters occurs in the millisecond time regime, the pancreatic acinar cells secrete digestive enzymes over minutes following a secretory stimulus. Being slow secretory cells, pancreatic acinar cells were ideal for investigation of the molecular steps involved in cell secretion. In the mid-1990s, AFM studies were carried out on live pancreatic acinar cells to evaluate at high resolution the structure and dynamics of the apical plasma membrane in both resting cells and following stimulation of cell secretion. To our surprise, isolated live pancreatic acinar cells in physiological buffer, when imaged using the AFM, reveal new cellular structures. At the apical plasma membrane, a group of circular ‘pits' measuring 0.4–1.2 μm in diameter and containing smaller ‘depressions' were observed. Each depression measures between 100 and 180 nm in diameter, and typically three to four depressions are found within a pit. The basolateral membrane in acinar cells is devoid of such structures. High-resolution AFM images of depressions in live acinar cells further reveal a cone-shaped morphology, and the depth of each cone measures 15–35 nm. Subsequent studies over the years demonstrate the presence of depressions in all secretory cells that were examined. Analogous to pancreatic acinar cells, examination of resting GH (growth hormones)-secreting cells of the pituitary (Cho et al., 2002e) and chromaffin cells of the adrenal medulla (Cho et al., 2002d) also reveals the presence of pits and depressions at the cell plasma membrane. The presence of depressions or porosomes in neurons, astrocytes, β-cells of the endocrine pancreas and in mast cells has also been elucidated, demonstrating their universal presence.
Exposure of pancreatic acinar cells to a secretagogue (mastoparan) results in a time-dependent increase (25–45%) in diameter and relative depth of depressions. Studies demonstrate that depressions return to their resting size upon completion of cell secretion (Schneider et al., 1997; Cho et al., 2002b). No demonstrable change in pit size is detected after stimulation of secretion (Schneider et al., 1997). Enlargement of depression diameter and an increase in its relative depth after exposure to secretagogue correlated with secretion. Additionally, exposure of pancreatic acinar cells to cytochalasin B, a fungal toxin that inhibits actin polymerization and secretion, results in a 15–20% decrease in depression size and a consequent 50–60% loss in secretion (Schneider et al., 1997). Results from these studies suggest depressions to be the fusion pores in pancreatic acinar cells. Furthermore, these studies demonstrate the involvement of actin in regulation of both the structure and function of depressions. Similarly, depression in resting GH cells measure 154±4.5 nm (mean±S.E.) in diameter, and exposure to a secretagogue results in a 40% increase in depression diameter (215±4.6 nm; P<0.01), with no appreciable change in pit size. The enlargement of depression diameter during cell secretion and subsequent decrease accompanied by loss in secretion following exposure to actin depolymerizing agents (Schneider et al., 1997) also suggested depressions to be secretory portals. A direct determination that depressions are indeed the portals through which secretory products are expelled from cells was unequivocally demonstrated via immuno-AFM studies (Cho et al., 2002b) (Figure 3). Localization at depressions of gold-conjugated antibody to secretory proteins finally provided the direct evidence that secretion occur through depressions. ZG contain the starch digesting enzyme amylase. AFM micrographs of the specific localization of gold-tagged amylase-specific antibodies (Figure 3) at depressions, following stimulation of cell secretion (Cho et al., 2002b; Jena et al., 2003), conclusively demonstrated depressions as the cellular secretory portal. Similarly, in somatotrophs of the pituitary gland, gold-tagged GH-specific antibodies were found to selectively localize at the depression openings following stimulation of secretion (Cho et al., 2002e), demonstrating them to be secretory portals in these cells. Over the years, the term ‘fusion pore' has been loosely used to refer to plasma membrane dimples that originate following a secretory stimulus, or to the continuity or channel established between opposing lipid membrane during membrane fusion. Therefore, for clarity, the term ‘porosome' was assigned to depressions.
