Apoptosis plays a pivotal role in development, tissue homeostasis, cancer, immune defense, and response to weightlessness. It can be initiated by external signals via death receptors, but may also emerge from mitochondria. We exposed mitochondria-rich thyroid carcinoma cells (ONCO-DG1 cell line) and normal thyroid cells (HTU-5) to conditions of simulated microgravity. After 24 h, 10% of the cancer cells had entered a Fas-dependent apoptotic pathway, but destruction and redistribution of mitochondria, microtubuli disruption, and caspase-3 activation were also detected, demonstrating the activation of extrinsic as well as intrinsic pathways. Furthermore, ONCO-DG1 cells grown on the clinostat showed elevated amounts of Bax, but reduced quantities of bcl-2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling, 4',6-diamidino-2-phenylindole staining, and 85-kDa apoptosis-related cleavage fragments. These fragments resulted from enhanced 116-kDa poly(ADP-ribose)polymerase activity and apoptosis. Apoptosis was also detected in normal HTU-5 cells, as demonstrated by electron microscopy, activation of caspase-3, increases in Fas and Bax, and elevation of 85-kDa apoptosis-related cleavage fragments resulting from enhanced poly(ADP-ribose) polymerase activity. Gravitational unloading affects the mitochondria and thereby may trigger apoptosis in thyroid cells subjected to weightlessness by clinorotation.

Growth of a filamentous fungus endophyte, Neotyphodium, and its host plant, tall fescue, Festuca arundinacea, was examined during the seed germination process under pseudo-microgravity [correction of micrgravity] generated by three dimensional (3D-) clinorotation. The shoot growth of tall fescue infected with the endophyte was remarkably suppressed on a 3D-clinostat compared with that of the ground control. Without being infected, shoot growth of tall fescue was not strongly affected by the 3D-clinorotation. Many aggregated hyphae were observed in the plant seed incubated for 1-day on the 3D-clinostat [correction of clinost] than in those kept on the ground. These results indicate that the clinorotation induces responses in the endophyte and its host plant different from those under normal gravity.

Gravitropism is directed growth of a plant or plant organ in response to gravity and can be divided into the following temporal sequence: perception, transduction, and response. This article is a review of the research on the early events of gravitropism (i.e., phenomena associated with the perception and transduction phases). The two major hypotheses for graviperception are the protoplast-pressure and starch-statolith models. While most researchers support the concept of statoliths, there are suggestions that plants have multiple mechanisms of perception. Evidence supports the hypothesis that the actin cytoskeleton is involved in graviperception/transduction, but the details of these mechanisms remain elusive. A number of recent developments, such as increased use of the molecular genetic approach, magnetophoresis, and laser ablation, have facilitated research in graviperception and have allowed for refinement of the current models. In addition, the entire continuum of acceleration forces from hypo- to hyper-gravity have been useful in studying perception mechanisms. Future interdisciplinary molecular approaches and the availability of sophisticated laboratories on the International Space Station should help to develop new insights into mechanisms of gravitropism in plants.

Moss protonemata from several species are known to be gravitropic. The characterization of additional gravitropic species would be valuable to identify conserved traits that may relate to the mechanism of gravitropism. In this study, four new species were found to have gravitropic protonemata, Fissidens adianthoides, Fissidens cristatus, Physcomitrium pyriforme, and Barbula unguiculata. Comparison of upright and inverted apical cells of P. pyriforme and Fissidens species showed clear axial sedimentation. This sedimentation is highly regulated and not solely dependent on amyloplast size. Additionally, the protonemal tip cells of these species contained broad subapical zones that displayed lateral amyloplast sedimentation. The conservation of a zone of lateral sedimentation in a total of nine gravitropic moss species from five different orders supports the idea that this sedimentation serves a specialized and conserved function in gravitropism, probably in gravity sensing.

