Nucleolar aging in Tetrahymena during the cultural growth cycle.

Nucelolar morphology was studied by electron microscopy in control and actinomycin D-treated populations of Tetrahymena pyriformis (W) during the cultural growth cycle. Nucleoli exhibit an "aging" cycle concomitant with the cultural growth cycle, but independent of the individual cell cycle. Four different stages in the course of this aging process have been defined. Stage 1 occurs upon inoculation (low number of cells per milliliter) and lasts through lag and accelerating growth phases. In this stage, many small nucleoli are found at the nuclear periphery. In stages 2 and 3, nucleolar fusion begins. Stage 2 dominates the first half of logarithmic growth, and stage 3 dominates the second half. In late decelerating growth phase, the nucleoli enter stage 4. In this stage, only a few large nucleoli are present and these are apparently inactive in ribosome production. In stationary phase, where total RNA remains constant, only stage 4 nucleoli are present. The relative preponderance of granular vs. fibrous components in the nucleoli changes during this cycle, the granular component dominating stage 1 nucleoli and the fibrillar, stage 4 nucleoli. There is a shortening of the intermediate nucleolar stages in the treated cultures; fusion occurs early and is now pronounced. Not enough ribosomes accumulate to carry the treated cultures through the number of generations equivalent to those of the control, which produces a premature stationary phase.

Stimulation and inhibition of secretion in Paramecium: role of divalent cations.

The effects of Ca2+ and Mg2+ on exocytosis in Paramecium tetraurelia cells were examined with light microscopy, freeze fracture (FEM) and transmission electron microscopy (TEM) of thin-sectioned embedded cells. Picric acid-Ca2+-induced secretion in wild type (wt) cells was captured by "quick" fixation with OsO4, and TEM demonstrated membrane fusion occurring before trichocyst matrix (tmx) expansion. Cells stimulated with picric acid in the presence of high extracellular Mg2+ showed very few sites of membrane fusion and no tmx expansion, suggesting that Ca2+ is required for both membrane fusion and tmx expansion. Further information was obtained by comparing secretory responses of wt cells with a temperature-sensitive secretory mutant, nd 9. These cells when grown at the permissive temperature (18 degrees C) possess normal rosettes at the secretory site and secrete in response to picric acid-Ca2+, but when grown at 27 degrees C they lack rosettes and do not secrete (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). Quantitation of picric acid-Ca2+-induced secretion revealed that: (a) the number of tmx secreted by wt and nd 9 cells was independent of their cultural growth phase, (b) wt cells secreted the same number of tmx when grown either at 18 or 27 degrees C, and (c) nd 9 18 degrees C cells secreted the same number of tmx as wt 18 or 27 degrees C cells. Wild type and nd 9 cells had the same frequencies of occupied and unoccupied secretory sites as determined by quantitative analysis of freeze-fracture replicas. After stimulation with divalent cation ionophore A23187 and Ca2+, wt cells showed a significant reduction in the frequency of occupied sites. FEM and TEM studies revealed that A23187-Ca2+ induced tmx expansion and normal fusion of the plasma and trichocyst membranes in wt and nd 9 18 degrees C cells, but induced tmx expansion without concomitant membrane fusion in nd 9 27 degrees C cells. The lack of membrane fusion in nd 9 27 degrees C cells suggests that the molecules represented by rosette particles are required specifically for membrane fusion.

Paramecium secretory granule content: quantitative studies on in vitro expansion and its regulation by calcium and pH.

