Filamentous actin in Paramecium cells: mapping by phalloidin affinity labeling in vivo and in vitro

In living Paramecium cells, microinjected rhodaminyl (R)-phalloidin rapidly labels a thin cortical layer. This can be more clearly resolved with microinjected and fixed cells (allowing for better resolution) as well as with isolated pellicles (surface membrane complexes with trichocysts, microfilaments, and mitochondria attached). Labeling of a longitudinal and perpendicular pattern, reflecting the relief of the cell surface, and labeling of ciliary basal bodies then becomes clearly visible. Other structures labeled by R-phalloidin are the surfaces of food vacuoles of different sizes and, although inconsistently, the borders of the buccal cavity. Small acidic compartments (as identified by acridine orange fluorescence vital staining), probably representing acidosomes and small lysosomes, were not labeled. F-actin on food vacuole surfaces may somehow be involved in intracellular transport or fusion processes. No labeling was observed in association with the osmoregulatory system (contractile vacuoles and their ampullae and radial canals). The specificity of in vivo labeling obtained was supported by the abolition of R-phalloidin labeling when isolated pellicles were pretreated with unlabeled phalloidin or with DNAse I. It was also possible to discriminate among different layers of R-phalloidin binding in the cortex by detaching different layers of the surface complex from each other. Since localization of F-actin in ciliates has raised a considerable amount of dispute in the past, we also repeated all these experiments with RITC-labeled HMM, but we obtained essentially the same labeling pattern as with R-phalloidin. Ciliary basal bodies therefore clearly contain some F-actin. Our data shed some light on aspects of surface structuring and motility in these cells.     

Calmodulin in Paramecium tetraurelia: localization from the in vivo to the ultrastructural level

Monospecific polyclonal antibodies against Paramecium tetraurelia calmodulin were prepared and labeled for calmodulin localization on different levels of resolution: by microinjection into living cells; with isolated cell surface complexes (cortices); on the ultrastructural level, using Lowicryl sections of non-permeabilized cells (with colloidal gold-protein A labeling of antibodies bound); or using permeabilized and gently fixed cells for incubation with peroxidase- or microperoxidase-tagged antibodies. Sites selectively labeled above cytoplasmic background largely coincided, irrespective of the method used, although sensitivity, resolution, and liability to redistribution of antigen were quite different. (The methodological diversification applied allowed for their mutual control.) Nonspecific binding can be largely excluded, since all these methods gave negative results with pre-immune sera. We reached the following conclusions on sites with selective calmodulin binding (above cytoplasmic background level) in P. tetraurelia cells. A pool of calmodulin co-localized with F-actin, not only in the cortex (including fibrous materials around ciliary basal bodies) but also around food vacuoles (phagosomes) and, to a lesser degree, around the buccal cavity. Trichocyst docking sites on the cell membrane, and coated pits also displayed calmodulin labeling, thus indicating the potential involvement of calmodulin in exo-endocytosis processes. Calmodulin was also enriched on membranes of compartments with presumable ion (possibly Ca2+) transport capacity, such as trichocysts and the osmoregulatory system. Not selectively labeled were nuclei, mitochondria, and some small lysosomal organelles (as identified in vivo by rhodamine 123 or acridine orange fluorescence, respectively).     

Morphological docking of secretory vesicles

Calcium-dependent secretion of neurotransmitters and hormones is essential for brain function and neuroendocrine-signaling. Prior to exocytosis, neurotransmitter-containing vesicles dock to the target membrane. In electron micrographs of neurons and neuroendocrine cells, like chromaffin cells many synaptic vesicles (SVs) and large dense-core vesicles (LDCVs) are docked. For many years the molecular identity of the morphologically docked state was unknown. Recently, we resolved the minimal docking machinery in adrenal medullary chromaffin cells using embryonic mouse model systems together with electron-microscopic analyses and also found that docking is controlled by the sub-membrane filamentous (F-)actin. Currently it is unclear if the same docking machinery operates in synapses. Here, I will review our docking assay that led to the identification of the LDCV docking machinery in chromaffin cells and also discuss whether identical docking proteins are required for SV docking in synapses.     

