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.     

Control of membrane fusion in exocytosis. Physiological studies on a Paramecium mutant blocked in the final step of the trichocyst extrusion process

Previous studies on exocytosis in Paramecium using mutants affecting trichocyst extrusion permitted us to analyze the assembly and function of three intramembrane particle arrays ("ring" and "rosette" in the plasma membrane, "annulus" in the trichocyst membrane) involved in the interaction between these two membranes. Using a conditional mutation, nd9, which blocks rosette assembly and prevents exocytosis at the nonpermissive temperature, we have analyzed the effect of temperature on the secretory capacity of nd9 cells. By combining several techniques (physiological studies, microinjections, inhibition of fatty acid synthesis, and freeze-fracture analysis) we demonstrate (a) that the product of the mutated allele nd9 is not thermolabile but that its activity is dependent upon temperature-induced changes in the membrane lipid composition and (b) that the product of the nd9 locus is a diffusible cytoplasmic component whose interaction with both plasma membrane and trichocyst membrane is required for rosette assembly and exocytosis. The data provide physiological evidence for the existence of a molecular complex(es) linking the two membranes and involved in the control of membrane fusion; we discuss the possible nature and function of these links.     

Genetic Analysis of the Relationships between the Cell Surface and the Nuclei in PARAMECIUM TETRAURELIA

In Paramecium tetraurelia, a number of mutations have been shown to affect simultaneously cortical organization (attachment of trichocysts to the cortex) and nuclear division (Ruiz et al. 1976). In order to analyze the genetic and physiological basis of this correlation, we have isolated new mutations affecting the properties of the trichocysts and studied their genetic relationships with other previously known mutations. Of 24 to 28 loci controlling the biogenesis and properties of the trichocysts, mutations only in the 16 to 20 loci that control trichocyst attachment to the cortex result in nuclear defects. Cytological observations show that all of these mutants display the same set of nuclear abnormalities: in particular, rounded shape of the resting macronucleus, mispositioning and defective elongation of the dividing macronucleus and unequal repartition of the macro- and micronuclei. This common syndrome is independent of both the mutagenic origin and the mutated locus. Furthermore, by microinjection, it is possible to localize the site of action of the mutations in either the trichocyst compartment or the nontrichocyst compartment. It was found by this technique that the nuclear syndrome is also independent of the site of action of the mutation. All the genetic and physiological data support the conclusion that the nuclear defects are the consequence of the lack of trichocyst attachment to the cortex: in wild-type cells, trichocyst attachment would induce a membranar or perimembranar state necessary for correct nuclear positioning during cell division. In the absence of trichocyst attachment, the cortical control of nuclear division would be abolished. The possible involvement of cytoskeletal links between surface and nuclei is discussed.     

Nd6p, a novel protein with RCC1-like domains involved in exocytosis in Paramecium tetraurelia

In Paramecium tetraurelia, the regulated secretory pathway of dense core granules called trichocysts can be altered by mutation and genetically studied. Seventeen nondischarge (ND) genes controlling exocytosis have already been identified by a genetic approach. The site of action of the studied mutations is one of the three compartments, the cytosol, trichocyst, or plasma membrane. The only ND genes cloned to date correspond to mutants affected in the cytosol or in the trichocyst compartment. In this work, we investigated a representative of the third compartment, the plasma membrane, by cloning the ND6 gene. This gene encodes a 1,925-amino-acid protein containing two domains homologous to the regulator of chromosome condensation 1 (RCC1). In parallel, 10 new alleles of the ND6 gene were isolated. Nine of the 12 available mutations mapped in the RCC1-like domains, showing their importance for the Nd6 protein (Nd6p) function. The RCC1 protein is well known for its guanine exchange factor activity towards the small GTPase Ran but also for its involvement in membrane fusion during nuclear envelope assembly. Other proteins with RCC1-like domains are also involved in intracellular membrane fusion, but none has been described yet as involved in exocytosis. The case of Nd6p is thus the first report of such a protein with a documented role in exocytosis.     

