Manfred Lindau

High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism.

Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the ‘kiss-and-run’ mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.

Structure and function of fusion pores in exocytosis and ectoplasmic membrane fusion.

Several proteins involved in exocytosis have been identified recently, but it is still completely unclear which molecules perform the fusion event itself. Although in viral fusion the fusion proteins are known, even there the molecular mechanism remains controversial. Investigation of single fusion events by electrophysiological techniques together with fluorimetric measurements have now provided some insight into the properties of the first aqueous connection, the fusion pore. This pore has an initial size similar to an ion channel and allows movement of lipids only after it has substantially expanded, indicating that it is initially not a purely lipidic structure, but incorporates lipids when it expands. Although neurotransmitter release may occur through narrow transient fusion pores, the fusion pore of synaptic vesicles probably expands vey rapidly, making it unlikely that secretion is performed by rapid exo/endocytosis without full fusion under normal conditions. Recent recordings from small membrane patches have made it possible to resolve fusion events from vesicles as small as synaptic vesicles. Future experiments using excised patches may provide an approach to identify the molecular machinery of exocytotic membrane fusion.

The fusion pore

The secretory process requires many different steps and stages. Vesicles must be formed and transported to the target membrane. They must be tethered or docked at the appropriate sites and must be prepared for fusion (priming). As the last step, a fusion pore is formed and the contents are released. Release of neurotransmitter is an extremely rapid event leading to rise times of the postsynaptic response of less than 100 micro s. The release thus occurs during the initial formation of the exocytotic fusion pore. To understand the process of synaptic transmission, it is thus of outstanding importance to understand the molecular structure of the fusion pore, what are the properties of the initial fusion pore, how these properties affect the release process and what other factors may be limiting the kinetics of release. Here we review the techniques currently employed in fusion pore studies and discuss recent data and opinions on exocytotic fusion pore properties.

Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms.

Using the patch‐clamp technique, we studied the role of protein phosphorylation and dephosphorylation on the exocytotic fusion of secretory granules with the plasma membrane in horse eosinophils. Phorbol 12‐myristate 13‐acetate (PMA) had no effect on the amplitude and dynamics of degranulation, indicating that the formation of fusion pores is insensitive to activation of protein kinase C (PKC). Fusion pore expansion, however, was accelerated ∼2‐fold by PMA, and this effect was abolished by staurosporine. Elevating intracellular Ca2+ to 1.5 μM also resulted in a 2‐fold acceleration of pore expansion; this effect was not prevented by staurosporine, indicating that intracellular Ca2+ and activation of PKC accelerate fusion pore expansion via distinct mechanisms. However, fusion pores can expand fully even when PKC is inhibited. In contrast, the phosphatase inhibitor α‐naphthylphosphate inhibits exocytotic fusion and slows fusion pore expansion. These results demonstrate that, subsequent to its formation, fusion pore expansion is under control of proteins subject to functional changes based on their phosphorylation states.

Dissociation behavior of weak polyelectrolyte brushes on a planar surface.

Poly(acrylic acid) (PAA) brushes and poly(methacrylic acid) (PMAA) brushes on gold substrates were synthesized by surface-initiated atom-transfer radical polymerization of sodium acrylate and sodium methacrylate in water media at room temperature. Fourier transform infrared spectroscopy (FTIR) titration and contact angle titration methods were used in combination to investigate the dissociation behavior of these two brushes. Whereas FTIR titration gives effective bulk pKa values of the polyacid brushes (pKabulk of PAA brushes is 6.5-6.6 and pKabulk of PMAA brushes is 6.9-7.0), contact angle titration provides effective surface pKa of the brushes (pKasurf of PAA brushes is 4.4+/-0.01 and pKasurf of PMAA brushes is approximately 4.6+/-0.1). The difference between pKabulk and pKasurf suggests that acid groups further from the substrate surface are easier to ionize and have smaller pKa values. Although such behavior of weak polyelectrolyte brushes has been predicted by theoretical simulation, here we provide the first experimental evidence of this behavior.

Direct measurement of ion mobility in a conducting polymer.

Using planar junctions between the conducting polymer PEDOT:PSS and various electrolytes, it is possible to inject common ions and directly observe their transit through the film. The 1D geometry of the experiment allows a straightforward estimate of the ion drift mobilities.

Techniques and concepts in exocytosis : focus on mast cells

Int roduct ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430 A. First considerat ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434 1. Biophysical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434 2. Biocher•ical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434 B. Calc ium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

Focal exocytosis by eosinophils--compound exocytosis and cumulative fusion.

We have investigated the granule fusion events during exocytosis in horse eosinophils by time‐resolved patch‐clamp capacitance measurements. Stimulation with intracellular GTP gamma S leads to a stepwise capacitance increase by 4.0 +/− 0.9 pF. At GTP gamma S concentrations < 20 microM the step size distribution is in agreement with the granule size distribution in resting cells. Above 80 microM the number of steps is reduced and very large steps occur. The total capacitance increase, however, is unaffected. These results show that at high GTP gamma S concentrations granule‐‐granule fusion occurs inside the cell forming large compound granules, which then fuse with the plasma membrane (compound exocytosis). The electrical equivalent circuit of the cell during degranulation indicates the formation of a degranulation sac by cumulative fusion events. Fusion of the first granule with the plasma membrane induces fusion of further granules with this granule directing the release of all the granular material to the first fusion pore. The physiological function of eosinophils is the killing of parasites. Compound exocytosis and cumulative fusion enable the cells to focus the release of cytotoxic proteins to well defined target regions and prevent uncontrolled diffusion of this material, which would damage intact host cells.

Time-resolved capacitance measurements: Monitoring exocytosis in single cells.

Many cells release preformed material contained in secretory granules by exocytosis. Exocytosis is a specialized means of secretion in which the granules fuse with the plasma membrane and thereby discharge their contents through the fusion pores. This mechanism mediates, for example, the formation of the fertilization envelope in eggs, the release of neurotransmitters and neuropeptides by neurons, the release of a variety of enzymes and mediators by mast cells and granulocytes or the secretion of hormones by endocrine cells. Classical methods for investigating exocytosis usually measure release of secreted material.

The role of the C terminus of the SNARE protein SNAP-25 in fusion pore opening and a model for fusion pore mechanics

Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. It has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25Δ9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25Δ9 displayed smaller amperometric “foot-current” currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25Δ9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.