The Free Fall Machine--a ground-based facility for microgravity research in life sciences.

A device is described in which a biological specimen is periodically accelerated for a short period. Each event is followed by a variable period of free fall. Assuming that the g-dose (expressed g s) has to surpass a certain minimal value to be perceived by cells, and in addition, there is a minimal time threshold for sensing a change in gravity, it is conceivable that conditions are created in which cells do not detect the periodic acceleration, and only experience the periodic free-fall movement as a long-term weightlessness condition. Using the cell-cycle progression of the unicellular green alga Chlamydomonas as an example, it is shown that with this device effects can be generated which are similar to those observed in satellite flights.

Possible actions of gravity on the cellular machinery.

Since the first flight of the ESA Biorack on the German Spacelab Mission D1 in 1985 evidence has been obtained that biological cells and small unicellular organisms function differently under conditions of microgravity. However, there is still lack of scientific proof that these effects are caused by a direct influence on the cells in the weightlessness condition. The question how normal gravity may play a role in cellular activity is being addressed and the results show that gravity may provide important signals during certain state transitions in the cell. These would be gravity-sensitive windows in the biological process. Also, by amplification mechanisms inside the cell, the cell may assume a state that is typical for normal gravity conditions and would change in microgravity. Experimental tools are discussed that would provide the conditions to obtain evidence for direct action of gravity and for the possible existence of gravity-sensitive windows.

Effects on ontogenesis of Carausius morosus hit by cosmic heavy ions.

Among the biological problems that arise in long duration spaceflights, the effects of weightlessness and ionizing radiation appear to be the two main risk factors. Eggs of the stick insect Carausius morosus were exposed to spaceflight conditions during the 12.56 day Biosatellite mission Cosmos 1887. Five different ages were used, representing different sensitivities to radiation and different capacities for regeneration. During spaceflight the eggs continued their development. Already, in the Spacelab D1 mission in 1985, it has been shown that microgravity leads to a reduced hatching rate of eggs exposed during the early steps of development. When the eggs were hit by a heavy ion, a further but not significant reduction of the hatching rate was observed. Hatching was normal for eggs which were exposed on a 1 g reference centrifuge in space. Heavy ion hits caused body anomalies. The combined action of heavy ions and microgravity resulted in an unexpectedly high rate of anomalies. In the experiment on Cosmos 1887 these results were confirmed. Studies on the embryonic development before hatching showed no major difference between flight and ground control specimen, neither in speed of development nor in morphological anomalies. Hatching therefore seems to be the critical point in insect ontogenesis.

Influence of cosmic radiation and/or microgravity on development of Carausius morosus.

Eggs of Carausius morosus were exposed to spaceflight conditions in two spaceflight missions, the German 7 day Spacelab Mission D1 and the Soviet 12.56 day Biosatellite Mission "COSMOS 1887". During spaceflight the eggs continued their development. Eggs of five different ages representing different sensitivity to radiation and different capacity to regeneration were used to investigate the influence of cosmic radiation and/or microgravity on insect development. Using the Biostack concept--eggs in monolayers sandwiched between nuclear track detectors--and the 1 g reference centrifuge of BIORACK in D1 we were able to separate effects of heavy ions of the cosmic radiation from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, embryonic and larval growth kinetics and anomaly frequencies were determined. Microgravity leads to a reduced hatching rate of eggs exposed in the early stages of development. Hatching was normal in eggs which were exposed on the 1 g reference centrifuge. Hits by heavy ions caused body anomalies. The combined action of heavy ions and microgravity resulted in an unexpectedly high frequency of anomalies. These results obtained from the Spacelab Mission D1, were confirmed in an experiment onboard of COSMOS 1887. In addition to the previous analysis, embryonic development before hatching was followed which showed no major difference between flight and the ground control specimens. Since a reconfirmation of reduced hatching rates was observed in COSMOS 1887, too, the above results suggest some microgravity induced functional impairment of the hatching activity, rather than blockage in embryonic development.

Localization of cell surface glycoproteins in membrane domains associated with the underlying filament network.

To visualize the localization of cell surface constituents in relation to the plasma membrane-associated filament network, we developed a method based on a combination of immunogold labeling and dry-cleaving. For labeling we used trinitrophenyl-derivatized ligand, anti-TNP antibodies, and protein A-coated colloidal gold. Dry-cleaving (Mesland, D. A. M., H. Spiele, and E. Roos, 1981, Exp. Cell Res., 132: 169-184) involves cleavage of lightly fixed critical point-dried cells by means of adhesive tape. Since cells cleave close to the cell surface, the remaining layer is thin enough to be examined in transmission electron microscopy. Using this method, we studied concanavalin A-binding constituents on the medium-facing surface of H35 hepatoma cells. The distribution of the gold particles, which was partly dispersed and partly patchy, coincided strikingly with membrane-associated filaments, and label was virtually absent from areas overlying openings in the filament network. In stereo pairs we observed the label to be localized to areas of somewhat enhanced electron density at the plane of the membrane. These areas were interconnected in a pattern congruent with the filament network. Preliminary observations on wheat germ agglutinin receptors on the hepatoma cells as well as concanavalin A receptors on isolated hepatocytes yielded comparable results. It thus appears that surface glycoproteins, although seemingly randomly distributed as observed in thin sections, may actually be localized to particular membrane domains associated with underlying filaments.

Brief extraction with detergent induces the appearance of many plasma membrane-associated microtubules in hepatocytic cells.