Apical cells of moss protonemata represent a single-celled system that perceives and reacts to light (positive and negative phototropism) and to gravity (negative gravitropism). Phototropism completely overrides gravitropism when apical cells are laterally irradiated with relatively high red light intensities, but below a defined light intensity threshold gravitropism competes with the phototropic reaction. A 16 day-long exposure to microgravity conditions demonstrated that gravitropism is allowed when protonemata are laterally illuminated with light intensities below 140 nmol m-2s-1. Protonemata that were grown in darkness in microgravity expressed an endogenous tendency to grow in arcs so that the overall culture morphology resembled a clockwise spiral. However this phenomenon only was observed in cultures that had reached a critical age and/or size. Organelle positioning in dark-grown apical cells was significantly altered in microgravity. Gravisensing most likely involves the sedimentation of starch-filled amyloplasts in a well-defined area of the tip cell. Amyloplasts that at 1-g are sedimented were clustered at the apical part of the sedimentation zone in microgravity. Clustering observed in microgravity or during clino-rotation significantly differs from sedimentation-induced plastid aggregations after inversion of tip cells at 1-g.

During the construction phase of the International Space Station (ISS), early flight opportunities have been identified (including designated Utilization Flights, UF) on which early science experiments may be performed. The focus of NASA's and other agencies' biological studies on the early flight opportunities is cell and molecular biology; with UF-1 scheduled to fly in fall 2001, followed by flights 8A and UF-3. Specific hardware is being developed to verify design concepts, e.g., the Avian Development Facility for incubation of small eggs and the Biomass Production System for plant cultivation. Other hardware concepts will utilize those early research opportunities onboard the ISS, e.g., an Incubator for sample cultivation, the European Modular Cultivation System for research with small plant systems, an Insect Habitat for support of insect species. Following the first Utilization Flights, additional equipment will be transported to the ISS to expand research opportunities and capabilities, e.g., a Cell Culture Unit, the Advanced Animal Habitat for rodents, an Aquatic Facility to support small fish and aquatic specimens, a Plant Research Unit for plant cultivation, and a specialized Egg Incubator for developmental biology studies. Host systems (Figure 1A, B: see text), e.g., a 2.5 m Centrifuge Rotor (g-levels from 0.01-g to 2-g) for direct comparisons between g and selectable g levels, the Life Sciences Glovebox for contained manipulations, and Habitat Holding Racks (Figure 1B: see text) will provide electrical power, communication links, and cooling to the habitats. Habitats will provide food, water, light, air and waste management as well as humidity and temperature control for a variety of research organisms. Operators on Earth and the crew on the ISS will be able to send commands to the laboratory equipment to monitor and control the environmental and experimental parameters inside specific habitats. Common laboratory equipment such as microscopes, cryo freezers, radiation dosimeters, and mass measurement devices are also currently in design stages by NASA and the ISS international partners.

Determinations of plant or algal cell density (cell mass divided by volume) have rarely accounted for the extracellular matrix or shrinkage during isolation. Three techniques were used to indirectly estimate the density of intact apical cells from protonemata of the moss Ceratodon purpureus. First, the volume fraction of each cell component was determined by stereology, and published values for component density were used to extrapolate to the entire cell. Second, protonemal tips were immersed in bovine serum albumin solutions of different densities, and then the equilibrium density was corrected for the mass of the cell wall. Third, apical cell protoplasts were centrifuged in low-osmolarity gradients, and values were corrected for shrinkage during protoplast isolation. Values from centrifugation (1.004 to 1.015 g/cm3) were considerably lower than from other methods (1.046 to 1.085 g/cm3). This work appears to provide the first corrected estimates of the density of any plant cell. It also documents a method for the isolation of protoplasts specifically from apical cells of protonemal filaments.

To accommodate a spaceflight experiment with moss (SPM), experiment-unique equipment (EUE) was developed by engineers at Kennedy Space Center. The hardware allows sterile culture for an extended period of time in commercial petri dishes, lateral illumination of each culture with light of a specific wavelength (660 nm; other wavelengths are possible) and a range of intensities (0.05-5 micromoles photons m-2 s-1), incubation in complete darkness, and chemical fixation to terminate the experiment under conditions of microgravity. The use of a fixative required triple containment to protect the astronaut crew. An external panel on the experiment container allowed the timing of illumination and fixation to be controlled by the crew. Light quality is provided by light emitting diodes (LEDs) that are located in the lid of the outer container, the BRIC (Biological Research In Canisters)-LED. Each canister accommodates 6 Petri Dish Fixation Units (PDFUs), and each PDFU holds one 6 cm petri dish. All components are autoclavable. LED illumination is piped through a transparent glass rod. Each PDFU contains fixative in a reservoir that is released by the depression of an actuator. This hardware performed well during its first flight, the 16-day STS-87 mission in Nov./Dec., 1997 as part of the Collaborative USA and Ukrainian Experiment (CUE). It supported vigorous and sterile moss growth, cells were maintained in position and were well-fixed, and there was a vigorous and consistent response to light. Although here used for moss, in future flight experiments this unique new hardware can be used for many types of organisms normally grown in petri dishes, with or without a requirement for illumination.

