Gerd Knoll

Local trichocyst exocytosis provides an efficient escape mechanism for Paramecium cells.

More than 1000 secretory organelles (trichocysts) are docked at the plasma membrane of Paramecium cells. After stimulation, the trichocyst contents are expelled as needle-like structures in an exocytotic response. Neither the function nor the natural stimulus of trichocyst exocytosis are known in this well established system. Several hypotheses have been put forward during the last 100 years, but as yet, none has withstood critical experimental testing. Using video-aided light microscopic evaluation of the explosive trichocyst exocytosis we have obtained conclusive evidence for a defensive mechanism. When stimulated locally by a non-cytotoxic chemical secretagogue, cells were rapidly propelled in the opposite direction by vigorous local trichocyst discharge. The same phenomenon was observed during encounter with a predatory ciliate, Dileptus. Whereas exocytosis-competent paramecia escaped by rapid propulsion away from the attacking predator, cells non-competent for exocytosis were paralysed and engulfed. Thus, oriented locomotion by locally stimulated trichocyst exocytosis serves as a rapid escape mechanism of Paramecium.

Cryofixation of Dynamic Processes in Cells and Organelles

Electron microscope analysis of cells depends on fixation; the goal is to trap the native state of dynamic structures and distribution patterns of components (Plattner and Bachmann 1982). This intention of fixation is, of course, not restricted to applications in electron microscopy. Attempts to literally “freeze” the living structure by cryofixation can be traced back to the last century, and were extended to electron microscopy in its very early days (compiled by Rash 1983). The main problem with simply freezing a biological specimen in order to fix it, is destruction caused by ice crystals. This freezing damage hampers further analysis, in particular at the high resolution level obtained with the electron microscope. For this reason, chemical fixation procedures have routinely been used, although their limitations and risks have been well recognized (Fernandez-Moran 1964; Rebhun 1965). On the other hand, the drawbacks of chemical treatments have stimulated the development of rapid freezing techniques which reduce the formation of ice crystals (Van Harreveld and Crowell 1964).

A rapid calcium influx during exocytosis in Paramecium cells is followed by a rise in cyclic GMP within 1 s

The synchrony of trichocyst exocytosis in Paramecium allows temporal correlation of associated events. Using quenched flow we observed a Ca2+ influx concurrent with exocytosis within 80 ms after stimulation with the secretagogue aminoethyldextran. Cyclic AMP did not change in depency of stimulation. Cyclic GMP transiently increased after 500 ms, culminating at 2 s, and thus considerably lags behind exocytosis induction and influx of Ca2+. Both Ca2+ influx and rise in cGMP are known to be induceable also by Ba2+ or veratridine, allegedly via the opening of ciliary Ca2+ channels. However, only veratridine stimulated exocytosis. We conclude that both aminoethyldextran and veratridine induce an exocytosis‐associated Ca2+ influx, which is responsible for the rise in cGMP, through an as yet unknown pathway.

Reconstitution of Na+/K+-ATPase into phosphatidylcholine vesicles by dialysis of nonionic alkyl maltoside detergents

The reconstitution of Na+/K+-ATPase from outer medulla of rabbit kidney into large unilamellar liposomes was achieved through detergent removal by dialysis of mixed micellar solutions of synthetic dioleoyl phosphatidylcholine/octyl glucoside and Na+/K+-ATPase/decyl maltoside or decenyl maltoside. Tight, transport-active liposomes were formed when the lipid and the enzyme were solubilized separately in the nonionic detergents and mixed immediately before starting the dialysis. The two maltoside detergents with different structures of the hydrophobic part of the molecule proved to be well suited for the solubilization of Na+/K+-ATPase with high retention of enzyme activity; the inactivation of enzyme being evidently slower with the unsaturated decenyl maltoside. The diameters of the proteoliposomes, 110 and 170 nm, respectively, were also dependent on the structure of the maltoside detergent, the saturated decyl maltoside producing the bigger liposomes. After freeze-fracture, both preparations exhibited intramembranous particles as structural indicators of successful reconstitution. The electrogenic activity of the reconstituted enzyme was determined by fluorescence measurements with Oxonol VI and by tracer-flux measurements with 22Na+.

Calcium-sequestering organelles of Dictyostelium discoideum: changes in element content during early development as measured by electron probe X-ray microanalysis

Starving Dictyostelium discoideum amoebae aggregate within a few hours by chemotaxis towards the attractant cAMP to form a multicellular organism. The differentiating cells possess rapid and efficient calcium buffering and sequestration systems which enable them to restrict changes in the cytosolic free calcium concentration temporally and spatially during their chemotactic reaction and allow the continuous accumulation of Ca2+ during development. In order to identify and to characterize calcium storage compartments, we analyzed the element content of amoebae at three consecutive stages of differentiation. Determination of the element distribution was done using energy-dispersive X-ray microanalysis of freeze-dried cryosections of rapid-frozen cells. Amoebae were frozen in the vegetative and aggregation-competent state and after formation of aggregates. Aggregation-competent as well as aggregated cells contained mass dense granules with large amounts of calcium together with phosphorous and either potassium or magnesium: in aggregation-competent cells calcium was colocalized with potassium, whereas in aggregated cells the mass dense granules contained calcium and magnesium. Although mass dense granules were also present in undifferentiated, vegetative cells, they contained only low amounts of phosphorous and potassium together with little Ca and Mg. We conclude that during their differentiation D. discoideum cells use an intracellular storage compartment to sequester Ca and other cations constantly throughout development.

cAMP-induced changes in the cytosolic free Ca2+ concentration in Dictyostelium discoideum are light sensitive

The cytosolic free calcium concentration ([Ca2+]i) of the social amoeba Dictyostelium discoideum was analyzed after challenge with the chemoattractant cAMP. [Ca2+]i was measured by digital imaging in single cells loaded with the Ca2+ indicator Fura-2-dextran. Global stimulation with low concentrations of cAMP (0.1-1 microM) led to a global transient [Ca2+]i increase. This increase was abolished when cells were illuminated with high doses of light. However, after a short recovery period of several minutes, the cells again displayed the normal response. Inhibition of the [Ca2+]i elevation depended on the wavelength of illumination light. We compared the required recovery period of cells irradiated with either 340, 380, 405, 450 or 490 nm at defined intensities. Light of 405 nm had a pronounced effect; 340 nm alone or in combination with 380 nm was also effective, but to a lesser extent, whereas neither 450 nm nor 490 nm inhibited the [Ca2+]i increase, even at very high irradiance. The wavelength dependence matched the absorption spectrum of amoebae grown in darkness that contain a photopigment which seems to be responsible for phototaxis of single cells. Cells grown in darkness exhibited an increased sensitivity of the cAMP-induced [Ca2+]i transient towards light compared to light-grown cells. From these data we conclude that phototactic signaling could interfere with chemotactic signaling at the level of [Ca2+]i changes.

Exo-Endocytosis in Isolated Peptidergic Nerve Terminals Occurs in the Sub-Second Range

Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horserdish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in subsecond time peroids after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.