Gravitational response of the slime mold Physarum.

The acellular slime mold Physarum polycephalum is used as a model system to investigate the graviresponse of single cells which possess no receptors specialized for the perception of gravity. To obtain insights into the gravity-signal transduction mechanism the light response of the cell is used: Macroplasmodia of the slime mold show clear geo- and phototaxes. Gravity increases and white light decreases transiently the contraction frequency of plasmodial strands whereby both responses follow the same time pattern. Since mitochondria play a major role in changing the contraction rhythm in response to light and gravity stimuli, the simultaneous and subsequent inductions of the opposing light and gravity responses and their mutual influences on one another were investigated. The experiments were performed in weightlessness (0 g)--simulated on the fast-rotating clinostat as well as in actual weightlessness during the IML-1 Space Shuttle mission. The results indicate that mitochondria (chondriome) are part of the acceleration-stimulus reaction chain in Physarum. Two models for a direct gravireceptor mechanism are discussed.     

Confirmation of gravisensitivity in the slime mold physarum polycephalum under near weightlessness

We have investigated Physarum polycephalum, a unicellular organism with no special gravity receptors, on its ability to react to gravity. The first experiments were 0 g-simulation experiments on the fast-rotating clinostat conducted with plasmodial strands of this acellular slime mold. In these earth-bound experiments the observed parameters were periodicity of the contractions and dilatations of the strand's ectoplasm as well as the periodicity and velocity of the striking cytoplasmic (endoplasmic) shuttle streaming. During 0 g-simulation these parameters showed significant changes indicating the existence of a gravisensitivity of the slime mold.

The Space-Shuttle experiment (ESA-Biorack in D 1-Mission) should demonstrate the validity of the 0 g-simulation on the fast-rotating clinostat. The experiment was designed in a way enabling the registration of the same parameters as on the clinostat (using the light microscope in combination with a photo diode and a cinecamera). Only one of the two planned measurement sessions was fully successful and provided us with data confirming the results gained on the fast-rotating clinostat: The slime mold showed under real near weightlessness in the D 1-Space Shuttle Mission a transient frequency increase in its contraction rhythmicity and a (steady) increase in the streaming velocity of its endoplasm.     

The role of calcium accumulation and the cytoskeleton in the perception and response of Coprinus cinereus to gravity.

The role of Ca2+ in the gravitropic perception and/or response mechanism of Coprinus cinereus was examined by treating stipes with inhibitors of Ca2+ transport and calmodulin. Inhibitors had no effect on gravity perception but significantly diminished gravitropism. It is concluded that, under the conditions tested, Ca2+ is not involved in gravity perception by Coprinus stipes, but does contribute to transduction of the gravitropic impulse. The results would be consistent with regulation of the gravitropic bending process requiring accumulation of Ca2+ within a membrane-bound compartment. Treatment of stipes with an actin inhibitor caused a significantly delayed response, a result not observed with the Ca2+ inhibitors. This suggests that cytoskeletal elements may be involved directly in perception of gravity by Coprinus stipes while Ca(2+)-mediated signal transduction may be involved in directing growth differentials.     

Classification of gravity effects on “free” cells

When cell physiologists detect gravity related reactions of their objects it is often difficult to decide where the receptors for the observed effects are located. Answering this question is necessary for any further analysis of a detected gravity effect on cells. In previous papers we have discussed direct and indirect gravity effects in relation to the smallest functional units where the primary receptor, which interacts with gravity, is positioned inside and outside of such a unit, respectively. So, in a first approximation we can conclude that in a multicellular aquatic organism, which changes its metabolism in weightlessness, the primary receptors of gravity are located inside the cells of that organism. A special approach is necessary when free living cells, the density of which may be higher than the one of the (liquid) medium, or even cells living on a free surface are observed. In these two cases also indirect effects have to be taken into account, which will be demonstrated with the aid of the slime mold . Additionally the environment of the organisms can be changed directly and indirectly by gravity.     

Ultrastructure and cytoskeleton of Chara rhizoids in microgravity.