Moss protonemata are among the few cell types known that both sense and respond to gravity and light. Apical cells of Ceratodon protonemata grow by oriented tip growth which is negatively gravitropic in the dark or positively phototropic in unilateral red light. Phototropism is phytochrome-mediated. To determine whether any gravitropism persists during irradiation, cultures were turned at various angles with respect to gravity and illuminated so that the light and gravity vectors acted either in the same or in different directions. Red light for 24h (> or = l40nmol m-2 s-1) caused the protonemata to be oriented directly towards the light. Similarly, protonemata grew directly towards the light regardless of light position with respect to gravity indicating that all growth is oriented strictly by phototropism, not gravitropism. At light intensities < or = l00nmol m-2 s-1, no phototropism occurs and the mean protonemal tip angle remains above the horizontal, which is the criterion for negative gravitropism. But those protonemata are not as uniformly upright as they would be in the dark indicating that low intensity red light permits gravitropism but also modulates the response. Protonemata of the aphototropic mutant ptr1 that lacks a functional Pfr chromophore, exhibit gravitropism regardless of red light intensity. This indicates that red light acts via Pfr to modulate gravitropism at low intensities and to suppress gravitropism at intensities < or = 140nmol m-2 s-1.

Plants are immobile; therefore, they are oriented in space due to growth movements--tropisms. The latter occur in response to environmental stimuli such as gravity (gravitropism), light (phototropism), chemical compounds or water (chemo- and hydrotropisms). Gravity is the only force that was impossible to control. The moss protonemata are among the limited group of plant objects with tip growth. What is unique about this structure is that protonemal apical cells both sense and respond to gravity. It is considered that the apical cell perceives gravity through amyloplasts (Sack, 1993; Chaban, 1996). Although the dynamics of protonemata negative gravitropism in different moss species was studied in detail, the role of gravity in both the structural polarity of apical cells and the formation of protonematal mat with circular symmetry is completely unexplored. Using the unique possibility to fly the moss on the space shuttle (STS-87) we aimed in this study to analyze the character of the interaction of gravity with light and endogenous factors in the pattern of protonemata space orientation.

A complete review of the scientific literature on experiments involving fungi in space is presented. This review begins with balloon experiments around 1935 which carried fungal spores, rocket experiments in the 1950's and 60's, satellite and moon expeditions, long-time orbit experiments and Spacelab missions in the 1980's and 90's. All these missions were aimed at examining the influence of cosmic radiation and weightlessness on genetic, physiological, and morphogenetic processes. During the 2nd German Spacelab mission (D-2, April/May 1993), the experiment FUNGI provided the facilities to cultivate higher basidiomycetes over a period of 10 d in orbit, document gravimorphogenesis and chemically fix fruiting bodies under weightlessness for subsequent ultrastructural analysis. This review shows the necessity of space travel for research on the graviperception of higher fungi and demonstrates the novelty of the experiment FUNGI performed within the framework of the D-2 mission.

The gravitropism of caulonemata of Pottia intermedia is described and compared with that of other mosses. Spore germination produces primary protonemata including caulonemata which give rise to buds that form the leafy moss plant, the gametophore. Primary caulonemata are negatively gravitropic but their growth and the number of filaments are limited in the dark. Axenic culture of gametophores results in the production of secondary caulonemata that usually arise near the leaf base. Secondary protonemata that form in the light are agravitropic. Secondary caulonemata that form when gametophores are placed in the dark for several days show strong negative gravitropism and grow well in the dark. When upright caulonemata are reorientated to the horizontal or are inverted, upward bending can be detected after 1 h and caulonemata reach the vertical within 1-2 d. Clear amyloplast sedimentation occurs 10-15 minutes after horizontal placement and before the start of upward curvature. This sedimentation takes place in a sub-apical zone. Amyloplast sedimentation also takes place along the length of upright and inverted Pottia protonemata. These results support the hypothesis that amyloplast sedimentation functions in gravitropic sensing since sedimentation occurs before gravitropism in Pottia and since the location and presence of a unique sedimentation zone is conserved in all four mosses known to gravitropic protonomata.

