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PubMed1985

Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium.

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.

Animals↗

PubMed1985

Synchronous exocytosis in Paramecium cells involves very rapid (less than or equal to 1 s), reversible dephosphorylation of a 65-kD phosphoprotein in exocytosis-competent strains.

Synchronous exocytosis in Paramecium cells involves the rapid (less than or equal to 1 s) dephosphorylation of a 65-kD phosphoprotein, which, after a lag phase of approximately 5 s, is reversed within approximately 20 s. Exocytosis inhibitors suppress this reaction; stimulatory and inhibitory effects are dose dependent. The dephosphorylation of the 65-kD phosphoprotein occurs only in exocytosis-competent strains, but not in mutant strains that cannot carry out membrane fusion, or that are devoid of secretory organelles or cannot transport them to the cell membrane. Since under all conditions analyzed the transient dephosphorylation of the 65-kD phosphoprotein strictly parallels the actual amount of exocytosed organelles, this process might be involved in exocytosis performance, perhaps in its initiation.

Animals↗

PubMed1986

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.

Adenosine Triphosphate↗

PubMed1987

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.

Animals↗

PubMed1988

Differential regulation of Paramecium ciliary motility by cAMP and cGMP.

cAMP and cGMP had distinct effects on the regulation of ciliary motility in Paramecium. Using detergent-permeabilized cells reactivated to swim with MgATP, we observed effects of cyclic nucleotides and interactions with Ca2+ on the swimming speed and direction of reactivated cells. Both cAMP and cGMP increased forward swimming speed two- to threefold with similar half-maximal concentrations near 0.5 microM. The two cyclic nucleotides, however, had different effects in antagonism with the Ca2+ response of backward swimming and on the handedness of the helical swimming paths of reactivated cells. These results suggest that cAMP and cGMP differentially regulate the direction of the ciliary power stroke.

Animals↗

PubMed1989

Microtubule dynamics investigated by microinjection of Paramecium axonemal tubulin: lack of nucleation but proximal assembly of microtubules at the kinetochore during prometaphase.

Microtubule (MT) dynamics in PtK2 cells have been investigated using in vivo injection of unmodified Paramecium ciliary tubulin and time-lapse fixation. The sites of incorporation of the axonemal tubulin were localized using a specific antibody which does not react with vertebrate cytoplasmic tubulin (Adoutte, A., M. Claisse, R. Maunoury, and J. Beisson. 1985. J. Mol. Evol. 22:220-229), followed by immunogold labeling, Nanovid microscopy, and ultrastructural observation of the same cells. We confirm data from microinjection of labeled tubulins in other cell types (Soltys, B. J., and G. G. Borisy. 1985. J. Cell Biol. 100:1682-1689; Mitchison, T., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527; Schulze, E., and M. Kirschner. 1986. J. Cell Biol. 102:1020-1031). In agreement with the dynamic instability model (Mitchison, T., and M. Kirschner. 1984. Nature (Lond.). 312:237-242), during interphase, fast (2.6 microns/min) distal growth of MTs occurs, together with new centrosomal nucleation. Most of the cytoplasmic MT complex is replaced within 15-30 min. During mitosis, astral MTs display the same pattern of renewal, but the turnover of the MT system is much faster (approximately 6 min). We have concentrated on the construction of the kinetochore fibers during prometaphase and observe that (a) incorporation of tubulin in the vicinity of the kinetochores is not seen during prophase and early prometaphase as long as the kinetochores are not yet connected to a pole by MTs; (b) proximal time-dependent incorporation occurs only into preexisting kinetochore MTs emanating from centrosomes. Consequently, in undisturbed prometaphase cells, the kinetochores probably do not act as independent nucleation sites. This confirms a model in which, at prometaphase, fast probing centrosomal MTs are grabbed by the kinetochores, where tubulin incorporation then takes place.

Animals↗

PubMed1990

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. Stürz, 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.

