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B H Satir

Publications and source records attributed to B H Satir.

At least 37 records · Page 2Linked to original sources
PubMed1986

Protein secretion in Tetrahymena thermophila. Characterization of the major proteinaceous secretory proteins.

The contents of mucocysts of the ciliated protozoan Tetrahymena thermophila comprise about 12 proteins, ranging in relative mobility (Mr) from approximately 160,000 to 8,000. There are at least four families of sulfhydryl-linked mucocyst polypeptides. One of these families includes a prominent Mr 34,000 protein, as determined by one- and two-dimensional gel electrophoresis. The Mr 34,000 protein is resolved into two species in isoelectric focusing gels, with apparent pI values of 4.8 and 4.9; most of the other mucocyst proteins also exhibit acidic apparent isoelectric points. The identity of the major Mr 34,000 protein as a bona fide mucocyst component is substantiated by indirect immunofluorescent localization of this protein in a linear punctate pattern coincident with the localization of mucocysts in these cells; this pattern of localization can be abolished by stimulation of synchronous secretion and is absent in a mutant strain devoid of these secretory organelles (Maihle, N. J., and Satir, B. H. (1985a) J. Cell Sci. 78, 49-65.

Fluorescent Antibody Technique↗
PubMed1986

Identification of a biochemical marker for the secretory pathway in Tetrahymena thermophila.

Tetrahymena thermophila is a ciliated protozoan studied by investigators from a wide range of disciplines as a model system for a variety of specialized eukaryotic cell functions. The proteinaceous secretory products of T thermophila have been isolated and characterized and in this study we identify the major secretory product, a 34,000 Mr polypeptide, and use an antiserum prepared against this secretory protein to (1) demonstrate that this 34,000 Mr polypeptide is truly a secreted protein in Tetrahymena and (2) monitor the synthesis and transport of this protein by indirect immunofluorescence and light microscopy during mucocyst biogenesis.

Animals↗
PubMed1986

Conjugation rescue of an exocytosis-competent membrane microdomain in Tetrahymena thermophila mutants.

Conjugation-rescue experiments with two Tetrahymena thermophila mutants (exo-) incapable of exocytosis (SB255, SB258) have been used to dissect regulatory steps in assembly of a functional membrane microdomain, the fusion rosette. "Rescue" refers to the recovery of a secretory activity. Exo- mutants fail to secrete mucus normally (form capsules) when stimulated by the secretagogue alcian blue and are blocked before the assembly of a functional fusion rosette in the cell membrane. Two criteria are used to assay recovery of the wild-type (exo+) phenotype: the conjugant's ability to form capsules when stimulated and the presence of assembled rosettes, which disperse upon stimulation. Conjugation of exo+ X SB258 results in restoration of secretion in 60% of the mutant conjugants and reappearance of assembled rosettes. Secretory capacity is restored in the SB258 cell within one-half hour of firm pair formation. This restoration is not due to new gene expression or continued protein synthesis, since it occurs when SB258 is crossed to a "star" strain (A*), which has defective micronuclei and therefore cannot contribute wild-type genes, and restoration occurs in the presence of cycloheximide during conjugation. Conjugation of exo+ X SB255 reveals a real but inefficient restoration of exocytic capacity in the exo- conjugant and a significant decrease of exocytic capacity in the exo+ conjugant. SB255 X SB258 crosses also show a low but significant rescue of exocytic competence, indicating that different components of the exocytic mechanism are affected in the two mutants. This cross leads to restoration of rosette assembly and function in one of the partners, presumably SB258. These results provide data about some of the steps necessary for rosette assembly and suggest that transferable factors that promote and/or inhibit exocytosis are present in these cells.

Cell Membrane↗
PubMed1985

Effect of Cerophyl growth medium on exocytosis in Tetrahymena thermophila.