Evidence for defects in membrane traffic in Paramecium secretory mutants unable to produce functional storage granules

The ciliated protozoan Paramecium has a regulated secretory system amenable to genetic analysis. The secretory storage granules, known as trichocysts, enclose a crystalline matrix with a genetically determined shape whose biogenesis involves proteolytic maturation of a family of precursor molecules into a heterogeneous set of small acidic polypeptides that crystallize within the maturing vesicles. We have developed an original pulse-chase protocol for monoxenic Paramecium cultures using radiolabeled bacteria to study the processing of trichocyst matrix proteins in wild-type and mutant cells. In wild-type cells, proteolytic processing is blocked in the presence of monensin and otherwise rapidly completed after approximately 20 min of chase, suggesting that the conversion occurs in the trans-Golgi and/or in small vesicles soon after sorting to the regulated pathway, probably before crystallization begins. In trichless mutant cells, which contain no visible trichocysts, secretory proteins are synthesized but not processed and we report constitutive secretion of the uncleaved precursor molecules. The mutation thus appears to affect sorting to the regulated pathway and should prove useful for analysis of the sorting machinery and of the relationship between sorting and proteolytic processing of secretory proteins. In mutants bearing misshapen trichocysts with poorly crystallized contents (tam33, tam38, stubbyA), the proteolytic processing of the trichocyst matrix proteins appears to be normal, while both pulse-chase and morphological data indicate that intracellular transport is perturbed, probably between ER and Golgi. Precursor molecules are present in the mutant trichocysts but not in wild-type trichocysts and may account for the defective crystallization. Our analysis of these mutants suggests that the temporal coordination of intracellular traffic plays a regulatory role in granule maturation.     

N-ethylmaleimide-sensitive factor is required to organize functional exocytotic microdomains in paramecium

In exocytosis, secretory granules contact plasma membrane at sites where microdomains can be observed, which are sometimes marked by intramembranous particle arrays. Such arrays are particularly obvious when membrane fusion is frozen at a subterminal stage, e.g., in neuromuscular junctions and ciliate exocytotic sites. In Paramecium, a genetic approach has shown that the "rosettes" of intramembranous particles are essential for stimulated exocytosis of secretory granules, the trichocysts. The identification of two genes encoding the N-ethylmaleimide-sensitive factor (NSF), a chaperone ATPase involved in organelle docking, prompted us to analyze its potential role in trichocyst exocytosis using a gene-silencing strategy. Here we show that NSF deprivation strongly interferes with rosette assembly but does not disturb the functioning of exocytotic sites already formed. We conclude that rosette organization involves ubiquitous partners of the fusion machinery and discuss where NSF could intervene in this mechanism.     

Isolation of surface membranes from normal and exocytotic mutant strains of Paramecium tetraurelia. Ultrastructural and biochemical characterization

A density gradient centrifugation method for the isolation of the surface membrane complex from Paramecium tetraurelia cells is presented. The resulting "pellicles" consist predominantly of the somatic cell membrane and the underlying alveolar membranes. Marker enzyme activities for other cell components are low and SDS-polyacrylamid-gel electrophoreses indicate the presence of only minor amounts of ciliary and secretory proteins. Pellicles were prepared from different strains: (a) Exocytosis-capable strains with the normal set of exocytotic organelles ("trichocysts") docked to the cell membrane (strains 7S, K 401, and 9-18 degrees C), (b) exocytosis-uncapable strains (although with normal trichocyst attachment: nd 9-27 degrees C, nd 6, nd 7) and (c) strain from tam 38 with empty docking sites and rare, defective, free trichocysts. A Ca2+-stimulated ATPase was present in the pellicles from all strains with Km (CA2+) values between 0.19 to 0.88 mM Ca2+ and Vmax between 286 to 787 nMoles Pi/mg protein/min. Km and Vmax was identical for all strains of group (a). Vmax was significantly lower for all strains of group (b) and still lower for group (c). Similar group differences were found for Km (except for strain nd 6). Freeze-fracture analysis shows that the disruption of the membrane-to-membrane attachments during fractionation is paralleled by the disarrangment of the regular arrays ("rings", "rosettes") of membrane-integrated particles.     

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").     

The effective sites of some mutations affecting exocytosis in Paramecium tetraurelia

Summary Using a microinjection technique, the functional competence of the trichocysts or of the nontrichocyst cytoplasms of wild-type and mutant stocks of Paramecium tetraurelia was tested. The results indicate that the exocytic (trichocyst discharge) phenotype of P. tetraurelia depends upon the functional competence of the trichocysts themselves and also upon the function of apparently trichocyst-specific cytoplasmic components. Thus, the mutants tam8, ndA and ndB are shown to contain defective trichocysts, but have apparently functional cytoplasms which can properly utilize normal trichocysts if these are supplied. Conversely, the mutant nd9 contains apparently normal trichocysts but is deficient in some cytoplasmic component required for normal trichocyst discharge. Injections of genetically complementary cytoplasm apparently supply nd9 with the missing component and can thus repair the nd9 trichocyst exocytic phenotype.     