Gravitropic tip growth of Chara rhizoids is dependent on the presence and functional interaction between statoliths, cytoskeleton and the tip-growth-organizing complex, the Spitzenkorper. Microtubules are essential for the polar cytoplasmic zonation but are excluded from the apex and do not play a crucial role in the primary steps of gravisensing and graviresponse. Actin filaments form a dense meshwork in the subapical zone and converge into a prominent apical actin patch which is associated with the endoplasmic reticulum (ER) aggregate representing the structural center of the Spitzenkorper. The position of the statoliths is regulated by gravity and a counteracting force mediated by actomyosin. Reducing the acceleration forces in microgravity experiments causes a basipetal displacement of the statoliths. Rhizoids grow randomly in all directions. However, they express the same cell shape and cytoplasmic zonation as ground controls. The ultrastructure of the Spitzenkorper, including the aggregation of ER, the assembly of vesicles in the apex, the polar distribution of proplastids, mitochondria, dictyosomes and ER cisternae in the subapical zone is maintained. The unaltered cytoskeletal organization, growth rates and gravitropic responsiveness indicate that microgravity has no major effect on gravitropic tip-growing Chara rhizoids. However, the threshold value of gravisensitivity might be different from ground controls due to the altered position of statoliths, a possibly reduced amount of BaSO4 in statoliths and a possible adaptation of the actin cytoskeleton to microgravity conditions.     

Gravity effects on membrane processes.

Application of the Gouy-Chapman-Debye-Hückel (GCDH) theory to a model membrane in contact with electrolytes of various concentrations and composition predict density variations within an interfacial layer. Assuming that on cellular dimensions hydrodynamics can be applied (the objections are briefly discussed) two types of gravity effects can be defined, 1. convection along the surface of vertically oriented membranes and 2. surface potential variations by layer deformations at horizontally oriented membranes. Both effects should affect transport across the layer to the membrane surface and across the membrane. According to the theoretical predictions first experiments with gramicidin channels incorporated into artificial phosphatidylserine bilayer membranes show a significant difference in single channel currents in vertical and horizontal membranes. The complexity of biological membrane functions requires investigation of isolated membrane surface reactions and transport systems to study the gravisensitivity for each process separately.     

Calcium signaling in plant cells in altered gravity.

Changes in the intracellular Ca2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus-response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80th, a review highlighting the performed research and the possible significance of such Ca2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface tension --> alterations in the physicochemical properties of the membrane --> changes in membrane permeability, --> ion transport, membrane-bound enzyme activity, etc. --> metabolism rearrangements --> physiological responses. An analysis of data available on biological effects of altered gravity at the cellular level allows one to conclude that microgravity environment appears to affect cytoskeleton, carbohydrate and lipid metabolism, cell wall biogenesis via changes in enzyme activity and protein expression, with involvement of regulatory Ca2+ messenger system. Changes in Ca2+ influx/efflux and possible pathways of Ca2+ signaling in plant cell biochemical regulation in altered gravity are discussed.     

Gravitational biology and the mammalian circadian timing system.

Mammals have evolved under the influence of many selective pressures. Two of these pressures have been the static force of gravity and the daily variations in the environment due to the rotation of the earth. It is now clear that each of these pressures has led to specific adaptations which influence how organisms respond to changes in either gravity or daily time cues. However, several unpredicted responses to altered gravitational environments occur within the homeostatic and circadian control systems. These results may be particularly relevant to biological and medical issues related to spaceflight. This paper demonstrates that the homeostatic regulation of rat body temperature, heart rate, and activity become depressed following exposure to a 2 G hyperdynamic field, and recovers within 5-6 days. In addition, the circadian rhythms of these same variables exhibit a depression of rhythm amplitude; however, recovery required a minimum of 7 days.     

The expression of a circadian rhythm in two strains of Chlamydomonas reinhardii in space.

During the D1 mission the endogenous circadian rhythm of the photoaccumulation response persisted in two strains of Chlamydomonas. The amplitude was about twice as high in space as on the ground indicating that a larger fraction of cells was able to contribute to the expression of the rhythm. On the ground, cells usually enter the light cone of the illuminated area in the recording cuvette on the upper edge and leave it, due to gravity, on the lower one in a pulsating manner. This sometimes produces high frequency oscillations of light extinction on the ground. In space there were no such fluctuations; instead, cells swam into the light and stayed there harvesting more light energy for photosynthesis than did control cells. This probably enhanced the survival rate and increased the fraction of motile cells which contribute to the photoaccumulation. A more sophisticated evaluation technique allowed determination of the phase in the short period strain; it was delayed by two hours compared to the control. In an acetate free wildtype sample a rhythm with a period of about 24 hours was also detected.     

