Exocytosis Assay

A variety of methods have been developed to study exocytosis. Neurotransmitter and hormone release can be measured by the electrical effects of released neurotransmitter or hormone on postsynaptic membrane receptors, such as the neuromuscular junction (NMJ see below), and directly by biochemical assay. Another direct measure of exocytosis is the increase in membrane area due to the incorporation of the secretory granule or vesicle membrane into the plasma membrane. This can be measured by increases in membrane capacitance (Cm). Cm is directly proportional to membrane area and is defined as Cm = QAJV, where Cm is the membrane capacitance in farads (F), Q is the charge across the membrane in coulombs (C), V is voltage (V) and Am is the area of the plasma membrane (cm2). The specific capacitance, Q/V, is the amount of charge that must be deposited across 1 cm2 of membrane to change the potential by IV. The specific capacitance, mainly determined by the thickness and dielectric constant of the phospholipid bilayer membrane, is approximately 1 pF/cm2 for intracellular organelles and the plasma membrane. Therefore, the increase in plasma membrane area due to exocytosis is proportional to the increase in Cm. 

Selected entries from Methods in Enzymology [vol, page(s)] Chelation, 238, 74, 76, 297 buffers [for analysis of exocytosis, 221, 132 preparation, 219, 186 modulation of cytosolic buffering capacity with quin2, 221, 159] fluorescence assay, 240, 724-725, 740-742 fluorescence imaging, 225, 531 238, 303-304, 322-325, 334-335 free intracellular levels after bacterial invasion, 236, 482-489 free calcium in solutions for membrane fusion analysis, calculation and control, 221, 149 homeostasis mechanisms, 238, 80 hormonal elevation, 238, 79 inositol phosphate effect on release, 238, 207 determination of cytosolic levels [computer methods, 238, 73-75 with fura-2, 238, 73, 146 with indo-1, 238, 298, 316-317 with quin-2, 238, 297] hormone effects, 238, 79 ionomycin effects, 238, 79 membrane depolarization effects,... 

Sanchez-Prieto J, Shira TS, Evans D, Ashton A, Dolly JO, Nicholls DG (1987) Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes. In Eur. J. Biochem. 165 675-81 Schiavo G, Montecucco C (1995) Tetanus and botulism neurotoxins isolation and assay. Methods Enzymol. 248 643-52... 

Fig. 1. Comparison of various SLO preparations using Ca -stimulated exocytosis from PC 12 cells. The experiment was performed as described in section 2.2 of this chapter using either SLO purified from the culture supernatant, SLO fusion protein, or the fusion protein of the alanine mutant. In this assay the SLO fusion protein was slightly more effective than the mutant protein...

Assay of exocytosis for cells attached to culture plates... 

For each BoNT serotype, the dichain form constimtes the active configuration of the neurotoxin the isolated LC and HC are devoid of systemic toxicity. The absence of toxicity is consistent with findings that the LC cannot gain access to the cytosol unless it is coupled to the HC and that the HC lacks the ability to inhibit neurotransmitter release (Stecher et al., 1989 Goodnough et al., 2002). The isolated LC does, however, remain enzymatically active as evidenced by its ability to inhibit exocytosis from permeabilized chromaffin cells (Stecher et al., 1989), by its ability to cleave SNARE proteins in cell-free assays (Adler et al., 1998), and by its capacity to inhibit ACh release in skeletal muscle when delivered by liposomes (de Paiva and Dolly, 1990). It is not clear whether any portion of the HC is translocated along with the LC, and if so, whether it exerts a role in enhancing the catalytic activity or stability of the LC. 

D. ASSAY FOR EXOCYTOSIS FROM BOVINE ADRENAL CHROMAFFIN CELLS IN PRIMARY CULTURE... 

Amperometry at single PC 12 cells has also been used in conjunction with a genetic cell transfection protocol to examine the effects of toxin expression on basal and evoked exocytosis. PC 12 cells have been transfected with the specific endoprotease Botulinum neurotoxin Cl light chain (BoNT/Cl), which cleaves the proteins syntaxin and SNAP-25 [5], The molecular dissection of the mechanisms underlying exocytosis has been motivated by the SNARE hypothesis, which postulates that exocytosis requires the assembly of the plasma membrane proteins syntaxin 1, SNAP-25, and the vesicle associated membrane protein (VAMP) into a complex [5], This SNARE complex then acts as a receptor for cytosolic components of the proposed fusion machinery. Direct evidence for the role of the SNARE proteins in neurotransmission comes from molecular genetic studies in which syntaxin and VAMP have been shown to be required for neurotransmission in Drosophila [47 9] and Caenorhabditis elegans [50,51]. To assess the effects of the disruption of SNARE proteins on exocytosis in PC 12 cells, amperometry has been used in conjunction with a genetic cell transfection assay to establish a... 

Biological Applications Detecting FRET in cells monitoring fast neuronal activity and signaling quantifying plasma membrane expression cytotoxicity assay membrane fusion assay probe for endocyto-sis probe for exocytosis ... 

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