In response to a change in the direction of gravity, morphogenetic changes of fruiting bodies of fungi are usually observed as gravitropism. Although gravitropism in higher fungi has been studied for over 100 years, there is no convincing evidence regarding the graviperception mechanism in mushrooms. To understand gravitropism in mushrooms, we isolated differentially expressed genes in Pleurotus ostreatus (oyster mushroom) fruiting bodies developed under three-dimensional clinostat-simulated microgravity. Subtractive hybridization, cDNA representational difference analysis was used for gene analysis and resulted in the isolation of 36 individual genes (17 upregulated and 19 downregulated) under clinorotation. The phenotype of fruiting bodies developed under simulated microgravity vividly depicted the gravitropism in mushrooms. Our results suggest that the differentially expressed genes responding to gravitational change are involved in several potential cellular mechanisms during fruiting body formation of P. ostreatus.

Abstract Spaceflight experiments have suggested a possible effect of microgravity on the plasmid transfer among strains of the Gram-positive Bacillus thuringiensis, as opposed to no effect recorded for Gram-negative conjugation. To investigate these potential effects in a more affordable experimental setup, three ground-based microgravity simulators were tested: the Rotating Wall Vessel (RWV), the Random Positioning Machine (RPM), and a superconducting magnet. The bacterial conjugative system consisted in biparental matings between two B. thuringiensis strains, where the transfer frequencies of the conjugative plasmid pAW63 and its ability to mobilize the nonconjugative plasmid pUB110 were assessed. Specifically, potential plasmid transfers in a 0 g position (simulated microgravity) were compared to those obtained under 1 g (normal gravity) condition in each device. Statistical analyses revealed no significant difference in the conjugative and mobilizable transfer frequencies between the three different simulated microgravitational conditions and our standard laboratory condition. These important ground-based observations emphasize the fact that, though no stimulation of plasmid transfer was observed, no inhibition was observed either. In the case of Gram-positive bacteria, this ability to exchange plasmids in weightlessness, as occurs under Earth's conditions, should be seen as particularly relevant in the scope of spread of antibiotic resistances and bacterial virulence. Key Words: Bacillus thuringiensis-Conjugation-Microgravity-pAW63. Astrobiology 9, 797-805.

Agaricus bisporus mushrooms were grown in compost amended with either arsenic-contaminated mine waste or an arsenate solution, to a final concentration of approximately 200 microg g(-1). Fungi were cultivated at a small-scale mushroom facility in Vineland (ON), where the controlled environment allowed for a large number of fruiting bodies (mushrooms) to be produced. The total arsenic concentrations as well as speciation were examined for each treatment over several harvests (breaks). Total concentrations were determined by acid digestion and inductively coupled plasma mass spectrometry (ICP-MS) detection and ranged from 2.3 to 16 microg g(-1) dry mass in treatment mushrooms. Arsenic compounds were extracted from mushrooms with methanol/water (1:1 v/v), and separated by high-performance liquid chromatography (HPLC, anion/cation exchange) before detection with ICP-MS. Fruiting bodies from all treatments contained arsenite, dimethylarsinic acid (DMA), and arsenobetaine (AB), and to a lesser extent arsenate and trimethylarsine oxide (TMAO). The ratio of arsenic compounds did not vary greatly over the first three harvests. AB was absent in compost not inoculated with A. bisporus supporting the hypothesis that AB is a product of fungal, not microbial, arsenic metabolism. X-ray absorption spectroscopy results lead us to hypothesize that AB plays a role in nutrient translocation within the fruiting body, as well as maintaining turgor pressure to ensure the mushroom cap remains elevated for maximum spore dispersal.