Ca2+-dependent secretion in Paramecium involves the exocytic release of a paracrystalline secretory product, the trichocyst matrix, which undergoes a characteristic structural change from a highly condensed storage form (Stage I) to an extended needle-like structure (Stage III) during release. We studied trichocyst matrix expansion in vitro to examine factors regulating the state of secretory organelle content. A new method for the isolation of membrane-free, condensed (Stage I) trichocyst matrices is described. These highly purified, condensed matrices were used to develop a rapid quantitative, spectrophotometric assay for matrix expansion to examine factors regulating the Stage I and Stage III transition. Expansion from Stages I to III was elicited in vitro by addition of Ca2+ and we found that at neutral pH, expansion required a Ca2+ concentration slightly above 10(-6)M. Previous studies indicate that calmodulin (CaM) antagonists inhibit matrix expansion in vivo. However, in vitro matrix expansion is normal even when trichocyst matrices are preincubated in CaM antagonists before stimulation. Thus, matrix components themselves are unlikely to be the site of CaM antagonist action in vivo. In vitro matrix expansion is also modulated by pH. Decreasing pH to 6.0 inhibits expansion, i.e., expansion requires higher Ca2+ concentration. Conversely, increasing pH to greater than 7.0 promotes expansion, allowing it to occur at a lower Ca2+ concentration. The pH sensitivity of the Ca2+ binding sites of the matrix suggests that, in vivo, the interior of the trichocyst vesicle may be maintained at an acidic pH. Exposure of cells to acridine orange, a fluorescent amine that accumulates in acidic intracellular compartments, leads to its uptake and concentration within trichocysts. Thus intratrichocyst pH appears to be acidic in vivo and may serve as a regulatory or "safety" mechanism to inhibit premature expansion.

Membrane fusion in a model system. Mucocyst secretion in Tetrahymena.

The freeze-fracture, freeze-etch technique can be employed to reveal new details of the process of fusion of two unit membranes For this study, mucocyst discharge in Tetrahymena pyriformis provides a model system with certain general implications The undischarged mature mucocyst is a saclike, membrane-bound, secretory vesicle containing crystalline material The organelle tip finds its way toward a special site, a rosette of 150 A diameter particles within the plasma membrane. To match this site, the mucocyst membrane forms an annulus of 110 A diameter particles, above whose inner edge the rosette particles sit. Discharge of some mucocysts is triggered by fixation. As discharge proceeds, the organelle becomes spherical and its content changes from crystalline to amorphous. The cytoplasm between the two matching membrane sites is squeezed away and the membranes fuse Steps in membrane reorganization can be reconstructed from changes in rosette appearance in the fracture faces. First, a depression in the rosette-the fusion pocket-forms. The rosette particles spread at the lip as the pocket deepens and enlarges from 60 to 200 nm. The annulus particles then become visible at the lip, indicating completed fusion of the A fracture faces of mucocyst and plasma membranes The remaining B faces of the two membranes have opposite polarities When the content of the mucocyst is released, the edges of these faces join so that the unit membrane runs uninterruptedly around the lip and into the pocket.

A freeze-fracture study of early membrane events during mast cell secretion.

The early membrane events taking place during mast cell secretion were followed in transmission and freeze-fracture electron microscopy. In order to slow down exocytosis and capture intermediate stages of membrane fusion, special conditions of incubation and stimulation were used. These were as follows: (a) the use of incubation media with altered ionic composition, and (b) stimulation with a low dosage of polymyxin B sulfate (4 microgram/ml) at low temperature (18 degrees C) for very short incubation times (30-60 s), with or without the presence of formaldehyde (0.8%). Under these conditions, unetchable circular impressions are found on the E face of the plasma membrane, 80-100 nm in diameter, with particles associated with their perimeters. In granule-to-granule fusion, the zone involved is demarcated by one or two rows of particles on the E face. In addition, raised circular areas of varying diameters (43-87 nm) surrounded by similar particles, also found on the E face, may represent potential sites before completion of fusion. Neither the circular impressions on the plasma membrane nor the sites on the granule membrane are permanent, but their appearance coincides with initiation of membrane fusion.

Plasma membrane folds on the mast cell surface and their relationship to secretory activity.

Changes in the surface morphology of secreting mast cells have been followed by scanning electron microscopy. Mast cells isolated from the rat peritoneal cavity have folds of plasma membrane that form snake-like ridges on their surfaces. Fold length varies considerably from cell to cell, whereas fold width and depth appear to remain relatively constant. To assess the possible relationship between secretory activity and surface folding, a seimquantitative method was used for measuring fold length in control and secreting populations. A positive correlation is found between secretion of histamine and the extent of membrane folds on the mast cell surface. The source of the membrane required for fold formation is probably secretory granule membrane incorporated into the plasma membranene as a result of exocytosis. Furthermore, a distinct cell type devoid of surface folds, designated as a raspberry-type cell, is found to occur as an integral part of a normal population of mast cells. This cell type is resistant to stimulation by polymyxin.