The porosome structure at the cytosolic compartment of the plasma membrane in the exocrine pancreas (Jeremic et al., 2003) and neurons (Cho et al., 2004) has also been determined at near-nanometre resolution in live tissues. To determine the morphology of porosomes at the cytosolic compartment of pancreatic acinar cells, isolated plasma membrane preparations in near physiological buffered solution have been imaged at high resolution using AFM. These studies reveal scattered circular discs measuring 0.5–1 μm in diameter, with inverted cup-shaped structures within (Jeremic et al., 2003). The inverted cups at the cytosolic compartment of isolated pancreatic plasma membrane preparations range in height from 10 to 15 nm. On several occasions, ZG ranging in size from 0.4 to 1 μm in diameter were observed in association with one or more of the inverted cups, suggesting the circular discs to be pits, and the inverted cups to be porosomes. To confirm that the cup-shaped structures are porosomes, where secretory vesicles dock and fuse, immuno-AFM studies were performed. Target membrane proteins, SNAP-23 (Oyler et al., 1989) and syntaxin (Bennett et al., 1992) (t-SNARE) and secretory vesicle-associated membrane protein v-SNARE or VAMP (vesicle-associated membrane protein) (Trimble et al., 1988), are part of the conserved protein complex involved in fusion of opposing bilayers in the presence of calcium (Malhotra et al., 1988; Wilson et al., 1992; Gaisano et al., 1997; Cho et al., 2002a, 2005; Jeremic et al., 2004a, 2004b, 2006; Cho and Jena, 2007; Cook et al., 2008; Potoff et al., 2008). Since ZG dock and fuse at the plasma membrane to release vesicular contents, it was hypothesized that if porosomes are the secretory sites, then plasma membrane-associated t-SNAREs should localize there. The t-SNARE protein SNAP-23 had previously been reported in pancreatic acinar cells (Gaisano et al., 1997). A polyclonal monospecific SNAP-23 antibody recognizing a single 23-kDa protein in immunoblots of pancreatic plasma membrane fraction, when used in immuno-AFM studies, demonstrated selective localization to the base of the cup-shaped structures. These results demonstrate that the inverted cup-shaped structures in inside-out isolated pancreatic plasma membrane preparations are indeed porosomes, where secretory vesicles dock and fuse to release their contents during cell secretion (Jena et al., 2003). The size and shape of the immunoisolated porosome complex has also been determined using both negative-staining EM (electron microscopy) and AFM (Cho et al., 2004, 2007, 2008; Jeremic et al., 2003) (Figure 4).
The immunoisolated porosome complex has also been both structurally and functionally reconstituted into liposomes and lipid bilayer membrane (Jeremic et al., 2003; Cho et al., 2004, 2007, 2008). Transmission electron micrographs of pancreatic porosomes reconstituted into liposomes exhibit a 150- to 200-nm cup-shaped basket-like morphology, similar to that observed in its native state when co-isolated with ZG. To test the functionality of the isolated porosome complex, purified porosomes obtained from exocrine pancreas or neurons were subjected to reconstitution in lipid membrane of the electrophysiological setup (EPC9) before being challenged with isolated ZG or synaptic vesicles. The electrical activity of the reconstituted membrane as well as the transport of vesicular contents from the cis to the trans compartments of the bilayer chambers were monitored. Results from these experiments demonstrate that the lipid membrane-reconstituted porosomes are indeed functional (Jeremic et al., 2003; Cho et al., 2004), since in the presence of calcium, isolated secretory vesicles dock and fuse to transfer intravesicular contents from the cis to the trans compartment of the bilayer chamber. ZG fused with the porosome-reconstituted bilayer, as demonstrated by an increase in capacitance and conductance, and a time-dependent transport of the ZG enzyme amylase from cis to the trans compartment of the bilayer chamber. Amylase is detected using immunoblot analysis of the buffer in the cis and trans chambers. As observed in immunoblot assays of isolated porosomes, chloride channel activity is also present in the reconstituted porosome complex. Furthermore, the chloride channel inhibitor, DIDS, was found to inhibit current activity through the porosome-reconstituted bilayer, demonstrating a requirement of the porosome-associated chloride channel activity in porosome function. Similarly, the structure and biochemical composition of the neuronal porosome, and the docking and fusion of synaptic vesicles at the neuronal porosome complex have also been elucidated. In summary, these studies demonstrate porosomes to be permanent supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release intravesicular contents to the outside. Porosomes are therefore the universal secretory machinery in cells (Jena, 2008, 2009).
