PubMed2000
The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca(2+)](i) transient and exocytosis of dense core vesicles ("trichocysts") in Paramecium cells, when applied at usual concentrations (</=10 microm) in presence of extracellular Ca(2+) ([Ca(2+)](o) = 50 microm). When [Ca(2+)](o) is kept at 30 nm (<[Ca(2+)](rest)(i)), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca(2+)(o). Upon readdition of Ca(2+)(o) or some other appropriate Me(2+)(o) at 90 microm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca(2+), Sr(2+) or Mn(2+) are vesicular, but when formed in presence of Mg(2+), for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg(2+)](o) = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface ("frustrated exocytosis") within approximately 15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of approximately 35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of approximately 35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg(2+)](o) to 3 mm, or by application of the anti-calmodulin drug, R24517 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me(2+)] to maintain docking sites in a functional state, (ii) requirement of Ca(2+)(o) or of some other Me(2+)(o) to drive membrane resealing during exo-endocytosis, (iii) requirement of an "empty" signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a "filled" signal for trichocysts to undergo detachment and redocking (with fluorescence) after "frustrated exocytosis".