For fusion to occur the repulsive forces between two interacting phospholipid bilayers must be reduced. In model systems, this can be achieved by increasing the surface tension of at least one of the membranes. However, there has so far been no evidence that the secretory granule membrane is under tension. We have been studying exocytosis by using the patch-clamp technique to measure the surface area of the plasma membrane of degranulating mast cells. When a secretory granule fuses with the plasma membrane there is a step increase in the cell surface area. Some fusion events are reversible, in which case we have found that the backstep is larger than the initial step, indicating that there is a net decrease in the area of the plasma membrane. The decrease has the following properties: (i) the magnitude is strongly dependent on the lifetime of the fusion event and can be extensive, representing as much as 40% of the initial granule surface area; (ii) the rate of decrease is independent of granule size; and (iii) the decrease is not dependent on swelling of the secretory granule matrix. We conclude that the granule membrane is under tension and that this tension causes a net transfer of membrane from the plasma membrane to the secretory granule, while they are connected by the fusion pore. The high membrane tension in the secretory granule may be the critical stress necessary for bringing about exocytotic fusion.
The development of mechanical force in skeletal muscle fibres is brought about by rapid increases in the intracellular calcium concentration (Ca2+ transients) which can be detected by optical methods. Local stimulation experiments and ultrastructural evidence suggest that, at a microscopic level, these Ca2+ transients are generated by the release of Ca2+ ions from the terminal cisternae of the sarcoplasmic reticulum in response to the depolarization of the transverse tubules (t-tubules). Nevertheless, to date, there is no functional information on the exact location at which Ca2+ release takes place. The present experiments were designed to obtain direct evidence about dynamic changes in localization and microscopic distribution of Ca2+ in a single sarcomere using two independent novel methodologies: confocal spot detection of Ca2+ transients and Ca2+ imaging with pulsed laser excitation.
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