Rapid and long-distance secretion of membrane components is critical for hyphal formation in filamentous fungi, but the mechanisms responsible for polarized trafficking are not well understood. Here, we demonstrate that in Candida albicans, the majority of the Golgi complex is redistributed to the distal region during hyphal formation. Randomly distributed Golgi puncta in yeast cells cluster toward the growing tip during hyphal formation, remain associated with the distal portion of the filament during its extension, and are almost absent from the cell body. This restricted Golgi localization pattern is distinct from other organelles, including the endoplasmic reticulum, vacuole and mitochondria, which remain distributed throughout the cell body and hypha. Hyphal-induced positioning of the Golgi and the maintenance of its structural integrity requires actin cytoskeleton, but not microtubules. Absence of the formin Bni1 causes a hyphal-specific dispersal of the Golgi into a haze of finely dispersed vesicles with a sedimentation density no different from that of normal Golgi. These results demonstrate the existence of a hyphal-specific, Bni1-dependent cue for Golgi integrity and positioning at the distal portion of the hyphal tip, and suggest that filamentous fungi have evolved a novel strategy for polarized secretion, involving a redistribution of the Golgi to the growing tip.

In addition to shoots and roots, the gravity (g)-vector orients the growth of specialized cells such as the apical cell of dark-grown moss protonemata. Each apical cell of the moss Ceratodon purpureus senses the g-vector and adjusts polar growth accordingly producing entire cultures of upright protonemata (negative gravitropism). The effect of withdrawing a constant gravity stimulus on moss growth was studied on two NASA Space Shuttle (STS) missions as well as during clinostat rotation on earth. Cultures grown in microgravity (spaceflight) on the STS-87 mission exhibited two successive phases of non-random growth and patterning, a radial outgrowth followed by the formation of net clockwise spiral growth. Also, cultures pre-aligned by unilateral light developed clockwise hooks during the subsequent dark period. The second spaceflight experiment flew on STS-107 which disintegrated during its descent on 1 February 2003. However, most of the moss experimental hardware was recovered on the ground, and most cultures, which had been chemically fixed during spaceflight, were retrieved. Almost all intact STS-107 cultures displayed strong spiral growth. Non-random culture growth including clockwise spiral growth was also observed after clinostat rotation. Together these data demonstrate the existence of default non-random growth patterns that develop at a population level in microgravity, a response that must normally be overridden and masked by a constant g-vector on earth.

INTRODUCTION Microgravity provides unique sensory inputs to the vestibular and oculomotor systems. We sought to determine the effects of long-term spaceflight on sensing of spatial orientation. METHODS Two cosmonauts participated in experiments on human vestibulo-visual interactions during a long-term mission (178 d) in the MIR station in 1995. During circular optokinetic stimulation (OKS) the tonic torsional eye position (torsional beating field, TBF) and the subjective visual vertical (SVV) were recorded on several days of the space mission as well as pre- and post-flight. A reference data set was obtained from healthy subjects on Earth, in whom the TBF was measured in upright and in prone positions. RESULTS Neither cosmonaut showed changes in the SVV or the TBF values during the first days in microgravity. On flight day 149, cosmonaut A showed an increase of both values, which continued to rise by 4- and 10-fold until the end of the flight (TBF: 8.1 degrees; SVV: 216.8 degrees). This cosmonaut reported that the increase was accompanied by a loss of spatial orientation. In contrast, cosmonaut B's values remained at pre-flight levels (TBF: 1.6 degrees; SVV: 4.4 degrees). Post-flight values of the TBF did not significantly differ from pre-flight values for either cosmonaut. Subjects showed an increase of the TBF by more than a factor of 2 in prone position (range -7.7 degrees to +10.2 degrees) compared with upright position (range -3.7 degrees to +3.4 degrees). CONCLUSIONS Pre-flight, post-flight and during the first part of the flight, both cosmonauts exhibited values similar to those of normal subjects in an upright position. The increased TBF values of cosmonaut A from flight day 110 on were within the range of the normal subjects in prone (face-down) position, when the gravity vector cannot be used to stabilize the TBF against the rotating stimulus (the axis of rotation is parallel to the gravity vector). The increasing deviations of cosmonaut A's SVV values in-flight suggest the presence of an internal body reference system, which weakened throughout the flight and thus lost its stabilizing effect.