3. SNARE-induced membrane fusion
As outlined in the preceding section, membrane fusion in live cells is mediated via a specialized set of proteins present in opposing bilayers (Oyler et al., 1989; Cho et al., 2002a; Jeremic et al., 2004b). Target membrane proteins, SNAP-25 and syntaxin (t-SNAREs) and secretory vesicle-associated protein (v-SNARE) are all part of the conserved protein complex involved in fusion of opposing lipid membranes. The structure and arrangement of the membrane-associated full-length SNARE complex was first determined using AFM (Trimble et al., 1988) (Figure 5). Results from the study demonstrate that t-SNAREs and v-SNARE, when present in opposing bilayers, interact in a circular array to form supramolecular ring complexes, each measuring a few nanometres. The size of the ring complex is directly proportional to the curvature of the opposing bilayers (Cho et al., 2005). In the presence of calcium, the ring complex helps in establishing continuity between the opposing bilayers (Jeremic et al., 2004a, 2004b; Potoff et al., 2008). In contrast, in the absence of membrane, soluble v- and t-SNAREs fail to assemble in such a specific and organized pattern, nor form such conducting channels. Once v-SNAREs and t-SNAREs residing in opposing bilayers meet, the resulting SNARE complex overcomes the repulsive forces between opposing bilayers, bringing them closer, to within a distance of 2.8–3 Å, thereby allowing calcium bridging of the opposing phospholipids head groups and leading to local dehydration and membrane fusion (Jeremic et al., 2004a, 2004b; Potoff et al., 2008).
VAMP and syntaxin are both integral membrane proteins, with the soluble SNAP-25 associating with syntaxin. Hence, the key to our understanding of SNARE-induced membrane fusion requires determination of the atomic arrangement and interaction between membrane-associated v- and t-SNAREs. Ideally, the atomic co-ordinates of membrane-associated SNARE complex using X-ray crystallography would help elucidate the chemistry of SNARE-induced membrane fusion in cells. So far, such structural details at the atomic level of membrane-associated t-/v-SNARE complex have not been realized. This has been primarily due to solubility problems of membrane-associated SNAREs, compounded by the fact that v-SNAREs and t-SNAREs need to reside in opposing membranes when they meet in order to assemble in a physiologically relevant SNARE complex. The remaining option has been the use of NMR spectroscopy; however, NMR has also been of little help, since the size of t-/v-SNARE ring complexes are beyond the maximum limit for NMR studies. Regardless of these setbacks, AFM has provided for the first time at nanometre to subnanometre resolution, an understanding of the structure, assembly and disassembly of membrane-associated t-/v-SNARE complexes in physiological buffer solution (Cho et al., 2002a, 2005; Jeremic et al., 2004a, 2004b, 2006; Cho and Jena, 2007; Cook et al., 2008; Potoff et al., 2008). A bilayer electrophysiological setup allowed measurements of membrane conductance and capacitance during fusion of v-SNARE-reconstituted liposomes with t-SNARE-reconstituted membrane, and vice versa (Figures 5A and 5B). Results from these studies demonstrated that t-SNAREs and v-SNAREs interact and assemble in a circular array when present in opposing membranes, and in the presence of calcium form conducting channels (Cho et al., 2002a). The interaction of t-/v-SNARE proteins to form such a conducting channel is strictly dependent on the presence of t-SNAREs and v-SNAREs in opposing bilayers. Addition of purified recombinant v-SNARE to a t-SNARE-reconstituted lipid membrane, results in non-physiological interactions that have no influence on the electrical properties of the membrane (Cho et al., 2002a). However, in the presence of calcium, when v-SNARE vesicles are added to a t-SNARE-reconstituted membrane or vice versa, SNAREs assemble in a ring conformation. The resultant increase in membrane capacitance and conductance demonstrate the establishment of continuity between the opposing t-SNARE- and v-SNARE-reconstituted bilayers. These results confirm that t- and v-SNAREs are required to reside in opposing membranes, as they exist in the physiological state in cells, to allow appropriate t-/v-SNARE interactions leading to membrane fusion in the presence of calcium. Studies using SNARE-reconstituted liposomes and bilayers (Jeremic et al., 2004a, 2004b) further demonstrate: (i) a low fusion rate (τ = 16 min) between t- and v-SNARE-reconstituted liposomes in the absence of Ca2+; and (ii) exposure of t-/v-SNARE liposomes to Ca2+ drives vesicle fusion on a near physiological relevant time-scale (τ∼10 s), demonstrating in the combination of Ca2+ and SNAREs the minimal fusion machinery in cells (Jeremic et al., 2004a, 2004b). Native and synthetic vesicles exhibit a significant negative surface charge, primarily due to the polar phosphate head groups generating a repulsive force that prevents the aggregation and fusion of opposing vesicles. In cells, SNAREs provide direction and specificity to bring opposing bilayers closer, to within a distance of 2–3 Å (Jeremic et al., 2004a, 2004b), enabling Ca2+ bridging and membrane fusion. The bound Ca2+ then leads to the expulsion of water between the bilayers at the bridging site, leading to lipid mixing and membrane fusion. Hence, SNAREs, besides bringing opposing bilayers closer, dictate the site and size of the fusion area during cell secretion. The size of the t-/v-SNARE complex is dictated by the curvature of the opposing membranes (Cho et al., 2005), hence the smaller the vesicle, the smaller the channel formed.
