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Biology Articles » Cell biology » Discovery of the Porosome: revealing the molecular mechanism of secretion and membrane fusion in cells » Figures

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

Figures

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Fig. 1. On the far left is an AFM micrograph depicting ‘pits’ (yellow arrow) and ‘depressions’ within (blue arrow), at the plasma membrane in live pancreatic acinar cells. On the right is a schematic drawing depicting depressions, at the cell plasma membrane, where membrane-bound secretory vesicles dock and fuse to release vesicular contents [2].

 

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Fig. 2. Dynamics of depressions following stimulation of secretion. The top panel shows a number of depressions within a pit in a live pancreatic acinar cell. The scan line across three depressions in the top panel is represented graphically in the middle panel and defines the diameter and relative depth of the depressions; the middle depressions is represented by red arrowheads. The bottom panel represents percent of total cellular amylase release in the presence and absence of the secretagogue Mas 7. Notice an increase in the diameter and depth of depressions, correlating with an increase in total cellular amylase release at 5 min after stimulation of secretion. At 30 min after stimulation of secretion, there is a decrease in diameter and depth of depressions, with no further increase in amylase release over the 5 min time point. No significant increase in amylase secretion or depressions diameter were observed in resting acini or those exposed to the nonstimulatory mastoparan analog Mas 17 [2, 40].

 

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Fig. 3. Depressions in live secretory cell of the exocrine pancreas (A,B), the growth hormone secreting cell of the pituitary (C), and in the chromaffin cell (D). Note the ‘pit’ (white arrow heads) with four depressions (yellow arrow head). A high resolution AFM micrograph is shown in Fig. B. Bars = 40 nm for Fig. A and B. [4,5,41].

 

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Fig. 4 Depressions are fusion pores or porosomes. Porosomes dilate to allow expulsion of vesicular contents. (A and B) AFM micrographs and section analysis of a pit and two out of the four fusion pores or porosomes, demonstrating enlargement following stimulation of secretion. (C) Exposure of live cells to gold conjugated-amylase antibody (Ab) results in specific localization of immuno-gold to the porosome opening. Amylase is one of the proteins within secretory vesicles of the exocrine pancreas. (D) AFM micrograph of a fixed pancreatic acinar cell, demonstrating a pit and porosomes within, immunogold-labeling amylase at the site. Blue arrowheads point to immunogold clusters and the yellow arrowhead points to a porosome [3].

 

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Fig. 5. Electron micrograph of Porosomes in neurons. A. Electron micrograph of a synaptosome demonstrating the presence of 40-50 nm synaptic vesicles. B-D. Electron micrograph of neuronal porosomes which are 10-15 nm cup-shaped structures at the presynaptic membrane (yellow arrow head), where synaptic vesicles transiently dock and fuse to release vesicular contents. [Kelly, M.L., Cho, W-J., Jeremic, A., Lazrishvili, I.L., Bikashvili, T.Z., Zhvania, M., and Jena, B.P., unpublished observation].

 

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Fig. 6. AFM micrographs of porosomes in β-cell of the endocrine pancreas (A,B) and mast cell (C,D). Note the 100-130 nm porosomes in the β-cell (B) and the 70-80 nm porosomes in the mast cell (D), demonstrated using AFM section analysis. [Jena, B.P., unpublished observation].

 

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Fig. 7. Morphology of the cytosolic side of the porosome revealed in AFM studies on isolated pancreatic plasma membrane (PM) preparations. (A). AFM micrograph of isolated PM preparation reveals the cytosolic end of a pit with inverted cup-shaped structures, the porosome. Note the 600 nm in diameter ZG at the left hand corner of the pit. (B). Higher magnification of the same pit showing clearly the 4-5 porosomes within. (C). The cytosolic end of a single porosome is depicted in this AFM micrograph. (D). Immunoblot analysis of 10μg and 20 μg of pancreatic PM preparations, using SNAP-23 antibody, demonstrates a single 23 kDa immunoreactive band. (E,F). The cytosolic side of the PM demonstrating the presence of a pit with a number of porosomes within, shown prior to (E) and following addition of the SNAP-23 antibody (F). Note the increase in height of the porosome cone base revealed by section analysis (bottom pannel), demonstrating localization of SNAP-23 antibody at the base of the porosome [8].

 

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Fig. 8. Electron micrograph depicting a porosome (red arrow head) close to a microvilli (MV) at the apical plasma membrane (PM) of a pancreatic acinar cell. Note association of the porosome membrane (yellow arrow head), and the zymogen granule membrane (ZGM) (red arrow head) of a docked zymogen granule (ZG), the membranebound secretory vesicle of exocrine pancreas. Also a cross section of the ring at the mouth of the porosome is seen (blue arrow head) [11].

 

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Fig. 9. Transmission electron micrograph of porosome associated with zymogen granules, the secretory vesicle in exocrine pancreas. (A) An isolated zymogen granule associated with a porosome, reveals (B) clearly the lateral and vertical structures in the complex. The porosome membrane (PM) and the vesicle membrane (VM) of the zymogen granule is clearly seen. Scale = 100 nm. [8, 11].

