J.CellSd. 77,1-17(1985) Printed in Great Britain © Company of Biologists Limited 1985 SYNCHRONOUS EXOCYTOSIS IN PARAMECIUM CELLS. VI. ULTRASTRUCTURAL ANALYSIS OF MEMBRANE RESEALING AND RETRIEVAL H. PLATTNER*. R. PAPE, B. HAACKE, K. OLBRICHT, C. WESTPHAL AND H. KERSKEN Faculty of Biology, University ofKonstanz, P.OJi. 5560, D-7750 Konstanz, Federal Republic of Germany SUMMARY After the synchronous induction of exocytosis of secretory organelles (trichocysts) in Paramecium tetraurelia cells the process of membrane resealing and retrieval could be followed under syn- chronous conditions. The characteristic aggregates of membrane intercalated particles (MIPs) contained within the freeze-fractured cell membrane (rings and rosettes) and trichocyst membranes (annulus MIPs), in addition to collar striations on the top of trichocyst membranes, served as endogenous ultrastructural markers. This allowed us to follow the re-arrangement of membrane constituents during and after exocytosis with high temporal and spatial precision. Membrane specificity is maintained to a considerable extent (~ 99-5 %), as judged from the rare occurrence of aberrant resealing (according to freeze-fracture data) and from the rather minute shift of glycocalyx components (according to electron staining experiments) during normal membrane resealing. Coated pits are not involved in membrane retrieval (155 ghosts analysed); since the membrane regions involved in exocytotic fusion are backed by apposed materials, probably proteins, this may restrain membrane constituents from intermixing. Another factor for maintaining membrane specificity is the fact that resealing of the exocytotic opening occurs much more rapidly than in most other systems. The retrieval operates with a half-life of 3 (strain 75) to 9min (K401); the involve- ment of cortical microtubules in the retrieval can be largely excluded, since only two microtubules (of unidentified origin) were seen to approach ghost structures in 4074 cases analysed during this period of intense ghost retrieval. Phalloidtn microinjected at a dose that blocked all cytoplasmic streaming (before synchronous exocytosis was induced) did not abolish membrane resealing and retrieval, which, therefore, may be passive processes. INTRODUCTION In a variety of cell types exocytosis is followed by retrieval of the secretory organelle membrane, which in turn may undergo recycling via elements of the Golgi apparatus (for reviews, see Farquhar, 1981; Holtzman, 1981). Nevertheless, important aspects of what occurs immediately after the release of secretory contents still await further elucidation. In particular, as recently summarized by Farquhar (1981), the following questions remain to be clarified. (1) What is the precise nature of the membrane removed from the cell surface? (2) What is the nature of the carrier, and are coated vesicles involved? (3) Are there stable, non-perturbing labels available for tracing the retrieval process? •Author for correspondence. Key words: exocytosis, freeze-fracture, membranes, Paramecium, secretion. 2 H. Plattner and others Although the answers to these questions may be different for different cell types, they appear to be particularly easy to approach in the case of our model system, the ciliated protozoon Paramecium tetraurelia. Each Paramecium cell has more than 1000 secretory organelles (trichocysts), most of which are dischargeable within one or perhaps several seconds by polyamine trigger compounds (Plattner et al. 1984; Plattner, Sturzl & Matt, 1985). This process entails exocytotic membrane fusion, resealing (Olbricht, Plattner & Matt, 1984) and subsequent retrieval (Haacke & Plattner, 1984). Distinct structural elements are present in the fusogenic zones of the cell membrane (Janisch, 1972; Plattner, Miller & Bachmann, 1973) and in or on the trichocyst tip membrane (Allen & Hausmann, 1976; Beisson et al. 1976; Hausmann & Allen, 1976; Plattner et al. 1973), which can be used as endogenous markers. These structural elements are in the form of: (1) membrane-intercalated particles (MIPs) visible after freeze-fracturing, and (2) membrane-associated materials (Westphal & Plattner, 1981a, b) seen in ultrathin sections, when tannic acid is applied as a mordant dye according to Fujiwara & Linck (1982). In contrast, most other exocytotic systems require exogenous markers (e.g. antibodies, lectins or affinity labels) for tracing the fate of certain membrane areas, and these tags may well influence the processes to be analysed (Farquhar, 1981; Helenius & Mellman, 1983; Holtzman, 1981). In the Paramecium cell the resealing of ghost membranes after trichocyst discharge has been described previously by us (Haacke & Plattner, 1984; Olbricht et al. 1984) and others (Allen & Hausmann, 1976; Hausmann & Allen, 1976). However, in none of these publications has the degree of specificity of membrane separation or any possible involvement of clathrin coats, microtubules or microfilaments been analysed in any detail. The present paper sets out to examine these aspects. MATERIALS AND METHODS Cell cultures Paramecium tetraurelia, strains 7S or K401, which are both equipped with a normal exocytotic apparatus (Plattner et al. 1980) were used as monoxenic cultures in a dry lettuce medium inoculated with Enterobacter aemgenes. 