A unique set of chemical and physical properties of the Ca2+ ion make it ideal for participation in the membrane fusion reaction. Calcium ion exists in its hydrated state within cells. The properties of hydrated calcium have been extensively studied using X-ray diffraction and neutron scattering, in combination with molecular dynamics simulations (Portis et al., 1979; McIntosh, 2000; Bako et al., 2002; Chialvo and Simonson, 2003). The molecular dynamics simulations include three-body corrections compared with ab initio quantum mechanics/molecular mechanics/molecular dynamics simulations. First principle molecular dynamics has also been used to investigate the structural, vibrational and energetic properties of [Ca(H2O)n]2+ clusters, and the hydration shell of the calcium ion (Bako et al., 2002). These studies demonstrate that hydrated calcium [Ca(H2O)n]2+ has more than one shell around the Ca2+, with the first hydration shell having six water molecules in an octahedral arrangement (Jena, 2008). In studies using light scattering and X-ray diffraction of SNARE-reconstituted liposomes, it has been demonstrated that fusion proceeds only when Ca2+ ions are available between the t- and v-SNARE-apposed proteoliposomes (Jeremic et al., 2004a, 2004b). Mixing of t- and v-SNARE proteoliposomes in the absence of Ca2+ leads to a diffuse and asymmetric diffractogram in X-ray diffraction studies, a typical characteristic of short-range ordering in a liquid system (McIntosh, 2000). In contrast, when t-SNARE and v-SNARE proteoliposomes in the presence of Ca2+ are mixed, it leads to a more structured diffractogram, with ∼12% increase in X-ray scattering intensity, suggesting an increase in the number of contacts between opposing bilayers, presumably established through calcium–phosphate bridges, as previously suggested (Portis et al., 1979; Jeremic et al., 2004a, 2004b). The ordering effect of Ca2+ on interbilayer contacts observed in X-ray studies (Malhotra et al., 1988) is in good agreement with light, AFM and spectroscopic studies, suggesting close apposition of PO-lipid head groups in the presence of Ca2+, followed by formation of Ca2+–PO bridges between the adjacent bilayers (Laroche et al., 1991; Jeremic et al., 2004a, 2004b). X-ray diffraction studies show that the effect of Ca2+ on bilayer orientation and interbilayer contacts is most prominent in the area of 3 Å, with additional appearance of a new peak at position 2.8 Å, both of which are within the ionic radius of Ca2+ (Jeremic et al., 2004b). These studies further suggest that the ionic radius of Ca2+ may make it an ideal player in the membrane fusion reaction. Hydrated calcium [Ca(H2O)n]2+ with a hydration shell having six water molecules and measuring ∼6 Å, however, would be excluded from the t-/v-SNARE apposed interbilayer space; hence, calcium has to be present in the buffer solution when t-SNARE vesicles and v-SNARE vesicles meet. Indeed, studies demonstrate that if t- and v-SNARE vesicles are allowed to mix in a calcium-free buffer, there is no fusion following post-addition of calcium (Jeremic et al., 2004a).
How does calcium work? Calcium bridging of apposing bilayers may lead to the release of water from the hydrated Ca2+ ion, leading to bilayer destabilization and membrane fusion. Additionally, the binding of calcium to the phosphate head groups of the apposing bilayers may also displace the loosely co-ordinated water at the PO-lipid head groups, resulting in further dehydration and leading to destabilization of the lipid bilayer and membrane fusion. Recent studies in our laboratory (Potoff et al., 2008), using molecular dynamics simulations in the isobaric–isothermal ensemble to determine whether Ca2+ was capable of bridging opposing phospholipid head groups in the early stages of the membrane fusion process, demonstrate that this indeed is the case. Furthermore, the distance between the oxygen atoms of the opposing PO-lipid head groups bridged by calcium was in agreement with the 2.8 Å distance previously determined using X-ray diffraction measurements. The hypothesis that there is loss of co-ordinated water both from the hydrated calcium ion and oxygen of the phospholipid head groups in opposing bilayers following calcium bridging is further demonstrated in this study.