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Fig. 10. SNAP-23 associated proteins in pancreatic acinar cells. Total pancreatic homogenate was immunoprecipitated using the SNAP-23 specific antibody. The precipitated material was resolved using 12.5% SDS-PAGE, electrotransferred to nitrocellulose membrane and then probed using antibodies to a number of proteins. Association of SNAP-23 with syntaxin2, with cytoskeletal proteins actin, α-fodrin, and vimentin, and calcium channels β3 and α1c, together with the SNARE regulatory protein NSF, is demonstrated (arrow heads). Lanes showing more than one arrowhead suggest presence of isomers or possible proteolytic degradation of the specific protein [8].

 

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Fig. 11. Negatively stained electron micrograph and atomic force micrograph of the immunoisolated porosome complex. (A). Negatively stained electron micrograph of an immunoisolated porosome complex from solubilized pancreatic plasma membrane preparations, using a SNAP-23 specific antibody. Note the three rings and the 10 spokes that originate from the inner smallest ring. This structure represents the protein backbone of the porosome complex, since the three rings and the vertical spikes are observed in electron micrographs of cells and porosome co-isolated with ZGs. Bar = 30 nm. (B). The electron micrograph of the fusion pore complex, cut out from (A), and (C) an outline of the structure presented for clarity. (D-F). Atomic force micrograph of the isolated pore complex in near physiological buffer. Bar = 30 nm. Note the structural similarity of the complex, imaged both by EM (G) and AFM (H). The EM and AFM micrographs are superimposable (I). [11].

 

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Fig. 12. Electron micrographs of reconstituted Porosome or fusion pore complex in liposomes, showing a cup-shaped basket-like morphology. (A). A 500 nm vesicle with an incorporated porosome is shown. Note the spokes in the complex. The reconstituted complex at greater magnification is shown in Fig. B-D. Bar = 100 nm. [11].

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Fig. 13. Lipid bilayer-reconstituted Porosome complex is functional. (A). Schematic drawing of the bilayer setup for electrophysiological measurements. (B). Zymogen Granules (ZGs) added to the cis side of the bilayer fuse with the reconstituted Porosomes, as demonstrated by an increase in capacitance and current activities, and a concomitant time dependent release of amylase (a major ZG content) to the trans side of the membrane. The movement of amylase from the cis to the trans side of the chamber was determined by immunoblot analysis of the contents in the cis and the trans chamber over time. (C). As demonstrated by immunoblot analysis of the immunoisolated complex, electrical measurements in the presence and absence of chloride ion channel blocker DIDS, demonstrate the presence of chloride channels in association with the complex [11].

 

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Fig. 14. AFM micrographs and force plots of mica and lipid surface and of SNAREs on lipid membrane. (A) AFM performed on freshly cleaved mica (left) and on lipid membrane formed on the same mica surface (right), demonstrating differences in the force-versus-distant curves. Note the curvilinear shape exhibited in the force-versus-distant curves of the lipid surface in contrast to mica. Three dimensional AFM micrographs of neuronal t-SNAREs deposited on the lipid membrane (B), and after the addition of v-SNARE (C). Section analysis of the SNARE complex in (B) and (C) is depicted in (D). Note that the smaller curve belonging to the t-SNARE complex in (B) is markedly enlarged after addition of v-SNARE. Artificial bilayer lipid membranes are nonconducting either in the presence or absence of SNAREs (E, F). Current verses time traces of bilayer membranes containing proteins involved in docking and fusion of synaptic vesicles while the membranes are held at -60 mV (current/reference voltage). (E) When t-SNAREs are added to the planar lipid bilayer containing the synaptic vesicle protein, VAMP-2, no occurrence of current spike for fusion event at the bilayer membrane is observed (n = 7). (F) Similarly, no current spike is observed when t-SNAREs (syntaxin 1A-1 and SNAP25) are added to the cis side of a bilayer chamber, following with VAMP-2. Increasing the concentration of t-SNAREs and VAMP-2 protein. [12].

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Fig. 15. Pore-like structures are formed when t-SNAREs and v-SNARE in opposing bilayers interact. (A) Unfused v-SNARE vesicles on t-SNARE reconstituted lipid membrane. (B) Dislodgement or fusion of v- SNARE-reconstituted vesicles with a t-SNARE-reconstituted lipid membrane, exhibit formation of pore-like structures due to the interaction of v- and t-SNAREs in a circular array. The size of the pores range between 50 and 150 nm (B-D). Several 3D AFM amplitude images of SNAREs arranged in a circular array (C) and some at higher resolution (D), illustrating a pore-like structure at the center is depicted. Scale bar is 100 nm. Recombinant t-SNAREs and v-SNARE in opposing bilayers drive membrane fusion. (E) When t-SNARE vesicles were exposed to v-SNARE reconstituted bilayers, vesicles fused. Vesicles containing nystatin/ergosterol and t- SNAREs were added to the cis side of the bilayer chamber. Fusion of t-SNARE containing vesicles with the membrane observed as current spikes that collapse as the nystatin spreads into the bilayer membrane. To determine membrane stability, the transmembrane gradient of KCl was increased, allowing gradient driven fusion of nystatin-associated vesicles [12].