7S cells grew within 3 days, and K401 cells within 4 days, to early stationary phase. Such cells were triggered for trichocyst release with aminoethyldextran (AED) in the presence of exogenous Ca2+ as indicated before (Plattner et al. 1984, 1985). Electron microscopy For ultrathin sectioning cells were fixed in different ways. For standard preparations we used glutaraldehyde, followed by osmium tetroxide as described previously (Plattneret al. 1984); tannic acid impregnation after saponin permeabilization was according to Haacke & Plattner (1984). In some experiments exogenous Ca^+ was chelated by adding 4mM-EGTA. In other experiments, living cells were exposed for 10s to cationized ferritin (Miles-Yeda, Rehovot, Israel), which could be used as a surface label and simultaneously - like other polycationic compounds (Plattner et al. 1985) - as a trigger agent. Routinely, cell pellets were dehydrated in acetone and embedded in Spurr's (1969) resin. After polymerization at 344 K, ultrathin sections of ~70nm thickness were made and double-stained with uranyl acetate and alkaline lead citrate. Some sections were subjected to polysaccharide staining according to Farragiana & Marinozzi (1979). For an analysis of mem- brane dynamics cells were quickly frozen in the living state as indicated by Olbricht et al. (1984). A Balzers BAF 300 freeze-fracture machine was used. The designation of fracture faces was as indicated by Plattner & Zingsheim (1983). Membrane reseating and retrieval 3 In addition, 10 cells were microinjected with rhodaminylated phalloidin (a gift from Th. Wieland) under a Zeiss I CM 405 inverted microscope with fluorescence optics and a video-enhanced image-intensifying system, as specified in more detail elsewhere (Kersken, Vilmart-Seuwen, Momayezi & Plattner, unpublished). Phalloidin doses were deliberately chosen high enough (up to 50 ^g/ml cell volume) for any cell motility phenomena to be totally blocked; only then (mostly after 30 min) were cells triggered with AED, and 10 s or 10 min afterwards they were fixed with glutaral- dehyde, supplemented with EGTA, tannic acid and saponin (see above). Individual cells were then processed to produce ultrathin sections as outlined above. Sections and replicas were analysed in a Zeiss EM 10 electon microscope (80kV). The different structures presented in Tables 1 and 2 were documented and counted in a random sampling procedure. RESULTS For the normal ultrastructure of trichocyst docking sites, we refer to Plattner et al. (1973) and Westphal & Plattner (1981a, b). A summary of the salient features is given in the scheme at the end of this paper. For the normal course of ultrastructural changes during and after AED-induced sychronous trichocyst release, concerning aspects not covered in this paper, we also refer to previous work (Haacke & Plattner, 1984; Olbricht et al. 1984; Pape & Platt- ner, 1985). Freeze-fracturing revealed that exocytosis involves the dispersal of rosette MIPs from within the rings to outside the fusion zone, not before but precisely during the formation of the exocytotic opening (Olbricht et al. 1984). Elements of the trichocyst tip membrane, the annulus MIPs and collar striations, become only tran- siently visible from the outside (Figs 1,2); when normal membrane detachment and Figs 1—4. Resealing stages obtained from AED-triggered freeze-fractured, unfixed cells. Fig. 1 is a PF view of an exocytotic opening; Fig. 2 is a somewhat later stage; an, annulus; co, collar; 'RI presumably indicates the ring, which is difficult to identify due to the expansion of the exocytotic site during discharge, while trichocyst and cell membrane are fused perfectly with each other. Fig. 3 is interpreted as an early {oval ring) and Fig. 4 as a late resealed stage (parenthesis). Bars, 0-1 /an. 4 H. Planner and others Table 1. Freeze-fracture appearance ofexocytosis sites at different times after trigger- ing ofexocytosis Time Stage 0 Is 1 min Resting stage 71-4 56-7 5-3 (ring + rosette) Oval ring* 1-6 4-2 3-6 (early resealed stage) Parenthesis] 18-0 16-8 82-2 (late resealed stage) Other stagesj 9-0 22-3 8-9 Number of docking sites analysed (n) 1656 978 225 The presence of methylcellulose retarded exocytotic processes in these freeze-fracture