Exocytosis Of Single Granules Research Articles

Imaging the Disassembly of Syntxin Cluster during the Exocytosis of Single Secretory Granules

During the exocytosis of synaptic vesicles or of secretory granules, three so-called SNARE proteins mediate membrane fusion. One of the SNARE proteins, Syntaxin (Syx) is known to reside in the plasma membrane. Our previous work has shown that Syx makes a submicron size cluster where a granule docks at the plasma membrane. By labeling Syx clusters and granules in different colors, we now show that the Syx cluster disassembles when the adjacent granule undergoes exocytosis.As a model system, we used Ins-1 cells, a cell line derived from pancreatic beta cells. Cells expressed tissue plasminogen activator-EGFP (tPA-EGFP) to mark granules in green, and Syx-mCherry to mark Syx in red. Cells were voltage clamped in the whole-cell mode and exocytisis was evoked by depolarizing pulse. Exocytosis of single granules was observed with TIRF. Some granules (“responders”) brightened suddenly, as their exocytosis caused the release of protons and the de-quenching of tPA-EGFP. Other granules remained quiescent and served as controls (non-responders). The fluorescence of Syx-mCherry was measured at the same times and locations as that of the granules. Before exocytosis, the Syx-mCherry signal was significantly brighter beneath responders than beneath non-responders. This is consistent with the idea that syntaxin nanodomains facilitate exocytosis. After exocytosis, the Syx-mCherry signal beneath responders diminished, while no large change occurred for non-responders. Some “responders” were not visible before fusion, suggesting that they were located apart from the plasma membrane. Even for such granules, Syx-mCherry signal was observed before exocytosis and diminished after exocytosis. These results indicate that Syx clusters beneath granules are an advantage for exocytosis but then disassembled at the time of granule fusion.

Exocytosis of single chromaffin granules in cell-free inside-out membrane patches.

In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.

Optical detection of synaptic vesicle exocytosis and endocytosis

Recent advances in optical methods have catalyzed a detailed study of individual visualized synapses in several model systems. Quantal events at small central synapses, as well as single granule exocytosis in secretory cells, have been detected using quantitative fluorescence imaging. Sensitive detection of exocytosis and endocytosis at individual synapses has advanced our knowledge of synaptic vesicle trafficking.

Capacitance Flickers and Pseudoflickers of Small Granules, Measured in the Cell-Attached Configuration

We have studied exocytosis of single small granules from human neutrophils by capacitance recordings in the cell-attached configuration. We found that 2.2% of the exocytotic events were flickers. The flickers always ended with a downward step. This indicates closing of the fusion pore. During flickering, the fusion pore conductance remained below 1 nS, and no net membrane transfer was detectable. After fusion pore expansion beyond 1 nS the pore expanded irreversibly, leading to rapid full incorporation of the granule/vesicle into the plasma membrane. Following exocytosis of single granules, a capacitance decrease directly related to the preceding increase was observed in 7% of the exocytotic events. This decrease followed immediately after irreversible pore expansion, and is presumably triggered by full incorporation of the vesicle into the patch membrane. The capacitance decrease could be interpreted as endocytosis triggered by exocytosis. However, the gradual decrease could also reflect a decrease in the “free” patch area following incorporation of an exocytosed vesicle. We conclude that non-stepwise capacitance changes must be interpreted with caution, since a number of factors go into determining cell or patch admittance.

Widespread association of annexin II with intracellular membrane systems and involvement of annexin II in granule--granule fusion in addition to granule--plasma membrane fusion in anterior pituitary cells

The subcellular localization in anterior pituitary secretory cells of annexin II, one of the Ca2+-dependent phospholipid-binding proteins, was examined by immunohistochemistry and immunoelectron microscopy. Annexin II was associated with the plasma membrane, the membranes of secretory granules and cytoplasmic organelles, such as rough endoplasmic reticulum, mitochondria and vesicles, and with the nuclear envelope. Annexin II was frequently detected at the contact sites of secretory granules with other granules and with the plasma membrane. The anterior pituitary and adrenal medulla were treated with Clostridium perfringens enterotoxin, which induces Ca2+ influx, and examined under an electron microscope. The anterior pituitary cells showed multigranular exocytosis, i.e. multiple fusions of secretory granules with each other and with the plasma membrane, but adrenal chromaffin cells, which lack annexin II on the granule membranes, never showed granule--granule fusion and only single granule exocytosis. From these results, we conclude that, in anterior pituitary secretory cells, annexin II is involved in granule--granule fusion in addition to granule--plasma membrane fusion. © 1998 Chapman & Hall

Multigranular exocytosis induced by phospholipase A2-activators, melittin and mastoparan, in rat anterior pituitary cells.

Effects of phospholipase A2-activators, melittin and mastoparan, on rat anterior pituitary cells were studied by use of the electron microscope. Rat anterior pituitaries were incubated in HEPES buffer containing 20 micrograms/ml of melittin or the same dose of mastoparan for 5 min, 10 min and 20 min. Features indicating discharge of granule contents by exocytosis were increased with time, and the simultaneous extrusion of a number of secretory granules, named "multigranular exocytosis", was often recognized in addition to single-granule exocytosis at 10 min and 20 min. Most membrane pits, where the multigranular exocytosis as well as the single-granule exocytosis occurred, were coated. Moreover, a large number of vesicles coated or non-coated were distributed near the trans side of the Golgi apparatus of melittin-treated or mastoparan-treated cells after 20 min. These vesicles might be related to membrane internalized from the excess surface membrane derived from the limiting membrane of exocytosed granules. These observations indicate that phospholipase A2-activators induce hormone release involving membrane fusion between limiting membranes of secretory granules, and between granule-limiting membrane and plasma membrane in rat anterior pituitary cells.