``Frustrated Exocytosis'' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

Abstract

Abstract. 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") , 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 ‫ס‬ 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 ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼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, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me 2+ ] to maintain docking sites in a functional state, (ii) requirement of Ca o 2+ or of some other Me o 2+ 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".

Signalling in ciliates: long- and short-range signals and molecular determinants for cellular dynamics

“…When exocytotic membrane fusion is induced in Paramecium tetraurelia under conditions inhibiting decondensation/release of trichocyst contents, the trichocyst membrane reseals, the organelles become detached and undergo a new round of docking for successful exocytosis upon stimulation under standard conditions (Klauke & Plattner, ). This phenomenon of ‘frustrated exocytosis’ indicates the occurrence of additional signals provided by the filling state that mediate competence for docking of trichocysts at the cell membrane.…”

Trichocysts—Paramecium's Projectile‐like Secretory Organelles

“…), as to be discussed in more detail below. Fifth, in the extreme, when membrane fusion is induced, at low [Ca 2+ ] o , membranes are to be resealed, followed by detachment of trichocysts for a new secretory cycle—a process called “frustrated exocytosis” (Klauke and Plattner ); see Fig. and section “Exocytotic membrane fusion”, below.…”

“…The reattachment phase of a trichocyst labeled by arrow is shown between 9.6 and 25.6 s. Scale bars – 5 μm. From Klauke and Plattner ().…”

“…), probably by perturbing membrane lipids. However, at low [Ca 2+ ] o , trichocyst contents are not discharged, exocytosis sites are slowly resealed and trichocysts with their FM1‐43 labeled membrane and all their contents are detached (Klauke and Plattner ). This phenomenon has been called “frustrated exocytosis”.…”

My favorite cell—Paramecium

“…6. (75) In the presence of the fluorescent fusion indicator, FM1-43, membrane fusion can occur, while trichocyst contents discharge can be inhibited by lowering [Ca 2þ ] in the medium close to internal resting levels. Cells do recognize-by an unknown signal-that membranes had fused, but not that their trichocysts were not emptied.…”