Synchronous exocytosis in Paramecium cells. III. Rearrangement of membranes and membrane-associated structural elements after exocytosis performance

Aminoethyldextran (AED) was used to trigger the synchronous release of trichocysts from Paramecium tetraurelia cells (see [8]) by a mechanism involving exocytotic membrane fusion and resealing (see [5]). Ultrastructural changes were analyzed by quantitative evaluation of ultrathin sections. In resting cells the percentage of potential trichocyst-docking sites which are actually occupied by a trichocyst was 58%; 36% of potential docking sites contained ghosts and 6% a "plug" of electron-dense material. We derived from our data that paramecia would discharge permanently and spontaneously trichocysts (without AED) at a rate of 2-3 per min (which we then also verified by counting the spontaneous release rate) and that this value is equivalent to the docking rate. For the synchronous expulsion of trichocysts in response to AED we had determined that the degree of synchrony is more than a hundred times better than in most other systems (see [8]). We have determined the half-lives (HL) for different events involved in exocytosis and re-docking as follows: approximately 3 sec for trichocyst discharge, approximately 3 sec for the formation of ghosts, 8 min for the clearing of ghosts from the cell surface, 4 min for the formation of "plugs". Trichocysts are docked with a HL of 40 min and "plugs" (considered as receptor-type structures for trichocyst docking) disappear with a concomitant HL of 50 min. Evidently the clearing of ghosts allows for re-formation of "plugs" but the respective HL values signal that "plugs" may also be formed anew. The relatively slow decline of the percentage of "plugs" (after their azimuth 15 min after AED triggering) may also indicate the synthesis of new docking sites. After a period of over approximately 3 h following AED triggering, the original situation is roughly re-established and maintained over the whole period of population growth analyzed.     

Microinjection of pollen‐specific actin‐depolymerizing factor, ZmADF1, reorientates F‐actin strands in Tradescantia stamen hair cells

Maize actin-depolymerizing factor (ADF) binds both monomeric and filamentous actin and increases actin dynamics in vitro. To test its effects in vivo, recombinant pollen ADF1 was expressed in bacteria and microinjected into Tradescantia stamen hair cells. Initially, all cytoplasmic streaming ceased and the central, longitudinal transvacuolar strands were disrupted. After 20–45 min, streaming resumed but in the form of conspicuous transverse pathways of movement in the cortex. Staining the actin filaments by a second injection of fluorescein-conjugated phalloidin showed that the longitudinal actin cables seen in controls had been replaced by a thickening of the transverse cortical arrays, whose orientation matched the new pattern of streaming. Microinjection of rhodamine–tubulin confirmed that the microtubules also formed a transverse cortical array and it is suggested that the spatial cues for re-modelling the actin after ADF1 injection may be provided by the microtubular system.     

Secretory proteins in a ciliate cell: Identification of epitopes common to numerous polypeptides

Secretory vesicles of the ciliate Pseudomicrothorax dubius, called trichocysts, are separated into > 40 proteins by two-dimensional gel electrophoresis. The trichocyst, composed of a shaft and four arms, is in a condensed state when docked in the cell cortex, and it elongates into an extended state during exocytosis. Monoclonal antibodies (mAbs) were raised against trichocyst proteins. Their reactivities were analysed: I) on Western blots of extended, isolated trichocysts by immunolabeling; 2) on entire cells and extended trichocysts by indirect immunofluorescent binding assay (IFA); 3) on semi-thin sectioned cells by IFA; and 4) on ultra-thin sections of cells by immunogold labeling. mAb IV 4E5 labels major trichocyst proteins at 15–19, 22 and 24 kDa, pI 4.6?6.6. The epitope recognized by mAb IV 4E5 is common to as many as 30 proteins and suggests a family of proteins with possible sequence homology. By IFA, the shafts of extended trichocysts are labeled. The shafts of condensed trichocysts are labeled on both semi-thin sections in Lowicryl and ultrathin sections. On semi-thin Epon sections, the part of the trichocyst which is labeled is arm-like. mAb VI 2D12 labels three major trichocyst proteins at 31 kDa, pI 5.0?5.4. The arms of extended trichocysts are labeled by IFA, but are only weakly labeled on ultrathin sections. The shaft of extended trichocysts is labeled by IFA, and the shaft of condensed trichocysts is labeled on ultrathin sections.     