Animals↗

PubMed1991

Quenched flow analysis of exocytosis in Paramecium cells: time course, changes in membrane structure, and calcium requirements revealed after rapid mixing and rapid freezing of intact cells.

Synchronous exocytosis in Paramecium cells was analyzed on a subsecond time scale. For this purpose we developed a quenched flow device for rapid mixing and rapid freezing of cells without impairment (time resolution in the millisecond range, dead time approximately 30 ms). Cells frozen at defined times after stimulation with the noncytotoxic secretagogue aminoethyldextran were processed by freeze substitution for electron microscopic analysis. With ultrathin sections the time required for complete extrusion of secretory contents was determined to be less than 80 ms. Using freeze-fracture replicas the time required for resealing of the fused membranes was found to be less than 350 ms. During membrane fusion (visible 30 ms after stimulation) specific intramembranous particles in the cell membrane at the attachment sites of secretory organelles ("fusion rosette") disappear, possibly by dissociation of formerly oligomeric proteins. This hitherto unknown type of rapid change in membrane architecture may reflect molecular changes in protein-protein or protein-lipid interactions, presumably crucial for membrane fusion. By a modification of the quenched flow procedure extracellular [Ca++] during stimulation was adjusted to less than or equal to 3 x 10(-8) M, i.e., below intracellular [Ca++]. Only extrusion of the secretory contents, but not membrane fusion, was inhibited. Thus it was possible to separate both secretory events (membrane fusion from contents extrusion) and to discriminate their Ca++ requirements. We conclude that no Ca++ influx is necessary for induction of membrane fusion.

Animals↗

PubMed1994

Ca2+ release from subplasmalemmal stores as a primary event during exocytosis in Paramecium cells.

A correlated electrophysiological and light microscopic evaluation of trichocyst exocytosis was carried out the Paramecium cells which possess extensive cortical Ca stores with footlike links to the plasmalemma. We used not only intra- but also extracellular recordings to account for polar arrangement of ion channels (while trichocysts can be released from all over the cell surface). With three widely different secretagogues, aminoethyldextran (AED), veratridine and caffeine, similar anterior Nain and posterior Kout currents (both known to be Ca(2+)-dependent) were observed. Direct de- or hyperpolarization induced by current injection failed to trigger exocytosis. For both, exocytotic membrane fusion and secretagogue-induced membrane currents, sensitivity to or availability of Ca2+ appears to be different. Current responses to AED were blocked by W7 or trifluoperazine, while exocytosis remained unaffected. Reducing [Ca2+]o to < or = 0.16 microM (i.e., resting [Ca2+]i) suppressed electrical membrane responses triggered with AED, while we had previously documented normal exocytotic membrane fusion. From this we conclude that the primary effect of AED (as of caffeine) is the mobilization of Ca2+ from the subplasmalemmal pools which not only activates exocytosis (abolished by iontophoretic EGTA injection) but secondarily also spatially segregated plasmalemmal Ca(2+)-dependent ion channels (indicative of subplasmalemmal [Ca2+]i increase, but irrelevant for Ca2+ mobilization). The 45Ca2+ influx previously observed during AED triggering may serve to refill depleted stores. Apart from the insensitivity of our system to depolarization, the mode of direct Ca2+ mobilization from stores by mechanical coupling to the cell membrane (without previous Ca(2+)-influx from outside) closely resembles the model currently discussed for skeletal muscle triads.

Amiloride↗

PubMed1967

Micronuclear RNA synthesis in Paramecium caudatum.

In a generation time of 8 hr in Paramecium caudatum, the bulk of DNA synthesis detected by thymidine-(3)H incorporation takes place in the latter part of the cell cycle. The micronuclear cycle includes a G(1) of 3 hr followed by an S period of 3-3(1/2) hr. G(2) and division occupies the remaining period of the cycle. Macronuclear RNA synthesis detected by 5'-uridine-(3)H incorporation is continuous throughout the cell cycle. Micronuclear RNA synthesis is restricted to the S period. Ribonuclease removes 80-90% of the incorporated label. Pulse-chase experiments showed that part of the RNA is conserved and released to the cytoplasm during the succeeding G(1) period.