The putative Cryptococcus neoformans pheromone receptor gene CPRalpha was isolated and studied for its role in mating and filamentation. CPRalpha is MATalpha specific and located adjacent to STE12alpha at the MATalpha locus. It encodes a protein which possesses high sequence similarity to the seven-transmembrane class of G-protein-coupled pheromone receptors reported for other basidiomycetous fungi. Strains containing a deletion of the CPRalpha gene exhibited drastic reductions in mating efficiency but were not completely sterile. Delta cpr alpha cells displayed wild-type mating efficiency when reconstituted with the wild-type CPRalpha gene. Hyphal production on filament agar was not affected in the delta cpr alpha strain, indicating no significant role for CPRalpha in sensing environmental cues during haploid fruiting. The wild-type MATalpha CPRalpha strain produced abundant hyphae in response to synthetic MATa pheromone; however, the hyphal response to pheromone by delta cpr alpha cells was significantly reduced. Exposure of wild-type cells to synthetic MATa pheromone for 2 h induced MFalpha pheromone expression, whereas unexposed cells showed only basal levels of the MFalpha transcript. The delta cpr alpha cells, however, exhibited only basal levels of MFalpha message with or without pheromone exposure, suggesting that CPRalpha and MFalpha are components of the same signaling pathway.

In the turgid cells of plants, protists, fungi, and bacteria, walls resist swelling; they also confer shape on the cell. These two functions are not unrelated: cell physiologists have generally agreed that morphogenesis turns on the deformation of existing wall and the deposition of new wall, while turgor pressure produces the work of expansion. In 1990, I summed up consensus in a phrase:"localized compliance with the global force of turgor pressure." My purpose here is to survey the impact of recent discoveries on the traditional conceptual framework. Topics include the recognition of a cytoskeleton in bacteria; the tide of information and insight about budding in yeast; the role of the Spitzenkörper in hyphal extension; calcium ions and actin dynamics in shaping a tip; and the interplay of protons, expansins and cellulose fibrils in cells of higher plants.

Contrary to the rarity of totipotent cells in animals, almost every cell formed by a fungus can function as a "stem cell". The multicellular fruiting bodies of basidiomycete fungi consist of the same kind of filamentous hyphae that form the feeding phase, or mycelium, of the organism, and visible cellular differentiation is almost nonexistent. Mushroom primordia develop from masses of converging hyphae, and the stipe (or stem), cap, and gills are clearly demarcated within the embryonic fruiting body long before the organ expands and unfolds through water uptake and cell wall loosening. Though frequent references are made to gilled mushrooms in this article, the totipotent nature of fruiting body cells and lack of meristems is also applicable to basidiomycetes that spread their spore-producing tissues inside tubes (e.g., boletes), over spines and rippled surfaces, or form spores in cavities within the fruiting body. Even in the mature mushroom, every hypha retains its totipotency. Among animals, only sponges exhibit a similar degree of developmental flexibility, which is interesting, because these simple metazoans may be relatively close relatives of fungi.

The ability of plant organs to use gravity as a guide for growth, named gravitropism, has been recognized for over two centuries. This growth response to the environment contributes significantly to the upward growth of shoots and the downward growth of roots commonly observed throughout the plant kingdom. Root gravitropism has received a great deal of attention because there is a physical separation between the primary site for gravity sensing, located in the root cap, and the site of differential growth response, located in the elongation zones (EZs). Hence, this system allows identification and characterization of different phases of gravitropism, including gravity perception, signal transduction, signal transmission, and curvature response. Recent studies support some aspects of an old model for gravity sensing, which postulates that root-cap columellar amyloplasts constitute the susceptors for gravity perception. Such studies have also allowed the identification of several molecules that appear to function as second messengers in gravity signal transduction and of potential signal transducers. Auxin has been implicated as a probable component of the signal that carries the gravitropic information between the gravity-sensing cap and the gravity-responding EZs. This has allowed the identification and characterization of important molecular processes underlying auxin transport and response in plants. New molecular models can be elaborated to explain how the gravity signal transduction pathway might regulate the polarity of auxin transport in roots. Further studies are required to test these models, as well as to study the molecular mechanisms underlying a poorly characterized phase of gravitropism that is independent of an auxin gradient.