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

by Gerd Knoll, Claudia Braun, Helmut Plattner - J. Cell , 1991
"... Abstract. 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 ~30 ms). Cells frozen at defined time ..."
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Abstract. 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 ~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 <80 ms. Using freeze-fracture replicas the time required for resealing of the fused membranes was found to be <350 ms. During membrane fusion (visible 30 ms after stimulation) specific intramembranous particles in the cell membrane at the at-
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Calcium in ciliated protozoa: sources, regulation, and calcium-regulated cell functions

by Helmut Plattner, Norbert Klauke - Int. Rev. Cytol , 2001
"... In ciliates, a variety of processes are regulated by Ca2 � , e.g., exocytosis, endocytosis, ciliary beat, cell contraction, and nuclear migration. Differential microdomain regulation may occur by activation of specific channels in different cell regions (e.g., voltagedependent Ca2 � channels in cili ..."
Abstract - Cited by 29 (9 self) - Add to MetaCart
In ciliates, a variety of processes are regulated by Ca2 � , e.g., exocytosis, endocytosis, ciliary beat, cell contraction, and nuclear migration. Differential microdomain regulation may occur by activation of specific channels in different cell regions (e.g., voltagedependent Ca2 � channels in cilia), by local, nonpropagated activation of subplasmalemmal Ca stores (alveolar sacs), by different sensitivity thresholds, and eventually by interplay with additional second messengers (cilia). During stimulus–secretion coupling, Ca2 � as the only known second messenger operates at �5 �M, whereby mobilization from alveolar sacs is superimposed by ‘‘store-operated Ca2 � influx’ ’ (SOC), to drive exocytotic and endocytotic membrane fusion. (Content discharge requires binding of extracellular Ca2 � to some secretory proteins.) Ca2 � homeostasis is reestablished by binding to cytosolic Ca2�-binding proteins (e.g., calmodulin), by sequestration into mitochondria (perhaps by Ca2 � uniporter) and into endoplasmic reticulum and alveolar sacs (with a SERCA-type pump), and by extrusion via a plasmalemmal Ca2 � pump and a Na � /Ca2 � exchanger. Comparison of free vs total concentration, [Ca2�] vs [Ca], during activation, using time-resolved fluorochrome analysis and X-ray microanalysis, respectively, reveals that altogether activation requires a calcium flux that is orders of magnitude larger than that expected from the [Ca2�] actually required for local activation.
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Microdomain Ca 2 � Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca 2 � Influx

by Christian Erxleben, Norbert Klauke, Matthias Flötenmeyer, Marie-pierre Blanchard, Claudia Braun
"... Abstract. In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca 2 �-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of ..."
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Abstract. In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca 2 �-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current � time (As) closely parallels the number of exocytotic events. We suggest that Ca 2 � mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca 2 � imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca 2 � is quenched to or below resting intracellular Ca 2 � concentration ([Ca 2 � ] e � [Ca 2 � ] i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca 2 � ] e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca 2 � ] i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca 2 � mobilization from stores (providing close to threshold Ca 2 � levels) and Ca 2 � influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca 2 � flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense. In most systems analyzed so far, exocytosis is triggered by the increase of intracellular free Ca 2 � concentration ([Ca 2 � ] i) 1. In fast responding systems such as motor endplates, this increase occurs through an influx of extracellular Ca 2 � (Ca 2 � e), via voltage-dependent Ca 2 � channels at active zones where neurotransmitter vesicles are docked. In other systems, Ca 2 � is mobilized exclusively or
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A Ca 2� influx associated with exocytosis is specifically abolished in a Paramecium exocytotic mutant

by Daniel Kerboeuf, Jean Cohen, Aed Plattner, H. Matt, H. Kersken, B. Haake - J. Cell Biol , 1990
"... Abstract. A Paramecium possesses secretory organdies called trichocysts which are docked beneath the plasma membrane awaiting an external stimulus that triggers their exo~.tosis. Membrane fusion is the sole event provoked by the stimulation and can therefore be studied per se. Using 3 #M aminoethyl ..."
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Abstract. A Paramecium possesses secretory organdies called trichocysts which are docked beneath the plasma membrane awaiting an external stimulus that triggers their exo~.tosis. Membrane fusion is the sole event provoked by the stimulation and can therefore be studied per se. Using 3 #M aminoethyl dextran
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ATP keeps exocytosis sites in a primed state but is not required for membrane fusion: an analysis with Paramecium cells

by Helmut Kersken, Renate Stiirzl, Helmut Plattner , 1986
"... Abstract. We have tried to specify a widespread hypothesis ..."
Abstract - Cited by 16 (5 self) - Add to MetaCart
Abstract. We have tried to specify a widespread hypothesis
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Polyamine triggering of exocytosis in Paramecium involves an extracellular Ca2+ /(polyvalent cation)-sensing receptor, subplasmalemmal Ca-store mobilization and store-operated Ca2+ -influx via unspecific cation channels