Animals↗

PubMed1968

The mechanism of the nephridial apparatus of Paramecium multimicronucleatum. I. Expulsion of water from the vesicle.

Recent analysis of the mechanism of the nephridial apparatus of Paramecium multimicronucleatum by high-speed cinematography (300 fps at x 250) confirms the observations by electron microscopy (Schneider, 1960) that once the pore is opened, the vesicle is invaginated by adjacent cytoplasm and is emptied by collapsing under pressure from that cytoplasm, aided perhaps by pressure of the fibrils which anchor the ampullae to the excretory canal. There is no indication of active contraction of the vesicle or its membrane. There is no permanent pore to the vesicle. The vesicle is closed by a sealing of the ruptured membrane where it is in contact with the pellicular excretory canal. At onset of expulsion of vesicular fluid the membrane across the basal opening of the excretory canal is ripped along one semicircular portion of the excretory pore and is driven up against the opposite wall as a flap while the water rushes out. A constriction of the vesicular and cell membranes at the base of the excretory canal reseals the opening.

Animals↗

PubMed1969

The mechanism of the nephridial apparatus of Paramecium multimicronucleatum. II. The filling of the vesicle by action of the ampullae.

Our recent analysis of the nephridial apparatus of Paramecium multimicronucleatum by high-speed cinematography (300 fps at X 250) indicates that before the water expulsion vesicle ("contractile vacuole") is completely voided of fluid during expulsion, the ampullae surrounding and confluent with the vesicle swell with fluid entering from their respective nephridial tubules. Once the membranes of the excretory pore at the base of the excretory canal (leading from the vesicle proper to the outside) have constricted and resealed the excretory pore, the up till then constricted injection tubules of the ampullae which conduct fluid to the vesicle open as waves of contraction along the coacervate gel around the ampulla and proceed along each ampulla from distal to proximal end. The coacervate gel around any one ampulla does not necessarily contract in phase with that of any other ampulla. Each ampulla acts independently. The fluid from the ampullae is thus pumped sequentially, but not in predetermined order, into the water expulsion vesicle, refilling and distending it. Our previous studies (Organ et al., 1968a) suggest that an actomyosinoid ATP-using mechanism may be functional in the ampullary contractions.

Membranes↗

PubMed1971

Fine structure of membranous and microfibrillar systems in the cortex of Paramecium caudatum.

An electron microscope study of the cortex of Paramecium caudatum has revealed new details pertinent to several unresolved problems. The lateral boundaries of the alveoli do not regularly follow the crests of the polygonal ridges and thus their staining with silver cannot account for the external lattice seen by light microscopists. A granulo-fibrillar material is present, however, within the peaks of the ridges, which would account for the external lattice if so stained. Perforations are present between adjacent alveoli which make the whole mosaic of alveolar sacs within the cell's cortex continuous-both the membranes and the lumen. A microfibrillar system exhibiting a cross-striated pattern lies in the superficial cortex. These bands are inserted at their ends in the epiplasm and have a fine structure and arrangement suggesting a muscular function. The infraciliary lattice is a branching system of fibers with electron-opaque posts at the center of each branching locus. This system is distinct from the striated bands in morphology and in space. The epiplasm is discontinuous along the crests of the ridges, which may account for the pellicles' disposition to tear along these lines. A three-dimensional drawing is presented to show the interrelationships between the above membranous and microfibrillar systems.

Animals↗

PubMed1972

Isolation and partial characterization of macro- and micronuclei from Paramecium aurella.