In the presence of ATP, the highly stable, membrane-directed and self-assembled t-/v-SNARE complex can be disassembled by a soluble ATPase, the NSF (N-ethylmaleimide-sensitive factor). Careful examination of the partially disassembled t-/v-SNARE bundles within the complex using AFM, demonstrates a left-handed supercoiling of SNAREs. These results demonstrate that t-/v-SNARE disassembly requires the right-handed uncoiling of each SNARE bundle within the ring complex, demonstrating NSF to behave as a right-handed molecular motor (Cho and Jena, 2007). Furthermore, from recent studies carried out in the laboratory (Cook et al., 2008) using CD spectroscopy, we can report for the first time that both t-SNAREs and v-SNAREs 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. NSF, in the presence of ATP, disassembles the SNARE complex as reflected from the CD signals demonstrating elimination of α-helices within the structure. These results further demonstrate that NSF-ATP is sufficient for the disassembly of the t-/v-SNARE complex, providing a molecular understanding of SNARE-induced membrane fusion in cells.
4. Secretory vesicle swelling and content expulsion
As outlined above, isolated live pancreatic acinar cells in near physiological buffer when imaged using AFM at high force (200–300 pN), demonstrate the size and shape of the ZG lying immediately below the apical plasma membrane of the cell (Figure 1). Within 2.5 min of exposure to a physiological secretory stimulus (1 μM carbamylcholine), the majority of ZG within cells swell (Figure 1), followed by a decrease in ZG size by which time secretion is complete. These studies revealed in live cells for the first time intracellular swelling of secretory vesicles following stimulation of cell secretion and their deflation following partial discharge of vesicular contents (Kelly et al., 2004). No loss of secretory vesicles was observed throughout the experiment. Measurements of intracellular ZG size further revealed that different vesicles swell differently following a secretory stimulus. For example, the ZG marked by the red arrowhead swelled with a 23–25% increase in diameter, in contrast with the green arrowhead-marked ZG, which increased by only 10–11% (Figure 1). This differential swelling among ZG within the same cell may explain why following stimulation of secretion, some intracellular ZG demonstrate the presence of less vesicular content than others, and hence have discharged more of their contents.
To determine precisely the role of swelling in vesicle–plasma membrane fusion and the expulsion of intravesicular contents, an electrophysiological ZG-reconstituted lipid bilayer fusion assay was used. The ZG used in the bilayer fusion assays were characterized for their purity and their ability to respond to a swelling stimulus. As previously reported (Jena et al., 1997; Cho et al., 2002c), exposure of isolated ZG to GTP results in ZG swelling. Similar to that observed in live acinar cells (Figure 1), each isolated ZG responded differently to the same swelling stimulus. This differential response of isolated ZG to GTP has been further assessed by measuring changes in the volume of isolated ZG of different sizes (Kelly et al., 2004). ZG in the exocrine pancreas range in size from 0.2 to 1.3 μm in diameter (Jena et al., 1997), and not all ZG swell following a GTP challenge (Kelly et al., 2004). In most ZG, volume increases are 5–20%; however, larger increases of up to 45% are observed only in vesicles ranging from 250 to 750 nm in diameter. In the electrophysiological bilayer fusion assay, immunoisolated porosome complexes from the exocrine pancreas are functionally reconstituted (Jeremic et al., 2003) into the lipid membrane of the bilayer apparatus, where membrane conductance and capacitance can be continually monitored (Figure 6A) (Kelly et al., 2004). Reconstitution of the porosome in the lipid membrane results in a small increase in capacitance (Figure 6B), possibly due to an increase in membrane surface area. Isolated ZG, when added to the cis compartment of the bilayer chamber, fuse at the porosome-reconstituted lipid membrane (Figure 6A) and this is detected as a step increase in membrane capacitance (Figure 6B). Even after 15 min of ZG addition to the cis compartment of the bilayer chamber, little or no release of the intravesicular enzyme α-amylase is detected in the trans compartment of the bilayer chamber (Figures 6C and 6D). In contrast, exposure of ZG to 20 mM GTP induced swelling and results both in the potentiation of fusion as well as the robust expulsion of α-amylase into the trans compartment of the bilayer chamber observed in immunoblot assays (Figures 6C and 6D). These studies demonstrate that during cell secretion, secretory vesicle swelling is required for the efficient expulsion of intravesicular contents. This mechanism of vesicular expulsion during cell secretion may explain why partially empty vesicles are generated in cells following secretion. The presence of empty secretory vesicles could result from the multiple rounds of fusion–swelling–expulsion cycles a vesicle may undergo during the secretory process. These results reflect the precise and regulated nature of cell secretion.