Calmodulin antagonists inhibit secretion in Paramecium.

Secretion in Paramecium is Ca2+-dependent and involves exocytic release of the content of the secretory organelle, known as the trichocyst. The content, called the trichocyst matrix, undergoes a Ca2+-induced reordering of its paracrystalline structure during release, and we have defined three stages in this expansion process. The stage I, or fully condensed trichocyst, is the 4 microns-long membrane-bounded form existing prior to stimulation. Stage II, the partially expanded trichocyst, we define as an intermediate stage in the transition, preceding stage III, the fully expanded extruded form which is a 20-40 microns-long needlelike structure. These stages have been used to assay the effects of trifluoperazine (TFP) and W-7, calmodulin (CaM) antagonists, on trichocyst matrix expansion in vivo. TFP and W-7 are shown to reversibly block matrix release induced by picric acid. Ultra-structural examination reveals that one effect of this inhibition is reflected in the organelles themselves, which are prevented from undergoing the stage I-stage II transition by preincubation in 14 microM TFP or 35 microM W-7 before fixation. This inhibition of expansion by TFP can be moderated but not abolished by high extracellular Ca2+ (5 mM). The moderation by high Ca2+ can be eliminated by raising TFP concentration to 20 microM. A possible explanation for the ability to titrate the inhibition in this manner is that TFP is acting to block expansion by binding to the Ca2+-CaM complex. Brief exposure of cells to the Ca2+ ionophore A23187 and 5 mM Ca2+ following TFP treatment promotes matrix expansion, although in 14 microM TFP a residual level of inhibition remains. These results suggest that, following stimulation, CaM regulates secretion in Paramecium, possibly by controlling the Ca2+-dependent matrix expansion which accompanies exocytosis in these cells.

Parafusin, an exocytic-sensitive phosphoprotein, is the primary acceptor for the glucosylphosphotransferase in Paramecium tetraurelia and rat liver.

Parafusin, the major protein in Paramecium tetraurelia to undergo dephosphorylation in response to secretory stimuli, appears to be the primary acceptor for the glucosylphosphotransferase in this species based on five independent criteria: identical molecular size of 63 kD; identical isoelectric points in the phosphorylated state of pH 5.8 and 6.2; identical behavior in reverse-phase chromatography; immunological cross-reactivity with an affinity-purified anti-parafusin antibody; the presence of a phosphorylated sugar after acid hydrolysis. It appears likely that the dephosphorylation observed with secretion reflects the removal of alpha Glc-1-P from parafusin's oligosaccharides and is consistent, therefore, with a regulatory role for this cytoplasmic glycosylation event. The glucosylphosphotransferase acceptor in rat liver is also immunoprecipitated by the anti-parafusin antibody and is very similar in physical characteristics to the paramecium protein. This conservation suggests a role for parafusin in mammalian exocytosis as well, at a step common to both the regulated and constitutive secretory pathways.

Retention of antidiuretic hormone-induced particle aggregates by luminal membranes separated from toad bladder epithelial cells.

Aggregates of intramembrane particles appear in the luminal membranes of renal collecting duct and amphibian bladder cells after stimulation by antidiuretic hormone (ADH). We undertook this freeze-fracture study to determine whether particle aggregates, once in place, remain in the luminal membrane of the amphibian bladder after the membrane is physically separated from the rest of the cell. We found that the aggregates do remain in high yield in isolated membranes stabilized with a bifunctional imidoester (DTBP) followed by fixation with glutaraldehyde, or unfixed but stabilized with DTBP. These findings support the view that the particles are intrinsic membrane components and that their organization in the form of aggregates does not depend on the presence of the intact cell. In addition, the availability of isolated membranes containing particle aggregates provides a starting point for the isolation of the water-conducting proteins.