Cytoskeleton-secretory vesicle interactions during the docking of secretory vesicles at the cell membrane in Paramecium tetraurelia cells

Stationary-phase cells of Paramecium tetraurelia have most of their many secretory vesicles ("trichocysts") attached to the cell surface. Log-phase cells contain numerous unoccupied potential docking sites for trichocysts and many free trichocysts in the cytoplasm. To study the possible involvement of cytoskeletal elements, notably of microtubules, in the process of positioning of trichocysts at the cell surface, we took advantage of these stages. Cells were stained with tannic acid and subsequently analyzed by electron microscopy. Semithin sections allowed the determination of structural connections over a range of up to 10 micrometer. Microtubules emanating from ciliary basal bodies are seen in contact with free trichocysts, which appear to be transported, with their tip first, to the cell surface. (This can account for the saltatory movement reported by others). It is noteworthy that the "rails" represented by the microtubules do not directly determine the final attachment site of a trichocyst. Unoccupied attachment sites are characterized by a "plug" of electron-dense material just below the plasma membrane; the "plug" seems to act as a recognition or anchoring site; this material is squeezed out all around the trichocyst attachment zone, once a trichocyst is inserted (Westphal and Plattner, in press. [53]). Slightly below this "plug" we observed fasciae of microfilaments (identified by immunocytochemistry using peroxidase labeled F(ab) fragments against P. tetraurelia actin). Their arrangement is not altered when a trichocyst is docked. These fasciae seem to form a loophole for the insertion of a trichocyst. Trichocyst remain attached to the microtubules originating from the ciliary basal bodies--at least for some time--even after they are firmly installed in the preformed attachment sites. Evidently, the regular arrangement of exocytotic organelles is controlled on three levels: one operating over a long distance from the exocytosis site proper (microtubules), one over a short distance (microfilament bundles), and one directly on the exocytosis site ("plug").     

Control of exocytotic processes: cytological and physiological studies of trichocyst mutants in Paramecium tetraurelia

The trichocysts of Paramecium tetraurelia constitute a favorable system for studying secretory process because of the numerous available mutations that block, at various stages, the development of these secretory vesicles, their migration towards and interaction with the cell surface, and their exocytosis. Previous studies of several mutants provided information (a) on the assembly and function of the intramembranous particles arrays in the plasma membrane at trichocyst attachment sites, (b) on the autonomous motility of trichocysts, required for attachment to the cortex, and (c) on a diffusible cytoplasmic factor whose interaction with both trichocyst and plasma membrane is required for exocytosis to take place. We describe here the properties of four more mutants deficient in exocytosis ability, nd6, nd7, tam38, and tam6, which were analyzed by freeze-fracture, microinjection of trichocysts, and assay for repair of the mutational defect through cell-cell interaction during conjugation with wild-type cells. As well as providing confirmation of previous conclusions, our observations show that the mutations nd6 and tam6 (which display striking abnormalities in their plasma membrane particle arrays and are reparable through cell-cell contact but not by microinjection of cytoplasm) affect two distinct properties of the plasma membrane, whereas the other two mutations affect different properties of the trichocysts. Altogether, the mutants so far analyzed now provide a rather comprehensive view of the steps and functions involved in secretory processes in Paramecium and demonstrate that two steps of these processes, trichocyst attachment to the plasma membrane and exocytosis, depend upon specific properties of both the secretory vesicle and the plasma membrane.     

Aluminum toxicity studies in Vaucheria longicaulis var. macounii (Xanthophyta, Tribophyceae). II. Effects on the F-actin array