 

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Fig. 16. Opposing bilayers containing t- and v-SNAREs respectively, interact in a circular array to form conducting pores. (A) Schematic diagram of the bilayer-electrophysiology setup. (B) Lipid vesicle containing nystatin channels (red) and both vesicles and membrane bilayer without SNAREs, demonstrate no significant changes in capacitance and conductance. Initial increase in conductance and capacitance may be due to vesicle-membrane attachment. To demonstrate membrane stability (both bilayer membrane and vesicles), the transmembrane gradient of KCl was increased to allow gradient-driven fusion and a concomitance increase of conductance and capacitance. (C) When t- SNARE vesicles were added to a v-SNARE membrane support, the SNAREs in opposing bilayers arranged in a ring pattern, forming pores (as seen in the AFM micrograph on the extreme right) and there were seen stepwise increases in capacitance and conductance (-60 mV holding potential). Docking and fusion of the vesicle at the bilayer membrane, opens vesicle-associated nystatin channels and SNAREinduced pore formation, allowing conductance of ions from cis to the trans side of the bilayer membrane. Then further addition of KCl to induce gradient-driven fusion resulted in little or no further increase in conductance and capacitance, demonstrating that docked vesicles have already fused [12].

 

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Fig. 17. Fluorimetric fusion assays demonstrate the ability of Ca2+ to induce rapid lipid mixing of plain (AV) and SNARE-associated vesicles. Addition of 5 mM Ca2+ to liposomal solution significantly increases the fusion of plain and SNARE-associated vesicles (+P0.1, Student t-test between AV and t- /v-SNARE-AV, n=5). Incorporation of t-/v-SNAREs at the vesicles membrane increases the overall yield but does not alter the rate of Ca2+-induced membrane fusion (A). The graph depicts the first-order kinetics of SNAREs vesicle fusion in the presence and absence of Ca2+ (B). [30].

 

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Fig. 18. Conductance and capacitance measurements of SNARE-reconstituted lipid bilayers. The EPC9-electrophysiological set up is shown (A). In the presence of 5 mM EGTA, t-SNARE-associated vesicles containing nystatin channels at their membrane (represented as red structures at the vesicle membrane) interact with v-SNARE-reconstituted lipid bilayer without fusing (B). Note no change in conductance or capacitance following exposure of SNAREassociated lipid vesicles to the bilayer. The vesicles fuse, however, when 3 mM KCl is applied, demonstrating fusion of docked vesicles and presence of an intact bilayer (B). The green arrowhead indicates when the stirring is switched on to mix the addition. In the presence of 1 mM CaCl2, the t-SNARE-associated vesicles fuse with the v- SNAREreconstituted bilayer as depicted in a consequent increase in conductance and capacitance. Since a large majority of docked vesicles have fused, addition of 3 mM KCl has no further e.ect (C). Traces (B-C) are representative profiles from one of five separate experiments [30].

 

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Fig. 19. AFM micrograph or live pancreatic acinar cell demonstrating the size of ZGs within the cell, in resting (A) and following stimulation of secretion (B). Note the increase in size of the same granules immediately following a secretory stimuli (section analysis of two such ZGs are shown). [39].

 

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Fig. 20. Increase in size of ZGs in the presence of GTP. (A-C) Two-dimensional AFM images of the same granules after exposure to 20 mM GTP at time 0 (A), 5 minutes (B), and 10 minutes (C). (D-F) The same granules are shown in three-dimensions: the three-dimensional image of the granules at time 0, 5 minutes, and 10 minutes, respectively, after exposure to GTP. (G-I) The GTP-induced increase in size of another group of ZGs observed by confocal microscopy. Confocal images of the same ZGs at time 0, 5 minutes, and 10 minutes after GTP exposure are shown. (Bar = 1 μm.) Values represent one of three representative experiments. [31].

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Fig. 21. AQP1-specific antibody binds to the ZG membrane and blocks water traffic. (A) Immunoblot assay demonstrating the presence of AQP1 antibody in SLO-permeabilized ZG. Lanes: 1, AQP1 antibody alone; 2, nonpermeable ZG exposed to antibody; 3, permeable ZG exposed to AQP1 antibody. Immunoelectron micrographs of intact ZGs exposed to AQP1 antibody demonstrate little labeling (B and C). (Bar = 200 nm.) Contrarily, SLO-treated ZG demonstrate intense gold labeling at the luminal side of the ZG membrane (D and E). AQP1 regulates GTP-induced water entry in ZG. (F) Schematic diagram ofZGmembrane depicting AQP1-specific antibody binding to the carboxyl domain of AQP1 at the intragranular side to block water gating. (G,H, and K) AQP1antibody introduced into ZG blocks GTP-induced water entry and swelling (from G to H, after GTP exposure). (I-K) However, only vehicle introduced into ZG retains the stimulatory effect of GTP (from I to J, after GTP exposure). [32].

 

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Fig. 22. Schematic outline depicting the regulatory pathway and the main players involved in water channel aquaporin-1 regulation. [33].

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Source: J. Cell. Mol. Med. Vol 8, No 1, 2004 pp. 1-21


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