A step in embryonic axis specification in Xenopus laevis is simulated by cytoplasmic displacements elicited by gravity and centrifugal force.

Determination of the body pattern in Xenopus embryos is known to involve at least six steps. One of these steps can be experimentally simulated by inclining the fertilized egg with respect to gravity or centrifugal force (10-30 g x 4 min, directed 90 degrees to the animal-vegetal axis). In these eggs, the dorsal structures of the body axis form from the side of the egg that was uppermost in the gravitational or centrifugal field. This topography is seen even if the sperm entry point side (the prospective ventral side in control eggs) was uppermost. In addition, conjoined twin embryos form at very high frequencies in response to certain conditions of single or double centrifugation. Cytological analysis shows that the dorsal structures invariably form from the side(s) of the egg away from which vegetal cytoplasm was displaced. This is similar to the situation in the unperturbed egg, where the subcortical cytoplasm of the vegetal hemisphere rotates some 30 degrees relative to the surface, and the dorsal structures form from the side of the egg away from which the subcortical cytoplasm moved. The displacements elicited by centrifugation probably substitute for the normal displacements brought about by the subcortical rotation. These and other data suggest that the subcortical rotation is a crucial step in the process of axis determination. The subcortical rotation is an autonomous activity of the activated egg, and can displace cytoplasm against gravity. I believe that the subcortical rotation will function normally at microgravity, and I expect that overall development and axis polarity at microgravity will be normal. This will be tested in spaceflight.     

Effect of artificial electric fields on plants grown under microgravity conditions.

Ionic and structural hetorogeneity of cells, tissues, and organs of plants are associated with a spectrum of electric characteristics such as bioelectric potentials, electrical conductance, and bioelectric permeability. An important determinant for the plant function is electric properties of the cell membranes and organelles which maintain energy and substance exchange with the environment. Enzymes and other biologically active substances have a powerful charge at the molecular level. Finally, all molecules, including those of water, represent dipoles, and this determines their reactive capacity. A major determinant is the bioelectric polarity of a plant is genetically predetermined and cannot be modified. It is an intrinsic structural feature of the organism whose evolution advent was mediated by gravity. An illustrative presentation of polarity is the downward growth of the roots and upward growth of stems in the Earth's gravitation field. However, gravity is a critical, but not the sole determinant of the plant organism polarization. Potent polarizing effects are exerted by light, the electromagnetic field, moisture, and other factors. It is known that plant cultivation in an upturned position is associated with impairment of water and nutrient uptake, resulting in dyscoordination of physiological processes, growth and developmental retardation. These abnormalities were characteristic when early attempts were made to grow plants in weightlessness conditions.     

Interaction of gravity with other environmental factors in growth and development: an introduction.

The life of plants and other organisms is governed by the constant force of gravity on earth. The mechanism of graviperception, signal transduction, and gravireaction is one of the major themes in space biology. When gravity controls each step of the life cycle such as growth and development, it does not work alone but operates with the interaction of other environmental factors. In order to understand the role of gravity in regulation of the life cycle, such interactions also should be clarified. Under microgravity conditions in space, various changes are brought about in the process of growth and development. Some changes would be advantageous to organisms, but others would be unfavorable. For overcoming such disadvantages, it may be required to exploit some other environmental factors which substitute for gravity in some properties. In terrestrial plants, gravity can be replaced by light under certain conditions. The gravity-substituting factors may play a principal role in future space development.     

The biological clock of Neurospora in a microgravity environment.