Genetic analysis of membrane differentiation in Paramecium. Freeze-fracture study of the trichocyst cycle in wild-type and mutant strains.

Using a series of mutants of Paramecium tetraurelia, we demonstrate, for the first time, changes in the internal structure of the cell membrane, as revealed by freeze-fracture, that correspond to specific single gene mutations. On the plasma membrane of Paramecium circular arrays of particles mark the sites of attachment of the tips of the intracellular secretory organelles-trichocysts. In wild-type paramecia, where attached trichocysts can be expelled by exocytosis under various stimuli, the plasma membrane array is composed of a double outer ring of particles (300 nm in diameter) and inside the ring a central rosette (fusion rosette) of particles (76 nm in diameter). Mutant nd9, characterized by a thermosensitive ability to discharge trichocysts, shows the same organization in cells grown at the permissive temperature (18 degrees C), while in cells grown at the nonpermissive temperature (27 degrees C) the rosette is missing. In mutant tam 8, characterized by normal but unattached trichocysts, and in mutant tl, completely devoid of trichocysts, no rosette is formed and the outer rings always show a modified configuration called "parentheses", also found in wild-type and in nd9 (18 degrees C) cells. From this comparison between wild type and mutants, we conclude: (a) that the formation of parentheses is a primary differentiation of the plasma membrane, independent of the presence of trichocysts, while the secondary transformation of parentheses into circular arrays and the formation of the rosette are triggered by interaction between trichocysts and plasma membranes; and (b) that the formation of the rosette is a prerequisite for trichocyst exocytosis.

Presence and indirect immunofluorescent localization of calmodulin in Paramecium tetraurelia.

In this paper we demonstrate the presence and localization of calmodulin, a calcium-dependent regulatory protein, in the ciliated protozoan Paramecium tetraurelia. Calmodulin is demonstrated by several criteria: (a) the ability of whole cell Paramecium extracts to stimulate mammalian phosphodiesterase activity, (b) the presence of an acidic, thermostable, 17,000-dalton polypeptide whose mobility shifts in SDS polyacrylamide gel electrophoresis in the presence of Ca2+, and (c) the affinity of antibodies against mammalian calmodulin for a Paramecium component as demonstrated by both indirect immunofluorescent localization and radioimmunoassay. Indirect immunofluorescence studies reveal that Paramecium calmodulin is distributed in three distinct regions of the cell, i.e., (a) large, spherical cytoplasmic organelles representing perhaps the food vacuoles or other vacuolar inclusions of the cell, (b) along the entire length of oral and somatic cilia, and (c) along a linear punctate pattern corresponding to the kinetics (basal bodies) of the cell.

Paramecium fusion rosettes: possible function as Ca2+ gates.

The function of a specific intramembrane particle array, "the fusion rosette," an essential requirement for exocytosis of trichocysts in Paramecium, was probed with a temperature sensitive secretory mutant (nd9). The cells were grown at 27 degrees C, the nonpermissive, nonreleasing temperature at which fusion rosettes do not assemble. Exocytosis could be triggered, nonetheless, by addition of 40 micrometer ionophore A23187 and 15 mM Ca2+ but not Mg+. Rosette function is bypassed by this procedure, suggesting that during normal release, the rosette acts as a Ca2+ channel that allows development of a site-specific increase in Ca2+, which in turn induces fusion and release.

Paramecium genome survey: a pilot project.

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Parafusin is a membrane and vesicle associated protein that cycles at exocytosis.