In this article, the current understanding of the molecular machinery and mechanism of cell secretion is presented. Porosomes are specialized plasma membrane structures universally present in secretory cells, from exocrine and endocrine cells to neuroendocrine cells and neurons. Since porosomes in exocrine and neuroendocrine cells measure 100–180 nm, and only a 20–35% increase in porosome diameter is demonstrated following the docking and fusion of 0.2–1.2 μm in diameter secretory vesicles, it is concluded that secretory vesicles ‘transiently' dock and fuse at the base of the porosome complex to release their contents to the outside. Furthermore, isolated live cells in near physiological buffer when imaged with AFM, demonstrate the size and shape of the secretory vesicles lying immediately below the apical plasma membrane of the cell. After exposure to a secretory stimulus, secretory vesicles swell, followed by a decrease in vesicle size. No loss of secretory vesicles is observed after secretion, demonstrating transient fusion and partial discharge of vesicular contents during cell secretion. In agreement, studies demonstrate that “secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells” (Taraska et al., 2003), “single synaptic vesicles fusing transiently and successively without loss of identity” (Aravanis et al., 2003) and “zymogen granule exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity” (Thorn et al., 2004). This is in contrast with the general belief that, in mammalian cells, secretory vesicles completely merge at the cell plasma membrane, resulting in passive diffusion of vesicular contents to the cell exterior, and the consequent retrieval of excess membrane by endocytosis at a later time. In addition, a major logistical problem with the complete merger of secretory vesicle membrane at the cell plasma membrane is the generation of partially empty vesicles after cell secretion. It is fascinating how even single-cell organisms have developed such specialized secretory machinery, such as the secretion apparatus of Toxoplasma gondii, the contractile vacuole in Paramecium and the secretory structures of bacteria. Hence, it comes as no surprise that mammalian cells have evolved such highly specialized and sophisticated structures—the porosome complex for cell secretion. The discovery of the porosome, and an understanding of its structure and dynamics at nanometre resolution and in real time in live cells, its composition and its functional reconstitution in lipid membrane, and the molecular mechanism of SNARE-induced membrane fusion, have greatly advanced our understanding of cell secretion. It is evident that the secretory process in cells is a well co-ordinated, highly regulated and finely tuned biomolecular orchestra. Clearly, these findings could not have advanced without AFM, and therefore this powerful tool has greatly contributed to a new understanding of the cell. AFM has enabled the determination of live cellular structure–function at subnanometre to angstrom resolution, in real time, contributing to the birth of the new field of NanoCellBiology. Future directions will involve an understanding of the protein distribution and their arrangement at atomic resolution in the porosome complex, and a similar understanding of the structure of the t-/v-SNARE ring complex. Determination of the atomic structure of membrane-associated full-length SNAREs and their complexes, and the neuronal porosome complex, are being carried out using cryoelectron microscopy in the author's laboratory.
The author thanks the many students and collaborators who have participated in the various studies discussed in this article. The author thanks Won Jin Cho for help in formatting the manuscript.
Figures 1 and 6 have been reproduced from Kelly M et al. (2004). Vesicle swelling regulates content expulsion during secretion. Cell Biol Int, vol. 28, 709-16, with permission from Elsevier. Figures 2(C-F) and 5 have been reproduced from Jena BP (2009) Porosome: the secretory portal in cells. Biochemistry, vol. 48, 4009-18, with permission from the American Chemical Society. Figure 3 has been reproduced from Cho S-J et al (2002b) Structure and dynamics of the fusion pore in live cells. Cell Biol Int, vol. 26, 35-42, with permission from Elsevier. Figure 4(A-C) has been reproduced from Cho WJ, Ren, G and Jena, BP (2008) EM 3D contour maps provide protein assembly at the nanoscale within the neuronal porosome complex. J Microsc, vol 232, 106-11, with permission from Wiley-Blackwell.
Research in the author's laboratory was supported by
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Received 30 September 2009/10 October 2009; accepted 29 October 2009
Published online 16 December 209, doi:10.1042/CBI20090161
© 2010 The Author(s) Journal compilation. © 2010 Portland Press Ltd
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 2 Porosomes, previously referred to as ‘depressions', at the plasma membrane in a pancreatic acinar cell and at the nerve terminal
Figure 4 Nanoscale, three-dimensional contour map of protein assembly within the neuronal porosome complex