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Biology Articles » Cell biology » Molecular Machinery and Mechanism of Cell Secretion » Figures

Figures
- Molecular Machinery and Mechanism of Cell Secretion

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Figure 1. (A) 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 (3). (B) 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 membrane-bound secretory vesicle of exocrine pancreas. Also, a cross-section of the ring at the mouth of the porosome is seen (blue arrow head).

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Figure 2. AFM micrograph of depressions, or porosomes, or fusion pores in live secretory cell of the exocrine pancreas (A, B), the growth hormone-secreting cell of the pituitary (C), in the chromaffin cell (D), and neurons (E, F). Note the pit (white arrow heads) with four depressions (yellow arrow head). A high-resolution AFM micrograph is shown in B. Bars = 40 nm for A and B. Similarly, AFM micrographs of porosomes in ß-cell of the endocrine pancreas and mast cell have been observed. Electron micrograph of neuronal porosomes, which are 10–15-nm cup-shaped structures at the presynaptic membrane, where synaptic vesicles transiently dock and fuse to release vesicular contents (E). Atomic force micrograph of isolated neuronal porosome, reconstituted into lipid membrane (F).

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Figure 3. Depressions are fusion pores, or porosomes. Porosomes dilate to allow expulsion of vesicular contents. (A, 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 immunogold 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 (4).

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Figure 4. 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.

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Figure 5. AFM micrographs revealing the dynamics of docked synaptic vesicles at porosomes and the porosome architecture in greater detail. (A) AFM micrograph of five docked synaptic vesicle at porosomes. (B) Addition of 50 µM ATP dislodges two synaptic vesicles at the lower left and exposes the porosome patches (red arrowheads). This also reveals that a single synaptic vesicle may dock at more than one porosome complex. (C–G) AFM micrographs obtained at higher imaging forces (300–500 pN rather than

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Figure 6. 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 three-dimensional 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. 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.

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Figure 7. Wide-angle x-ray diffraction patterns of interacting SNARE vesicles. Representative diffraction profiles from one of four separate experiments using t- and v-SNARE-reconstituted lipid vesicles, both in the presence or absence of 5 mM Ca2+ are shown. Arrows mark appearance of a new peak in the x-ray diffractogram following addition of calcium.

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Figure 8. Light-scattering profiles of SNARE-associated vesicle interactions. (A, B) Addition of t-SNARE and v-SNARE vesicles in calcium-free buffer lead to a significant increase in light scattering. Subsequent addition of 5 mM Ca2+(marked by arrowhead) does not have any significant effect on light scattering ({square}). (A, C) In the presence of NSF-ATP (1 µg/ml) in assay buffer containing 5mM Ca2+, there was significantly inhibited vesicle aggregation and fusion ({triangleup}). (A, D) When the assay buffer was supplemented with 5mM Ca2+ before addition of t- and v-SNARE vesicles, it led to a 4-fold decrease in light scattering intensity due to Ca2+-induced aggregation and fusion of t-/v-SNARE apposed vesicles ({circ}). Light-scattering profiles shown are representatives of four separate experiments.

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Figure 9. Swelling of isolated ZGs. (A) Electron micrograph of isolated ZGs demonstrating a homogeneous preparation. Bar = 2.5 µm. (B, C) Isolated ZGs, on exposure to 20 µM GTP, swell rapidly. Note the enlargement of ZGs as determined by AFM-section analysis of two vesicles (red and green arrowheads). (D) Percentage ZG volume increase in response to 20 µM GTP. Note how different ZGs respond to the GTP-induced swelling differently (46).

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Figure 10. Fusion of isolated ZGs at porosome-reconstituted bilayer and GTP-induced expulsion of {alpha}-amylase. (A) Schematic diagram of the EPC9 bilayer apparatus showing the cis and trans chambers. Isolated ZGs, when added to the cis chamber, fuse at the bilayers-reconstituted porosome. Addition of GTP to the cis chamber induces ZG swelling and expulsion of its contents, such as {alpha}-amylase to the trans bilayers chamber. (B) Capacitance traces of the lipid bilayer from three separate experiments following reconstitution of porosomes (green arrowhead), addition of ZGs to the cis chamber (blue arrowhead), and the red arrowhead represents the 5-min time point after ZG addition. Note the small increase in membrane capacitance following porosome reconstitution and a greater increase when ZGs fuse at the bilayers. (C) In a separate experiment, 15 mins after addition of ZGs to the cis chamber, 20 µM GTP was introduced. Note the increase in capacitance, demonstrating potentiation of ZG fusion. Flickers in current trace represent current activity. (D) Immunoblot analysis of {alpha}-amylase in the trans chamber fluid at different times following exposure to ZGs and GTP. Note the undetectable levels of {alpha}-amylase even up to 15 mins following ZG fusion at the bilayer. However, following exposure to GTP, significant amounts of {alpha}-amylase from within ZGs were expelled into the trans bilayers chamber (n = 6; Ref. 46).

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Source: Experimental Biology and Medicine 230:307-319 (2005).


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