In this study, we test the hypothesis that exposure to environmentally significant concentrations of aluminum (Al, 80 μM) causes the microfilament array of Vaucheria longicaulis var. macounii vegetative filaments to become fragmented and disorganized. Changes in F-actin organization following treatment of vegetative filaments by Al are examined using vital staining with fluorescein phalloidin. In the cortical cytoplasm of the apical zone of pH 7.5 and pH 4.5 control cells, axially aligned bundles of F-actin lead to a region of diffuse, brightly stained material. Dimly stained focal masses are noted deeper in the cytoplasm of the apical zone whereas they are absent from the zone of vacuolation. The F-actin array is visualized in the cortical cytoplasm of the region of the cell, distal to the apical tip, which exhibits vigorous cytoplasmic streaming (zone of vacuolation) as long, axially aligned bundles with which chloroplasts and mitochondria associate. Thirty minutes following treatment with aluminum, and for the next 8-16 h, the F-actin array is progressively disorganized. The longitudinally aligned F-actin array becomes fragmented. Aggregates of F-actin, such as short rods, amorphous and stellate F-actin focal masses, curved F-actin bundles and F-actin rings replace the control array. Each of these structures may occur in association with chloroplasts or independently with no apparent association with organelles. Images are recorded which indicate that F-actin rings not associated with organelles may self-assemble by successive bundling of F-actin fragments. The fragmentation and bundling of F-actin in cells of V. longicaulis upon treatment with aluminum resembles those reported after diverse forms of cell disturbance and supports the hypothesis that aluminum-induced changes in the F-actin array may be a calcium-mediated response to stress.     

Secretory organelle docking at the cell membrane of Paramecium cells: dedocking and synchronized redocking of trichocysts

We present the first evidence that secretory organelle docking at the cell membrane can be reversed in vivo. In nondischarge (nd) mutants of Paramecium tetraurelia all trichocysts can be detached from the cell surface within 2-3 h by different means, including cytochalasin B (but not D), high cell density, or Ca2+ ionophores. Considering the well-established ultrastructural differences between nd and wild-type (wt) cells, one can conclude that trichocyst docking at the cell periphery involves two docking sites (I, II): Site I ties the organelles to the epiplasm, and site II is the connection to the cell membrane at the fusogenic zone (expressed only in wt cells); both sites are close to the cell surface and only 150 nm apart. When the trigger for detachment of cortically docked trichocysts (high cell density, cytochalasin B) is relieved, trichocysts are synchronously reattached at the cell membrane, within 40-50 min, with a rate of 20-40 organelles/min, which far exceeds spontaneous docking rates. This is therefore also the first report on synchronization of secretory organelle docking. It is shown by radioactive leucine labeling that the same organelles are redocked, because trichocyst biogenesis is minimal under the conditions of de/redocking used. Surprisingly not only redocking but also detachment of trichocysts from the cell surface can be abolished by inhibitors of protein synthesis. Since Ca2+ ionophores mimic the effects of other conditions sufficient to detach trichocysts from the cell surface, we assume that a protein-dependent mechanism sensitive to Ca2+ (or other ions in exchange) may operate in trichocyst detachment. The precise mechanism involved in attachment or detachment of trichocysts remains to be elucidated.     

Lectin binding sites in Paramecium tetraurelia cells. I. Labeling analysis predominantly of secretory components

Though all three lectins tested (ConA, RCA II, WGA) bound to the entire cell membrane, none bound selectively to the docking site of secretory organelles (trichocysts); the same results were achieved with FITC-conjugates, or, on the EM level, with peroxidase- or gold-labeling. Only WGA triggered the release of trichocysts and none of the lectins tested inhibited AED-induced synchronous exocytosis. When exocytosis was triggered synchronously in the presence of any of these three lectins (FITC-conjugates), the resulting ghosts trapped the FITC-lectins and the cell surface was immediately afterwards studded with regularly spaced dots (corresponding to the ghosts located on the regularly spaced exocytosis sites). These disappeared within about 10 min from the cell surface (thus reflecting ghost internalization with a half life of 3 min) and fluorescent label was then found in approximately 6-10 vacuoles, which are several microns in diameter, stain for acid phosphatase and, on the EM level, contain numerous membrane fragments (otherwise not found in this form in digesting vacuoles). We conclude that synchronous massive exocytosis involves lysosomal breakdown rather than reutilization of internalized trichocyst membranes and that these contain lectin binding sites (given the fact free fluorescent probes did not efficiently stain ghosts). Trichocyst contents were analyzed for their lectin binding capacity in situ and on polyacrylamide gels. RCA II yielded intense staining (particularly of "tips"), while ConA (fluorescence concentrated over "bodies") and WGA yielded less staining of trichocyst contents on the light and electron microscopic level. Only ConA- and WGA-staining was inhibitable by an excess of specific sugars, while RCA II binding was not. ConA binding was also confirmed on polyacrylamide gels which also allowed us to assess the rather low degree of glycosylation (approximately 1% by comparison with known glycoprotein standards) of the main trichocyst proteins contained in their expandable "matrix". Since RCA II binding could be due to its own glycosylation residues we looked for an endogenous lectin. The conjecture was substantiated by the binding of FITC-lactose-albumin (inhibitable by a mixture of glucose-galactose). This preliminary new finding may be important for the elucidation of trichocyst function.     