The circadian rhythm of conidiation in Neurospora crassa is thought to be an endogenously derived circadian oscillation; however, several investigators have suggested that circadian rhythms may, instead, be driven by some geophysical time cue(s). An experiment was conducted on space shuttle flight STS-9 in order to test this hypothesis; during the first 7-8 cycles in space, there were several minor alterations observed in the conidiation rhythm, including an increase in the period of the oscillation, an increase in the variability of the growth rate and a diminished rhythm amplitude, which eventually damped out in 25% of the flight tubes. On day seven of flight, the tubes were exposed to light while their growth fronts were marked. Some aspect of the marking process reinstated a robust rhythm in all the tubes which continued throughout the remainder of the flight. These results from the last 86 hours of flight demonstrated that the rhythm can persist in space. Since the aberrant rhythmicity occurred prior to the marking procedure, but not after, it was hypothesized that the damping on STS-9 may have resulted from the hypergravity pulse of launch. To test this hypothesis, we conducted investigations into the effects of altered gravitational forces on conidiation. Exposure to hypergravity (via centrifugation), simulated microgravity (via the use of a clinostat) and altered orientations (via alterations in the vector of a 1 g force) were used to examine the effects of gravity upon the circadian rhythm of conidiation.     

Lithium-induced changes in gravicurvature, statocyte ultrastructure and calcium balance of pea roots.

Calcium signaling has been implicated in plant graviperception. In order to investigate the role of intracellular calcium in the process, I used lithium ions (LiCl), which suppress inositol 1,4,5-trisphosphate (IP3) cycling and signaling by inhibiting inositol-1-phosphatase. After 4 h of gravistimulation, no curvature was observed in 81% of the roots of 5-day Pisum sativum seedlings pretreated with 5 mM LiCl. Structural features of statocyte ultrastructure in these roots were the following: loss of a cellular polarity, appearance of amyloplast clusters, condensed mitochondria, local dilations in a perinuclear space, increases in a relative volume of vacuoles. The intensity of a cytochemical reaction (pyroantimonate staining which detected Ca2+ ions) was moderate: the Ca2+ pyroantimonate deposits were observed in all organelles. There were few granules of this precipitate in a hyaloplasm of the statocytes. Mitochondria and vacuoles were found to contain more granules of the precipitate compared with the controls. Additionally, Ca(2+)-ATPase activity in the statocytes of pea roots pretreated with LiCl was approximately the same as in control roots. Data obtained by using inhibitor of inositol signaling suggest that the observed effects of LiCl on root gravicurvature and ultrastructure of root statocytes were due to effects on Ca2+ homeostasis, particularly on IP3-mediated release of intracellular Ca2+ which can be inhibited by inositol depletion. The work demonstrates the key role played by second messengers (Ca2+ and IP3) in a gravity perception and response.     

Early development of fern gametophytes in microgravity.

Dormant spores of the fern Ceratopteris richardii were flown on Shuttle mission STS-93 to evaluate the effects of micro-g on their development and on their pattern of gene expression. Prior to flight the spores were sterilized and sown into one of two environments: (1) Microscope slides in a video-microscopy module; and (2) Petri dishes. All spores were then stored in darkness until use. Spore germination was initiated on orbit after exposure to light. For the spores on microscope slides, cell level changes were recorded through the clear spore coat of the spores by video microscopy. After their exposure to light, spores in petri dishes were frozen in orbit at four different time points during which on earth gravity fixes the polarity of their development. Spores were then stored frozen in Biological Research in Canister units until recovery on earth. The RNAs from these cells and from 1-g control cells were extracted and analyzed on earth after flight to assay changes in gene expression. Video microscopy results revealed that the germinated spores developed normally in microgravity, although the polarity of their development, which is guided by gravity on earth, was random in space. Differential Display-PCR analyses of RNA extracted from space-flown cells showed that there was about a 5% change in the pattern of gene expression between cells developing in micro-g compared to those developing on earth.     

Cytoskeleton and gravity at work in the establishment of dorso-ventral polarity in the egg of Xenopus laevis

The establishment of polarities during early embryogenesis is essential for normal development. Amphibian eggs are appropriate models for studies on embryonic pattern formation. The animal-vegetal axis of the axially symmetrical amphibian egg originates during oogenesis and foreshadows the main body axis of the embryo. The dorso-ventral polarity is epigenetically established before first cleavage. Recent experiments strongly suggest that in the monospermic eggs of the anuran Xenopus laevis both the cytoskeleton and gravity act in the determination of the dorso-ventral polarity. In order to test the role of gravity in this process, eggs will be fertilized under microgravity conditions during the SL-D1 flight in 1985. In a fully automatic experiment container eggs will be kept under well-defined conditions and artificially fertilized as soon as microgravity is reached; eggs and embryos at different stages will then be fixed for later examination. Back on earth the material will be analysed and we will know whether fertilization under microgravity conditions is possible. If so, the relation of the dorso-ventral axis to the former sperm entry point will be determined on the whole embryos; in addition eggs and embryos will be analysed cytologically.     