In cultured H35 hepatoma cells membrane-associated cortical networks have a microtrabecular appearance as revealed by dry-cleaving. Filaments having diameters of 15 nm can be readily distinguished within these networks and have not been described previously. Microtubules are seldom observed to be part of this structure. Extraction of cells with 0.1% Saponin in microtubule-stabilizing buffer produces holes in the membrane and reorganization of the networks resulting in the loss of microtrabecular structure, the loss of 15 nm filaments and the appearance of abundant membrane-associated microtubules (about 1.25 micron per micron2 substrate-adherent membrane). These observations were confirmed by immunolabelling experiments with affinity-purified anti-tubulin immunoglobulin G. By both fluorescence microscopy and electron microscopy it was shown that labelled tubulin in the cortical networks became organized into microtubules upon treatment with detergent. By determination of the microtubule density, expressed as micron microtubule per micron2 membrane, the effects of various conditions on microtubule occurrence were determined. The Saponin-induced appearance of microtubules in the membrane-associated network could be inhibited by: 1% and 2% glutaraldehyde, 0 degrees C, millimolar Ca2+, absence of Mg2+ (subsequent reversal of inhibition by addition of Mg2+ was shown), and 20 microM-nocodazole (but not 20 microM-colchicine). In addition to Saponin, extraction with 0.1% Nonidet P-40 or 0.1% Triton X-100 also resulted in microtubule-containing cortical networks. However, 0.1% Triton N-101 was not effective, although holes were produced in the plasma membrane. These data provide evidence suggesting rapid polymerization of membrane-associated microtubule protein rather than detergent-induced displacement or collapse of existing microtubules. The arguments for this hypothesis and its implications are discussed.

Plasma membrane-associate filament systems in cultured cells visualized by dry-cleaving.

Substrate-attached critical-point-dried cells cleave along the level of the substrate-adherent membrane if removed by means of adhesive tape. The remaining membrane fragments on grids can be visualized three-dimensionally by means of stereo transmission electron microscopy. Attachment of cells may be achieved by active spreading of the cell, or artificially by poly-L-lysine adherence of prefixed cells. In 11 different cell types a filamentous network appears to remain associated with the cytoplasmic face of the membrane. In one hepatoma cell type virtually no filamentous network could be detected. Two general network morphologies are described: the hepatocytic network and the lymphoid network. Since no correspondence could be found between cytoplasmic structure and the structure of the membrane-associated network, and since cells generally cleave along the level of this network, excluding cell organelles, we conclude that it comprises a distinct structural system, analogous to the membrane skeleton of the red cell membrane.

Flagellar tip activation stimulated by membrane adhesions in Chlamydomonas gametes.

Membrane adhesions between the flagella of mating-type "plus" and "minus" gametes of Chlamydomonas reinhardi are shown to stimulate a rapid change in the ultrastructure of the flagellar tips, designated as flagellar tip activation (FTA). A dense substance, termed fibrous tip material (FTM), accumulates between the flagellar membrane and the nine single A microtubules of the tip. The A microtubules then elongate, growing into the distal region of the tip, increasing tip length by 30%. This study describes FTA kinetics during normal and mutant matings, presents experiments designed to probe its role in the mating reaction, and offers the following conclusions: (a) FTA is elicited by agents that cross-link flagellar membrane components (including natural sexual agglutinins, antiflagellar antisera, and concanavalin A) but not by flagellar adherence to polylysine-coated films. (b) FTA is reversed by flagellar disadhesion. (c) Gametes can undergo repeated cycles of FTA during successive rounds of adhesion/disadhesion. (d) FTA, flagellar tipping, and sexual signaling are simultaneously blocked by colchicine and by vinblastine, suggesting that tubulinlike molecules, perhaps exposed at the membrane surface, are involved in all three responses. (e) FTA is not blocked by short exposure to chymotrypsin, by cytochalasins B and D, nor by concanavalin A, even though all block cell fusion; the response is therefore autonomous and experimentally dissociable from later stages in the mating reaction. (f) Under no experimental conditions is mating-structure activation observed to occur unless FTA also occurs. This study concludes that FTA is a necessary event in the sexual signaling sequence, and presents a testable working model for its mechanism.

Membrane-membrane and membrane-ligand interactions in Chlamydomonas mating.

Our investigations of the mating reaction of Chlamydomonas revealed a surprisingly intricate series of interrelated events. Adhering sites are moved to the flagellar tips in a fashion highly reminiscent of the capping of surface ligands over the centriolar regions of lymphocytes (28). Tipping is prevented by the gam-1 mutation and by agents that interact with tubulin; the molecular mechanism(s) for the inhibition effects are currently being sought. Tip locking appears to be accompanied by the accumulation of a dense material beneath the tip membrane, a postulated alteration of axonemal structure, and an immobilization of component(s) involved in surface motility. Two mating signals are then transduced to the locked-in cells who respond by shedding cell walls, activating mating structures, and fusing together. Signal transmission and/or reception is sensitive to such agents as trypsin, chymotrypsin, and cold temperature. Once zygotic cell fusion has occurred, tip unlocking and a reversal of the tip activation response appear to occur in parallel. Since all of these events can occur within 30 sec, the mating reaction serves as an experimental paradigm for studying rapid cellular responses to specific membrane-membrane interactions.

Mating in Chlamydomonas eugametos. A scanning electron microscopical study.

A technique has been developed by which mating gametes of Chlamydomonas eugametos can be studied in the Scanning Electron Microscope. A detailed description of the mating process, from the initial flagellar agglutination until the release of free vis-à-vis pairs, is presented. Flagella appear to agglutinate at random points on their surface. This is followed by a rapid increase of the contact area such that they "line-up" tip to tip. Flagella always exhibit a typical position prior to cell fusion. After cell fusion the flagella of a pair separate rapidly while the female have shortened about 33%. In a vis-à-vis pair the plasma bridge has contracted. The observations are interpreted in terms of a specific reorganization of the sexuale aggregate.