Culturing the ciliate Tetrahymena thermophila in Cerophyl has provided an opportunity for studying the assembly and/or synthesis of the intramembrane particle array, the rosette, which marks the site of exocytosis in these cells. Cultures grown in this medium cease cell division after only 12h and enter 'stationary phase' earlier (12h of growth) relative to growth in standard medium (proteose peptone). In addition, the cell changes from the normally observed pear-shaped body to a thinner more ellipsoid form. Despite the initial similarities to starving cells, several differences are observed in the Cerophyl-grown cells. One is that cell size remains constant for at least 72h in contrast to starved cells. Secondly, in spite of this block in cell division, results from freeze-fracture replicas of the cell membrane of these cells show that they continue to assemble rosettes, the number of which increases approximately six times, from 0.34 rosette/microgram2 to 2.1 rosettes/microgram2. Addition of the protein synthesis inhibitor, cycloheximide (6h exposure), during growth in Cerophyl shows that 70% of rosettes can be assembled, despite the blockage of translation, by using pre-existing component(s) from a pool. The remaining 30% must involve de novo synthesis of one or more components; this percentage can be increased with longer exposure to the drug. Thirdly, an apparent increase in the number of mucocysts is observed by thin-section electron microscopy. At first (12-24h) only docked mucocysts seem to accumulate in the cell. However, by 36h a considerable increase seems to have taken place, particularly in the number of mucocysts located in the cytoplasm. In the cycloheximide-treated cells this increase in mucocysts begins to be blocked after 6h of exposure to the drug. These observations are in agreement with the results obtained from the freeze-fracture data on the concomitant increase in number of rosettes. This system therefore offers the first possibility of exploring the biosynthesis of these components.

Alcian Blue↗
PubMed1985

Protein secretion in Tetrahymena thermophila: characterization of the secretory mutant strain SB281.

The ciliated protozoon Tetrahymena thermophila contains membrane-bounded secretory organelles termed mucocysts, the release of which has previously been characterized ultrastructurally as a model system for the events occurring during membrane fusion and protein secretion. Recently, a series of secretory mutant strains of Tetrahymena has been isolated following mutagenesis of a parental wild-type strain designated SB210. In this study, the correlates of non-release in one unique mutant strain of this series, designated SB281, are described. SB281 appears to express a diminished (undetectable) level of the major 34000 Mr proteinaceous secretory product of Tetrahymena, as determined by Western immunoblot analysis and indirect immunofluorescence labelling. Thin-section electron-microscopic studies of these cells reveal that they possess no docked or free mature mucocysts. In addition, freeze-fracture electron microscopy demonstrates that an intramembrane particle array termed the rosette, present in the plasma membrane of wild-type cells above sites of docked mucocysts, is absent in the plasma membrane of mutant SB281 cells. A morphometric analysis of intramembrane particles in the plasma membrane of both wild-type and mutant cells indicates that both strains have a similar intramembrane particle density in both leaflets of the the plasma membrane. Although assembled rosettes are missing in the plasma membrane of mutant cells, a 15 nm intramembrane particle size class does exist in the plasma membrane of the mutant, but this size class is significantly reduced in number relative to wild-type.

Animals↗
PubMed1984

Trifluoperazine-induced changes in swimming behavior of paramecium: evidence for two sites of drug action.

Trifluoperazine (TFP), a drug that binds to Ca2+-calmodulin (CaM) complexes, altered swimming behavior not only in living paramecia, but also in reactivated, Triton-extracted "models" of the ciliate. By comparing the responses of living cells and models, we have ascertained that two sites of drug action exist in paramecium cilia. Swimming movements were recorded in darkfield stroboscopic flash photomicrographs; this permitted accurate quantitation of velocities and body-shape parameters. When living paramecia were incubated in a standard buffer containing 10 microM TFP, their speed of forward swimming fell over several minutes and their bodies shortened. Untreated paramecia backed up repeatedly and frequently upon transfer to a solution containing barium ions (the "barium dance"), but cells preincubated in TFP did not "dance." Instead they swam forward slowly for long periods of time without reversing and occasionally then exhibited abnormally prolonged reversals. W7 effects on swimming mimicked low doses of TFP, and the analog W5 did not visibly alter normal swimming patterns. These results suggest that TFP induces a decrease in the intracellular pCa of living paramecia, perhaps by reducing the efficiency of a calmodulin-activated calcium pump in the cell membrane. Paramecia extracted with Triton X-100 and reactivated to swim forward (7 greater than or equal to pCa greater than or equal to 6) were not affected by addition of up to 40 microM TFP to the reactivation medium. We conclude that the main drug effect in living cells is probably not at the axoneme. However, at low pCa, TFP directly affected the ciliary axoneme to shift its behavior to one characteristic of a higher pCa: TFP inhibited backward swimming in models reactivated at pCa less than 6; instead they swam forward or rocked in place. The mechanism of ciliary reversal in paramecium may therefore depend on an axonemal Ca2+-sensor, possibly bound CaM, which is affected by TFP only at low pCa, as has been postulated for other types of cilia.