In the unicellular eukaryote Paramecium tetraurelia, stimulation of exocytosis leads to Ca2+ activation of an alpha Glc-1-phosphodiesterase that dephosphoglucosylates the phosphoglycoprotein parafusin (PFUS). This process fails in exo mutant nd9 and also in the absence of Ca2+ influx upon stimulation suggesting that PFUS dephosphoglucosylation may be causally related to exocytosis. To further corroborate the hypothesis that PFUS is involved in the molecular events in exocytosis, we used laser confocal scanning microscopy and a PFUS specific peptide antibody to perform localization studies of PFUS in wild type (wt) and mutant Paramecium. In unstimulated wt cells, PFUS was associated both with the exocytic site of the cell membrane and with the membrane of the dense core secretory vesicles. Localization at these two sites was shown to be independent of each other since the exocytosis mutant (exo-) tam8, in which docking of its vesicles is blocked, still showed cell membrane staining. Immunofluorescence and immunoblotting of isolated intact secretory vesicles also revealed PFUS association. Upon stimulation of exocytosis, PFUS dissociated from both the dense core secretory vesicles and the cell membrane in a Ca(2+)-dependent manner. During recovery of exocytic capacity, PFUS reassociated with the newly formed secretory vesicles in the cytoplasm prior to their docking at the exocytic sites. Immunoblot analysis of PFUS during this time showed no changes in levels of the protein. Stimulation of exocytosis in wt in Mg2+ buffer or in the exo- temperature sensitive mutant (nd9) at the non-permissive temperature did not lead to dissociation of the PFUS. We conclude that PFUS is a novel critical component whose cycling probably participates in the molecular exocytic fusion machinery in these cells.

Mammalian homologue of the calcium-sensitive phosphoglycoprotein, parafusin.

Three specific antipeptide antibodies and oligonucleotide probes synthesized to internal sequences of parafusin have been used to search for mammalian counterpart(s) of this protein. Parafusin is an exocytic-sensitive phosphoglycoprotein from a unicellular eukaryote Paramecium that was recently cloned and sequenced (Subramanian et al., Proc. Natl. Acad. Sci. USA 91, 9832-9836 (1994)). Western and Southern blot analyses, polymerase chain reaction (PCR) and reverse transcriptase coupled PCR (RT-PCR) techniques have been used to examine rat liver and pancreas, human pancreas and a murine pancreatic beta-cell line (beta TC3) arising in transgenic mice. The parafusin-specific antibodies showed cross-reaction with a protein at approximately 63 kDa in 4 tissues, whereas a phosphoglucomutase-specific antibody also detected a second band of similar molecular weight in the beta TC3 cells. The presence of two bands shows that parafusin homologue(s) and phosphoglucomutase are separate entities. beta TC3 cells were shown to incorporate [beta 35]UDPGlc into the parafusin homologue in a Ca(++)-sensitive manner characteristic of parafusin. Southern blot analysis revealed that the parafusin-specific probe hybridized with restriction enzyme digests of rat DNA in distinct patterns different from those observed with a phosphoglucomutase-specific probe. Rat genomic DNA and mRNA from the beta TC3 cells were used as the templates for PCR and RT-PCR using internal parafusin primers. In both cases similarly sized products were obtained which hybridized in Southern analysis with a specific parafusion probe located within the amplified region. These results indicate that a parafusin homologue exists in mammalian cells.

A parafusin-related Toxoplasma protein in Ca2+-regulated secretory organelles.

We cloned a gene, PRPI, of Toxoplasma gondii encoding a 637-amino-acids protein having a calculated mass of 70 kDa. The sequence showed high homology to parafusin, a protein that in Paramecium tetraurelia participates in Ca2+-regulated exocytosis and is a paralog of phosphoglucomutase. We show that Toxoplasma gondii homogenate and an expressed recombinant PRP1 fusion protein cross-react with a specific peptide-derived antibody to parafusin in Western blots. Antibodies to the recombinant PRP1 showed cross-reaction with parafusin and recognized PRP1, as bands at M, 63 x 10(3) and 68 x 10(3), respectively. PRP1 is labeled when Toxoplasma gondii cells are incubated with inorganic 32P and appears as the major band on autoradiograms of SDS-PAGE gels. The localization of PRP1 was examined in secretory organelles of Toxoplasma gondii by deconvolution light microscopy followed by three dimensional reconstruction using pairwise combinations of specific antibodies. PRP1 localized to the apical third of the cell. It co-localized with micronemes, the only secretory organelle the secretion of which is Ca2+ dependent. Quantification of the co-localized stain suggests that only mature micronemes ready for exocytosis have PRP1. These findings suggest that PRP1, parafusin and other members of the phosphoglucomutase superfamily have a conserved role in Ca2+-regulated exocytic processes.