by N. Klauke, M. -p. Blanchard, H. Plattner , 2000
"... Abstract. The polyamine secretagogue, aminoethyldex-tran (AED), causes a cortical [Ca2+] transient in Para-mecium cells, as analyzed by fluorochrome imag-ing. Our most essential findings are: (i) Cortical Ca2+ signals also occur when AED is applied in presence of the fast Ca2+ chelator, BAPTA. (ii) ..."
Abstract - Cited by 14 (7 self) - Add to MetaCart
Abstract. The polyamine secretagogue, aminoethyldex-tran (AED), causes a cortical [Ca2+] transient in Para-mecium cells, as analyzed by fluorochrome imag-ing. Our most essential findings are: (i) Cortical Ca2+ signals also occur when AED is applied in presence of the fast Ca2+ chelator, BAPTA. (ii) Extracellular La3+ application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca2+]i transient (while injected La3+ causes a sustained fluorescence signal). (iii) Simply in-creasing [Ca2+]o causes a similar rapid, short-lived [Ca2+]i transient. All these phenomena, (i–iii), are com-patible with activation of an extracellular “Ca2+/ (polyvalent cation)-sensing receptor ” known from some higher eukaryotic systems, where this sensor (responding
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Facilitation of membrane fusion during exocytosis and exocytosis-coupled endocytosis and acceleration of “ghost” detachment in Paramecium by extracellular calcium. A quenched-flow/freeze-fracture analysis

by H. Plattner, C. Braun, J. Hentschel - J. Membrane Biol , 1997
"... Abstract. We had previously shown that an influx of extracellular Ca2+ (Ca2+e), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocy-totic membrane fusion in Paramecium. We now ana-lyze, by quenched-flow/freeze-fracture, to what extent Ca2+e contributes to exocytoti ..."
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Abstract. We had previously shown that an influx of extracellular Ca2+ (Ca2+e), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocy-totic membrane fusion in Paramecium. We now ana-lyze, by quenched-flow/freeze-fracture, to what extent Ca2+e contributes to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of ‘‘ghosts’ ’ — a process difficult to analyze by any other method or in any other system. Maximal exocy-totic membrane fusion (analyzed within 80 msec) occurs readily in the presence of [Ca2+]e ù 5 × 10−6 M, while normally a [Ca2+]e 4 0.5 mM is in the medium. A new finding is that exocytosis and endocytosis is significantly stimulated by increasing [Ca2+]e even beyond levels usu-ally available to cells. Quenching of [Ca2+]e by EGTA
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Seventeen a-subunit isoforms of Paramecium V-ATPase provide high specialization in localization and function

by Thomas Wassmer, Jean Cohen, Helmut Plattner, Howard Riezman - Mol. Biol. Cell , 2006
"... In the Paramecium tetraurelia genome, 17 genes encoding the 100-kDa-subunit (a-subunit) of the vacuolar-proton-ATPase were identified, representing by far the largest number of a-subunit genes encountered in any organism investigated so far. They group into nine clusters, eight pairs with>82 % am ..."
Abstract - Cited by 13 (6 self) - Add to MetaCart
In the Paramecium tetraurelia genome, 17 genes encoding the 100-kDa-subunit (a-subunit) of the vacuolar-proton-ATPase were identified, representing by far the largest number of a-subunit genes encountered in any organism investigated so far. They group into nine clusters, eight pairs with>82 % amino acid identity and one single gene. Green fluorescent protein-tagging of representatives of the nine clusters revealed highly specific targeting to at least seven different compartments, among them dense core secretory vesicles (trichocysts), the contractile vacuole complex, and phagosomes. RNA interference for two pairs confirmed their functional specialization in their target compartments: silencing of the trichocyst-specific form affected this secretory pathway, whereas silencing of the contractile vacuole complex-specific form altered organelle structure and functioning. The construction of chimeras between selected a-subunits surprisingly revealed the targeting signal to be located in the C terminus of the protein, in contrast with the N-terminal targeting signal of the a-subunit in yeast. Interestingly, some chimeras provoked deleterious effects, locally in their target compartment, or remotely, in the compartment whose specific a-subunit N terminus was used in the chimera. This article was published online ahead of print in MBC in Press
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Ca 2� release from subplasmalemmal stores as a primary event during exocytosis in Paramecium cells

by Christian Erxleben, Helmut Plattner - J. Cell Biol , 1994
"... Abstract. A correlated electrophysiological and light microscopic evaluation of trichocyst exocytosis was carded out with 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 ..."
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Abstract. A correlated electrophysiological and light microscopic evaluation of trichocyst exocytosis was carded out with 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
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Molecular aspects of membrane trafficking in Paramecium

by Helmut Plattner - Int. Rev. Cytol , 2003
"... Results achieved in tile molecular biology of Paramecium have shed new light on its elaborate membrane trafficking system. Paramecium disposes not only of the standard routes (endoplasmic reticulum---+ Golgi---+ Iysosomes or secretory vesicles; endo- and phagosomes---+ Iysosomes/digesting vacuoles), ..."
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Results achieved in tile molecular biology of Paramecium have shed new light on its elaborate membrane trafficking system. Paramecium disposes not only of the standard routes (endoplasmic reticulum---+ Golgi---+ Iysosomes or secretory vesicles; endo- and phagosomes---+ Iysosomes/digesting vacuoles), but also of some unique features, e.g. and elaborate phagocytic route with the cytoproct and membrane recycling to the cytopharynx, as well as the osmoregulatory system with multiple membrane fusion sites. Exocytosis sites for trichocysts (dense-core secretory vesicles), parasomal sacs (coated pits), and terminal cisternae (early endosomes) display additional regularly arranged predetermined fusion/fission sites, which now can be discussed on a molecular basis. Considering the regular, repetitive arrangements of membrane components, availability of mutants for complementation studies, sensitivity to gene silencing, and so on, Paramecium continues to be a valuable model system for analyzing membrane interactions. This review intends to set a new baseline for ongoing work along these lines.
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