Animals↗
PubMed1984

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.

Acridine Orange↗
PubMed1983

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.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

Isolation and ultrastructural characterization of secretory mutants of Tetrahymena thermophila.

Isolation of 14-secretory mutants (exo-) of Tetrahymena thermophila and ultrastructural characterization (freeze-fracture and thin-section) of two of these (SB255 and SB258) are described. The site of secretion is marked by an intramembrane particle array, the rosette, beneath which the secretory organelle rests. Using Alcian Blue (8GS) as a secretagogue, a screening procedure for exo- cells was developed. Of the resulting 14 clones isolated, 10 are stable and have a tight mutant phenotype. Two of these, SB255 and SB258, lack assembled rosettes. Electron microscopy shows that SB255 has a reduced total number of mucocysts, whereas SB258 appears to have the normal number. This study demonstrates a useful eukaryotic model with which to study by genetic dissection the regulatory mechanisms involved in membrane events in secretion.

Alcian Blue↗
PubMed1982

Protein phosphorylation/dephosphorylation and stimulus-secretion coupling in wild type and mutant Paramecium.

Axenic cultures of Paramecium tetraurelia take up 32Pi and phosphorylate a number of polypeptides as determined by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The most heavily labeled polypeptide has an apparent Mr of approximately 65,000. Wild type cells stimulated to secrete with picric acid, the standard secretagogue for these cells, show a marked reduction in labeling of the 65,000 Mr polypeptide. There is no change in the Coomassic blue staining protein pattern after addition of picric acid. Addition of picric acid to cells solubilized in sample buffer containing 10% sodium dodecyl sulfate, significantly lowers the pH but does not induce dephosphorylation of the 65,000 Mr polypeptide. Dephosphorylation of the 65,000 Mr polypeptide is further correlated with secretion in two types of experiments. 1) Preincubation of cells in Mg2+ (no added Ca2+) inhibits both secretion and dephosphorylation in response to picric acid. 2) A temperature-sensitive mutant, nd 9, when grown at 18 degrees C (permissive temperature) has the normal intramembrane particle array (rosette) at the secretory site and secretes and dephosphorylates the 65,000 Mr polypeptide in response to picric acid, but when grown at 27 degrees C (nonpermissive temperature) does not have assembled rosettes at the secretory site, and does not secrete nor dephosphorylate the 65,000 Mr polypeptide in response to picric acid. This represents the first correlation between a phosphoprotein and a physiological activity (secretion) in Paramecium. Our results show the presence of an in vivo stimulus-sensitive phosphoprotein of Mr 65,000 which appears related to Ca2+-mediated exocytosis. Inhibition of dephosphorylation occurs when secretion is blocked, either by Mg2+ or by a mutation affecting an intramembrane particle array, the rosette.

Animals↗
PubMed1982

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.

Animals↗
PubMed1981

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.

Animals↗
PubMed1980

The role of local design in membranes.

The IMP arrays are integral membrane multiprotein complexes that may be site-specific control centers regulating the local concentration of specific ions by functioning as pumps, gates, or channels. In the case of the fusion rosette, an increase in Ca2+ may trigger local membrane fusion; in other cases, Ca2+ or H+ may be important physiological regulators. Ca2+ dependent regulatory proteins, such as calmodulin, and other parameters prevent widespread dispersion of the ions and permit fine local regulation.

Animals↗
PubMed1978

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.

Biological Transport↗