Aspects of signal transduction in stimulus exocytosis-coupling in Paramecium

This paper deals with the detailed mechanisms of signal transduction that lead to exocytosis during regulative secretion induced by specific secretagogues in a eukaryotic cell, Paramecium tetraurelia. There are at least three cellular compartments involved in the process: I) the plasma membrane, which contains secretagogue receptors and other transmembrane proteins, II) the cytoplasms, particularly in the region between the cell and secretory vesicle membranes, where molecules may influence interactions of the membranes, and III) the secretory vesicle itself. The ciliated protozoan Paramecium tetraurelia is very well suited for the study of signal transduction events associated with exocytosis because this eukaryotic cell contains thousands of docked secretory vesicles (trichocysts) below the cell membrane which can be induced to release synchronously when triggered with secretagogue. This ensures a high signal-to-noise ratio for events associated with this process. Upon release the trichocyst membrane fuses with the cell membrane and the trichocyst content undergoes a Ca2+-dependent irreversible expansion. Secretory mutants are available which are blocked at different points in the signal transduction pathway. Aspects of the three components mentioned above that will be discussed here include a) the properties of the vesicle content, its pH, and its membrane; b) the role of phosphorylation/dephosphorylation of a cytosolic 63-kilodalton (kDa)Mr protein in membrane fusion; and c) how influx of extracellular Ca2+ required for exocytosis may take place via exocytic Ca2+ channels which may be associated with specific membrane microdomains (fusion rosettes).     

Interactions between Genes Involved in Exocytotic Membrane Fusion in Paramecium

Crosses between members of two independent collections of Paramecium tetraurelia mutants blocked in the final membrane fusion step of trichocyst release (nd mutants) allowed us to define 13 complementation groups comprising 23 alleles. The mutant nd9a was then used as a target in a mutagenesis experiment designed to screen both revertants and new mutants in order to identify interacting genes. This mutant was chosen because it is the best known of its class to date and seems to be altered in assembly of the material connecting the trichocyst membrane to the plasma membrane and in assembly of the "rosette," a complex array of intramembranous particles in the plasma membrane at the trichocyst insertion sites. No revertants were obtained but two new mutants deficient for rosette assembly were identified, nd16b and nd18, whose gene products appear to interact with that of nd9. Indeed, the double mutants grown at 18 degrees, a permissive temperature for each of the single mutants, are characterized by a deficiency in exocytosis and in rosette assembly, as are also double mutants combining other allelic forms of the same genes. Moreover, aberrant dominance relationships among alleles of nd9 and of nd16 indicate the existence of interactions between identical subunits, which most likely assemble into multimeric structures. The nd16 gene product was shown by microinjection experiments to be a cytosolic factor, as is the nd9 gene product. It is therefore tempting to propose that the nd16 gene product also belongs to the connecting material and is involved in rosette assembly, in cooperation with nd9 and nd18.     

The crystal lattice of Paramecium trichocysts before and after exocytosis by X-ray diffraction and freeze-fracture electron microscopy

Paramecium trichocysts are unusual secretory organelles in that: (a) their crystalline contents are built up from a family of low molecular mass acidic proteins; (b) they have a precise, genetically determined shape; and (c) the crystalline trichocyst contents expand rapidly upon exocytosis to give a second, extracellular form which is also an ordered array. We report here the first step of our study of trichocyst structure. We have used a combination of x-ray powder diffraction, freeze-etching, and freeze-fracture electron microscopy of isolated, untreated trichocysts, and density measurements to show that trichocyst contents are indeed protein crystals and to determine the elementary unit cell of both the compact intracellular and the extended extracellular form.     

A Ca2+ influx associated with exocytosis is specifically abolished in a Paramecium exocytotic mutant

A Paramecium possesses secretory organelles called trichocysts which are docked beneath the plasma membrane awaiting an external stimulus that triggers their exocytosis. Membrane fusion is the sole event provoked by the stimulation and can therefore be studied per se. Using 3 microM aminoethyl dextran (AED; Plattner, H., H. Matt, H.Kersken, B. Haake, and R. Sturz, 1984. Exp. Cell Res. 151:6-13) as a vital secretagogue, we analyzed the movements of calcium (Ca2+) during the discharge of trichocysts. We showed that (a) external Ca2+, at least at 3 X 10(-7) M, is necessary for AED to induce exocytosis; (b) a dramatic and transient influx of Ca2+ as measured from 45Ca uptake is induced by AED; (c) this influx is independent of the well-characterized voltage- operated Ca2+ channels of the ciliary membranes since it persists in a mutant devoid of these channels; and (d) this influx is specifically abolished in one of the mutants unable to undergo exocytosis, nd12. We propose that the Ca2+ influx induced by AED reflects an increase in membrane permeability through the opening of novel Ca2+ channel or the activation of other Ca2+ transport mechanism in the plasma membrane. The resulting rise in cytosolic Ca2+ concentration would in turn induce membrane fusion. The mutation nd12 would affect a gene product involved in the control of plasma membrane permeability to Ca2+, specifically related to membrane fusion.     