ATP keeps exocytosis sites in a primed state but is not required for membrane fusion: an analysis with Paramecium cells in vivo and in vitro

We have tried to specify a widespread hypothesis on the requirement of ATP for exocytosis (membrane fusion). With Paramecium tetraurelia cells, synchronously (approximately 1 s) exocytosing trichocysts, ATP pools have been measured in different strains, including wild type cells, "non-discharge" (nd), "trichless" (tl), and other mutations. The occurrence of a considerable and rapid ATP consumption also in nd and tl mutations as well as its time course (with a maximum 3-5 s after exocytosis) in exocytosis-competent strains does not match the actual extent of exocytosis performance. However, from in vivo as well as from in vitro experiments, we came to the conclusion that ATP might be required to keep the system in a primed state and its removal might facilitate membrane fusion. (For the study of exocytosis in vitro we have developed a new system, consisting of isolated cortices). In vivo as well as in vitro exocytosis is inhibited by increased levels of ATP or by a nonhydrolyzable ATP analogue. In vitro exocytosis is facilitated in ATP-free media. In vivo-microinjected ATP retards exocytosis in response to chemical triggers, whereas microinjected apyrase triggers exocytosis without exogenous trigger. Experiments with this system also largely exclude any overlaps with other processes that normally accompany exocytosis. Our data also explain why it was frequently assumed that ATP would be required for exocytosis. We conclude that membrane fusion during exocytosis does not require the presence of ATP; the occurrence of membrane fusion might involve the elimination of ATP from primed fusogenic sites; most of the ATP consumption measured in the course of exocytosis may be due to other effects, probably to recovery phenomena.     

Actin remodeling to facilitate membrane fusion

Actin and its associated proteins participate in several intracellular trafficking mechanisms. This review assesses recent work that shows how actin participates in the terminal trafficking event of membrane bilayer fusion. A recent flurry of reports defines a role for Rho proteins in membrane fusion and also demonstrates that this role is distinct from any vesicle transport mechanism. Rho proteins are well known to govern actin remodeling, which implicates this process as a condition of membrane fusion. A small but significant body of work examines actin-regulated events of intracellular membrane fusion, exocytosis and endocytosis. In general, actin has been shown to act as a negative regulator of exocytosis. Cortical actin filaments act as a barrier that requires transient removal to allow vesicles to undergo docking at the plasma membrane. However, once docked, F-actin synthesis may act as a positive regulator to give the final stimulus to drive membrane fusion. F-actin synthesis is clearly needed for endocytosis and intracellular membrane fusion events. What may seem like dissimilar results are perhaps snapshots of a single mechanism of membranous actin remodeling (i.e. dynamic disassembly and reassembly) that is universally needed for all membrane fusion events.     

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

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 [Ca²⁺] _i transient and exocytosis of dense core vesicles (``trichocysts') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>] <sub>o</sub> = 50 μm). When [Ca<sup>2+</sup>] <sub>o</sub> is kept at 30 nm (<[Ca<sup>2+</sup>]<sup>rest</sup> <sub>i</sub> ), 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<sup>2+</sup> <sub>o</sub> . Upon readdition of Ca<sup>2+</sup> <sub>o</sub> or some other appropriate Me<sup>2+</sup> <sub>o</sub> at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca<sup>2+</sup>, Sr<sup>2+</sup> or Mn<sup>2+</sup> are vesicular, but when formed in presence of Mg<sup>2+</sup>, 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<sup>2+</sup>] <sub>o</sub> = 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 [Mg²⁺] _o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me²⁺] to maintain docking sites in a functional state, (ii) requirement of Ca²⁺ _o or of some other Me²⁺ _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'. Received: 20 January 2000/Revised: 5 May 2000     