Polarity of root statocytes—Relevance for graviperception

During outgrowth of the radicle of cress ( L.) the statocytes of the root cap develop a structural polarity with the nucleus at the proximal cell pole and a complex of endoplasmic reticulum (ER) at the distal cell pole. Amyloplasts sediment upon this complex of ER. During all stages of development of the cytoskeleton (microtubules, microfilaments) is involved in positioning of the ER. The structural polarity of the statocytes develops independently of gravity, as indicated by corresponding results from fast and slow rotating clinostats and roots grown under microgravity in orbit. Disturbance of the structural polarity is possible by application of drugs, influencing microtubules and microfilaments. If, by rotation of roots on slow rotating clinostats or centrifugation, the structural polarity of the statocytes is changed, the ability of the roots to perceive gravity is affected also.     

Early development in the mouse: would it be affected by microgravity?

Gravity has been identified as a morphogenetic signal in Amphibian and bird embryonic development so it is plausible that it might be such in mammals as well. Since early mammalian development shows some apparently significant differences to these other groups, a brief summary of mouse embryogenesis will be given identifying events in which polarity is an important feature and consequently, in which gravity may be a causative factor. These include compaction and polarization during cleavage, establishment of the radial axis, the embryonic-abembryonic axis, the dorso-ventral axis, and the anterior-posterior axis, implantation, and the later rotation of the embryo. The experimental data on these morphogenetic steps will be discussed and an assessment of the possible involvement of gravity will be made.     

Gravitropism of axial organs in multicellular plants.

Gravitropism of plant organs such as roots, stems and coleoptiles can be separated into four distinct phases: 1. perception (gravity sensing), 2. transduction of a signal into the target region and 3. the response (differential growth). This last reaction is followed by a straightening of the curved organ (4.). The perception of the gravitropic stimulus upon horizontal positioning of the organ (1.) occurs via amyloplasts that sediment within the statocytes. This conclusion is supported by our finding that submerged rice coleoptiles that lack sedimentable amyloplasts show no graviresponse. The mode of signal transduction (2.) from the statocytes to the peripheral cell layers is still unknown. Differential growth (3.) consists of a cessation of cell expansion on the upper side and an enhancement of elongation on the lower side of the organ. Based on the facts that the sturdy outer epidermal wall (OEW) constitutes the growth-controlling structure of the coleoptile and that growth-related osmiophilic particles accumulate on the upper OEW, it is concluded that the differential incorporation of wall material (presumably glycoproteins) is causally involved. During gravitropic bending, electron-dense particles ('wall-loosening capacity') accumulate on the growth-inhibited upper OEW. It is proposed that the autotropic straightening response, which is in part due to an acceleration of cell elongation on the curved upper side, may be attributable to an incorporation of the accumulated particles ('release of wall-loosening capacity'). This novel mechanism of autotropic re-bending and its implications for the Cholodny-Went hypothesis are discussed.     

Calcium gradient in plant cells with polarized growth in simulated microgravity.

Plant cells characterized by apical growth, for example, root hairs and apical cells of moss protonema, are a convenient model to address the problem of gravity response mechanisms including initiation of cell polarity. The fluorescent calcium probe, chlorotetracycline, allowed us to display the calcium distribution gradient in these cells. Irradiation by red light led to a sharp decrease in the Ca2+ ion activity in cells. During clinostatting in darkness the pattern of calcium influx and distribution changes inconsiderably as compared with control; in root hairs calcium is detected mainly in their apices and bases as in control. Addition of chlorpromazine to the medium probably increases the influx and accumulation of Ca2+ ions. Under data obtained confirm speculations on the Ca2+ ion functional role for the apical growth of plant cells and may suggest the participation of gravity in redistribution or activation of ion channels, calcium channels included, in the plasmalemma.