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.     

Genetic characterization of the secretory mutants ofParamecium caudatum

Summary Two trichocyst-nondischarge (TND) mutants ofParamecium caudatum, tndl andtnd2, are unable to discharge the trichocyst matrix (tmx) in response to chemical stimuli, although they contain many docked trichocysts at predetermined sites in the cortex. Freeze-fracture electron microscopy (FEM) of the plasma membrane showed thattndl possess two typical intramembrane particle arrays at the trichocyst docking site in the cortex, the outer ring and the inner rosette, as observed in wild-type (WT) cells, whereastnd2 possess the ring but not the rosette. The tmx of both TND mutants are able to expand when they are freed and exposed to an extracellular medium containing 1.5 mM Ca²⁺. When mutant cells were treated with ionophore A23187 and Ca²⁺, tmx-expansion took place intnd2, but not intndl cells. Thetnd2 mutant could be rescued by an injection of the WT cytoplasm and also by partial cell fusion during conjugation with the WT andtndl cells. However, the secretion capacity oftndl was not restored either by a microinjection of the WT cytoplasm or by the conjugating pair formation. Freeze-fracture electron microscopy on the double homozygote fortndl andtndl genes, revealed only the parenthesis instead of the ring and the rosette, indicating that trichocysts do not dock to the cortical site. Double mutation at thetndl andtndl loci caused a decrease in the number of the trichocysts at the cortical site. These results suggest that cooperative action of the two TND genes is necessary for stable docking of the trichocysts to the cortical sites.Abbreviations FEM freeze-fracture electron microscopy - IMP intramembrane particle - TD trichocyst discharge: tmx trichocyst matrix - TND trichocyst nondischarge - WT wild-type     

Synchronous exocytosis in Paramecium cells. I. A novel approach

From a total number of approximately 1100-1300 secretory organelles ("trichocysts") in a Paramecium tetraurelia cell, approximately 90% are docked to the cell membrane. Approximately 90% of this subpopulation can be discharged from the cells within seconds, when exposed to the novel trigger agent aminoethyldextran (AED) at a concentration of 10(-6) M. No deleterious side effects were recognized with this trigger agent even over long time periods. By application of AED close to cells with the use of a micropipette we found that triggering of trichocyst release by AED involves a local, non-propagated effect and that all regions of the cell body are equally reactive. It requires exogenous Ca2+. It is independent of ciliary Ca2+ channels, since deciliated cells or ciliary mutations with "Ca2+-tight" cilia respond to AED with normal exocytosis performance. The massive and rapid occurrence of trichocyst release in response to AED allowed for a freeze-fracture analysis of intramembraneous changes (see Olbricht et al., Exp cell res 151 (1984) 14 [23]) which also shows the involvement of exocytosis) as well as for a long-term study of the re-attachment of trichocysts (see Haacke & Plattner, Exp cell res 151 (1984) 21 [10]) under synchronous conditions.     

Membrane behavior of exocytic vesicles. II. Fate of the trichocyst membranes in Paramecium after induced trichocyst discharge.

A specific exocytic process, the discharge of spindle trichocyts 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. 6,000-8,000) 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 the 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.     

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.     

Proteolytic cleavage and maturation of the crystalline secretion products of Paramecium

The secretory vesicles (trichocysts) of the unicellular eukaryote Paramecium provide a model system for genetic, cytological and biochemical studies of secretory processes. An additional interest in trichocysts lies in the crystalline organization of their content, before and after exocytosis. We have analysed the biosynthesis of the secreted proteins and the building up of their crystalline packing by a combination of methods using: (1) antibodies raised against the secreted products; (2) mutants blocked at different steps of the secretory pathway; and (3) the carboxylic ionophore monensin. Our results support the following conclusions: firstly, the secreted polypeptides are derived from higher molecular weight precursors by a proteolytic cleavage; and secondly, this post-translational maturation is required for the building up of the crystalline structure of the trichocyst contents.