Secretory organelle docking at the cell membrane of Paramecium cells: Dedocking and synchronized redocking of trichocysts

We present the first evidence that secretory organelle docking at the cell membrane can be reversed in vivo. In nondischarge (nd) mutants of Paramecium tetraurelia all trichocysts can be detached from the cell surface within 2–3 h by different means, including cytochalasin B (but not D), high cell density, or Ca²⁺ ionophores. Considering the well-established ultrastructural differences between nd and wild-type (wt) cells, one can conclude that trichocyst docking at the cell periphery involves two docking sites (I, II): Site I ties the organelles to the epiplasm, and site II is the connection to the cell membrane at the fusogenic zone (expressed only in wt cells); both sites are close to the cell surface and only 150 nm apart. When the trigger for detachment of cortically docked trichocysts (high cell density, cytochalasin B) is relieved, trichocysts are synchronously reattached at the cell membrane, within 40–50 min, with a rate of 20–40 organelles/min, which far exceeds spontaneous docking rates. This is therefore also the first report on synchronization of secretory organelle docking. It is shown by radioactive leucine labeling that the same organelles are redocked, because trichocyst biogenesis is minimal under the conditions of de/redocking used. Surprisingly not only redocking but also detachment of trichocysts from the cell surface can be abolished by inhibitors of protein synthesis. Since Ca²⁺ ionophores mimic the effects of other conditions sufficient to detach trichocysts from the cell surface, we assume that a protein-dependent mechanism sensitive to Ca²⁺ (or other ions in exchange) may operate in trichocyst detachment. The precise mechanism involved in attachment or detachment of trichocysts remains to be elucidated.     

Molecular identification of a SNAP-25-like SNARE protein in Paramecium

Using database searches of the completed Paramecium tetraurelia macronuclear genome with the metazoan SNAP-25 homologues, we identified a single 21-kDa Qb/c-SNARE in this ciliated protozoan, named P. tetraurelia SNAP (PtSNAP), containing the characteristic dual heptad repeat SNARE motifs of SNAP-25. The presence of only a single Qb/c class SNARE in P. tetraurelia is surprising in view of the multiple genome duplications and the high number of SNAREs found in other classes of this organism. As inferred from the subcellular localization of a green fluorescent protein (GFP) fusion construct, the protein is localized on a variety of intracellular membranes, and there is a large soluble pool of PtSNAP. Similarly, the PtSNAP that is detected with a specific antibody in fixed cells is associated with a number of intracellular membrane structures, including food vacuoles, the contractile vacuole system, and the sites of constitutive endo- and exocytosis. Surprisingly, using gene silencing, we could not assign a role to PtSNAP in the stimulated exocytosis of dense core vesicles (trichocysts), but we found an increased number of food vacuoles in PtSNAP-silenced cells. In conclusion, we identify PtSNAP as a Paramecium homologue of metazoan SNAP-25 that shows several divergent features, like resistance to cleavage by botulinum neurotoxins.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Early membrane retrieval following exocytosis in rat mast cells

The fate of the surplus membrane following exocytosis of mast cell granules was studied by the extracellular tracer Ruthenium red (Ru red). Isolated rat peritoneal mast cells were stimulated with 4 micrograms/ml polylysine, washed and maintained in a culture medium for 80 min. Mast cells were observed both with the light microscope after adding Ru red in physiological solution and with the electron microscope after fixation in Ru red-containing fixatives. Whereas all exocytotic cavities were found to be stained with Ru red immediately after stimulation, a gradual lack of staining was observed in the subsequent period. The exits of the cavities were sealed by membrane fusions which resulted in closed vacuoles containing exocytosed granule remnants. These vacuoles often fused with each other to form a few giant vacuoles. The overwhelming majority of the vacuoles were observed to be closed 30 to 80 min after stimulation. In one experiment a quantitative analysis was performed to assess the degree of membrane recapture by sealing of the exocytotic cavities. A considerable portion of the plasma membrane area was retrieved in this way as early as between 15 and 30 min after stimulation. We conclude that the dominant mechanism of membrane retrieval in the early period following exocytosis is the recapture of large membrane areas by sealing of exocytotic cavities.     

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

The Ca²⁺-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.