A method was developed for the isolation of macro- and micronuclei from Paramecium aurelia. This method utilized ionic and nonionic detergents to rupture the intact cells, calcium ions and spermidine were employed to protect the nuclei, and the nuclei were purified by centrifugation. Macronuclei consisted of 22% DNA, 10% RNA, and 68% protein. Micronuclei were composed of 9% DNA, 11% RNA, and 80% protein. DNA from both macro- and micronuclei had a density of 1.687 g/cc in CsCl and 1.417 g/cc in Cs(2)SO(4). These values corresponded to G + C content of about 23%. The RNA of macronuclei was examined by gel electrophoresis, and two high molecular weight species were identified having molecular Weights of 1.3 x 10(6) and 2.8 x 10(6) daltons. Three syngens were studied, and in each case the conditions for isolation of the nuclei were the same and no differences were observed in the properties of the nuclei.

Animals↗

PubMed1972

The effects of erythromycin and chloramphenicol on the ultrastructure of mitochondria in sensitive and resistant strains of Paramecium.

The effects on cell structure of 12 hr to 6 days of exposure to erythromycin or chloramphenicol, two antibiotics known to inhibit specifically the mitochondrial protein synthesizing system, have been studied in the ciliate Paramecium aurelia. A wild type strain (sensitive to both antibiotics) and three mutant strains carrying cytoplasmically inherited mutations conferring resistance to one or the other antibiotic have been used In sensitive cells both antibiotics lead to a progressive and profound alteration of mitochondrial structure evidenced by an elongation of the organelle, a considerable decrease in the number of cristae, and the appearance of some abnormal lamellar cristae and of rigid plates of periodic structure. The modifications of cell structure, then, are mainly restricted to mitochondrial cristae. The three resistant mutants studied, on the contrary, retain normal or nearly normal mitochondrial structure in the presence of the antibiotic to which they are resistant. This fact is in good agreement with the postulated location in the mitochondrial DNA of the resistance mutations studied. The results are discussed in the light of present knowledge concerning the function of the mitochondrial protein-synthesizing system.

Animals↗

PubMed1973

Synthesis of Paramecium surface proteins. I. Puromycin insensitive amino acid incorporation.

The synthesis of a surface protein has been studied in Paramecium through double-labeling experiments using [(14)C]- and [(3)H]leucine-labeled bacteria as the source of radioactive amino acid. Over a 4-5 h incubation period, the turnover rate was found to be higher than that of overall cell protein. In addition, the initial label is apparently utilized during the chase period, being incorporated into protein via a puromycin insensitive pathway.

Amino Acids↗

PubMed1976

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.

Animals↗

PubMed1976

Divalent cation affinity sites in Paramecium aurelia.

Sites with high calcium affinity in Paramecium aurelia were identified by high calcium (5 mM) fixation and electron microscope methods. Electron-opaque deposits were observed on the cytoplasmic side of surface membranes, particularly at the basal regions of cilia and trichocyst-pellicle fusion sites. Deposits were also observed on some smooth cytomembranes, within the axoneme of cilia, and on basal bodies. The divalent cations, Mg2+, Mn2+, Sr2+, Ni2+, Ba2+, and Zn2+, could be substituted for Ca2+ in the procedure. Deposits were larger with 5 mM Sr2+. Ba2+, and Mn2+ at ciliary transverse plates and the terminal plate of basal bodies. Microprobe analysis showed that Ca and C1 were concentrated within deposits. In some analyses, S and P were detected in deposits. Also, microprobe analysis of 5 mM Mn2+-fixed P. aurelia showed that those deposits were enriched in Mn and C1 and sometimes enriched in P. Deposits were seen only when the ciliates were actively swimming at the time of fixation. Locomotory mutants having defective membrane Ca-gating mechanisms and ciliates fixed while exhibiting ciliary reversal showed no obvious differences in deposition pattern and intensity. Possible correlations between electron-opaque deposits and the locations of intramembranous particles seen by freeze-fracture studied, as well as sites where fibrillar material associate with membranes are considered. The possibility that the action sites of calcium and other divalent cations were identified is discussed.

Animals↗