The kinetics of translocation and cellular quantity of protein kinase C in human leukocytes are modified during spaceflight.

Protein kinase C (PKC) is a family of serine/threonine kinases that play an important role in mediating intracellular signal transduction in eukaryotes. U937 cells were exposed to microgravity during a space shuttle flight and stimulated with a radiolabeled phorbol ester ([3H]PDBu) to both specifically label and activate translocation of PKC from the cytosol to the particulate fraction of the cell. Although significant translocation of PKC occurred at all g levels, the kinetics of translocation in flight were significantly different from those on the ground. In addition, the total quantity of [3H]PDBu binding PKC was increased in flight compared to cells at 1 g on the ground, whereas the quantity in hypergravity (1.4 g) was decreased with respect to 1 g. Similarly, in purified human peripheral blood T cells the quantity of PKCdelta varied in inverse proportion to the g level for some experimental treatments. In addition to these novel findings, the results confirm earlier studies which showed that PKC is sensitive to changes in gravitational acceleration. The mechanisms of cellular gravisensitivity are poorly understood but the demonstrated sensitivity of PKC to this stimulus provides us with a useful means of measuring the effect of altered gravity levels on early cell activation events.

Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat).

Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post-flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/ APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity.

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.

The distribution of protein kinase C in human leukocytes is altered in microgravity.

Protein kinase C (PKC) is an ubiquitous enzyme that mediates intracellular signal transduction in eukaryotes. Jurkat and U937 cells were exposed to microgravity during a Space Shuttle flight and stimulated with a radiolabeled phorbol ester (3H-PDBu) that specifically activates and labels several PKC isoforms. Both the total amount of 3H-PDBu labeling per cell and the relative distribution of labeling between subcellular compartments were altered in microgravity compared to onboard and ground 1 g control samples. The amount of total phorbol ester labeling per cell was increased approximately twofold in microgravity samples when compared with onboard 1 g samples for both cell lines. The subcellular distribution of PKC in the cytosol and nuclear fractions appeared to be correlated with the applied acceleration. In both cell types the relative amount of phorbol ester labeling in the nuclear fraction decreased with applied acceleration, whereas the labeling in cytosolic fraction increased with g level. No significant differences were observed between labeling levels in the membrane fraction in both cell types. Interleukin-1beta synthesis by U937 cells was markedly decreased in microgravity when compared to the onboard 1 g control, suggesting that the observed alterations in PKC distribution may have functional consequences. The results may have important implications for the effect of gravity on cellular signal transduction.

Kinetics of stipe gravitropism in the mushroom fungus Coprinus cinereus under the conditions of microgravity simulation provided by clinostat treatment.

The extent of the gravitropic response, measured as the angle of the stipe apex at maximum curvature, was dependent upon the gravitational exposure time. The reaction time did not depend on exposure time, and interruption of gravitational exposure by a period of clinostating allowed the gravitational stimulus to decay. It is concluded that the gravitropic impulse is an 'all-or-nothing' signal in Coprinus cinereus, that perception and response probably occur in the same tissue regions, and that sustained exposure to the unidirectional gravity vector is necessary for the normal gravitropic response. The presentation time was found to be 7 min. Immediately after reaching curvature, stipes placed on the clinostat after various gravity exposure times 'relaxed' by 5 degrees. This relaxation suggests that gravitropic bending is a two-stage process with an initial, reversible, phase of plastic bending followed by a 'fixation' phase providing the gravitropic stimulus has been maintained.

Gravitational biology of mushrooms: a flow-chart approach to characterising processes and mechanisms.

Flow charts are presented which systematise recently published work on gravitropic responses of the mushroom stipe of Coprinus cinereus. The hypothetical model represented by the charts suggests that the meiotic division is a pivotal point in the gravitational biology of the mushroom fruit body. The unilateral gravity vector seems to be required for formation of the tissues in which meiosis normally occurs, and stipes become gravitropically competent only after onset of meiosis. The gravitropism flow-chart also indicates that two signals emanate from the upper regions of the stipe, one promotes the process of gravitropic bending, and is followed by a second signal which compensates for excess bending and adjusts the stipe apex to the vertical. Formalisation of the various observations into flow-charts, even though comparatively simple at the moment, facilitates comparison with other species and concentrates attention on aspects requiring further experimental analysis.

Kinetics of stem gravitropism in Coprinus cinereus: determination of presentation time and "dosage-response" relationships using clinostats.

The sensitivity to gravitational stimulation of excised stems of the mushroom fruit body of Coprinus cinereus was determined using clinostat rotation to remove partially-stimulated stems from the normal unidirectional gravitational field. For the strain and conditions tested, the presentation time (the minimum time of stimulation required to elicit a gravitropic reaction) was determined to be 9.6 min. This is the first time the presentation time has been determined for a fungal gravitropic response. Constructional details are given of the clinostats employed in the research and their further use is discussed.

Kinetics and mechanics of stem gravitropism in Coprinus cinereus.

Using video recordings we have completed the first kinetic analysis of mushroom stem gravitropism. The stem became gravireceptive after completion of meiosis, beginning to bend within 30 minutes of being placed horizontal. Stem bending first occurred in the apical 15% of its length, then the position of the bend moved rapidly towards the base, traversing 40% of stem length in 2.5 h. Meanwhile, the stem elongated by 25%, mostly in its upper half but also in basal regions. If the apex was pinned horizontally the stem base was elevated but overshot the vertical, often curling through more than 300 degrees. When the base was pinned to the horizontal (considered analogous to the normal situation), 90% of the initial bend was compensated as the stem brought its apex accurately upright, rarely overshooting the vertical. The apex had to be free to move for this curvature compensation to occur. Stems transferred to a clinostat after some minutes gravistimulation showed curvature which increased with the length of initial gravistimulation, indicating that continued exposure to the unilateral gravity vector was necessary for continued bending. Such gravistimulated stems which bent on the clinostat subsequently relaxed back towards their original orientation. Reaction kinetics were unaffected by submergence in water, suggesting that mechanical events do not contribute, but submerged stems bent first at the base rather than apex. In air, the gravitropic bend appeared first near the apex and then moved towards the base, suggesting basipetal movement of a signal. In water, the pattern of initial bending was changed (from apex to base) without effect on kinetics. Taken together these results suggest that bending is induced by a diffusing chemical growth factor (whose extracellular propagation is enhanced under water) which emanates from the apical zone of the stem. The apex is also responsible for regulating compensation of the bend so as to bring the tip to the vertical. The nature of this latter stimulus is unknown but it is polarized (the apex must be free to move for the compensation to occur) and it may not require reference to the unilateral gravity vector.