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Gravity is a key factor for life on Earth. It is the only environmental factor that has remained constant throughout evolution, and plants use it to modulate important physiological activities; gravity removal or alteration produces substantial changes in essential functions. For root gravitropism, gravity is sensed in specialised cells, which are capable of detecting magnitudes of the g vector lower than 10-3. Then, the mechanosignal is transduced to upper zones of the root, resulting in changes in the lateral distribution of auxin and in the rate of auxin polar transport. Gravity alteration has consequences for cell growth and proliferation rates in root meristems, which are the basis of the developmental programme of a plant, in which regulation via auxin is involved. The effect is disruption of meristematic competence, i.e. the strict coordination between cell proliferation and growth, which characterises meristematic cells. This effect can be related to changes in the transport and distribution of auxin throughout the root. However, similar effects of gravity alteration have been found in plant cell cultures in vitro, in which neither specialised structures for gravity sensing and signal transduction, nor apparent gravitropism have been described. We postulate that gravity resistance, a general mechanism of cellular origin for developing rigid structures in plants capable of resisting the gravity force, could also be responsible for the changes in cell growth and proliferation parameters detected in non-specialised cells. The mechanisms of gravitropism and graviresistance are complementary, the first being mostly sensitive to the direction of the gravity vector, and the second to its magnitude. At a global molecular level, the consequence of gravity alteration is that the genome should be finely tuned to counteract a type of stress that plants have never encountered before throughout evolution. Multigene families and redundant genes present an advantage in that

Research in microgravity is indispensable to disclose the impact of gravity on biological processes and organisms.However, research in the near-Earth orbit is severely constrained by the limited number of flight opportunities.Ground-based simulators of microgravity are valuable tools for preparing spaceflight experiments, but they also facilitate stand-alone studies and thus provide additional and cost-efficient platforms for gravitational research. The various microgravity simulators that are frequently used by gravitational biologists are based on different physical principles. This comparative study gives an overview of the most frequently used microgravity simulators and demonstrates their individual capacities and limitations. The range of applicability of the various ground-based microgravity simulators for biological specimens was carefully evaluated by using organisms that have been studied extensively under the conditions of real microgravity in space. In addition, current heterogeneous terminology is discussed critically, and recommendations are given for appropriate selection of adequate simulators and consistent use of nomenclature. Key Words: 2-D clinostat—3-D clinostat—Gravity—Magnetic levitation—Random positioning machine—Simulated microgravity—Space biology.

Background Cell growth and cell proliferation are intimately linked in the presence of Earth’s gravity, but are decoupled under the microgravity conditions present in orbiting spacecraft. New technologies to simulate microgravity conditions for long-duration experiments, with stable environmental conditions, in Earth-based laboratories are required to further our understanding of the effect of extraterrestrial conditions on the growth, development and health of living matter. Results We studied the response of transgenic seedlings of Arabidopsis thaliana, containing either the CycB1-GUS proliferation marker or the DR5-GUS auxin-mediated growth marker, to diamagnetic levitation in the bore of a superconducting solenoid magnet. As a control, a second set of seedlings were exposed to a strong magnetic field, but not to levitation forces. A third set was exposed to a strong field and simulated hypergravity (2 g). Cell proliferation and cell growth cytological parameters were measured for each set of seedlings. Nucleolin immunodetection was used as a marker of cell growth. Collectively, the data indicate that these two fundamental cellular processes are decoupled in root meristems, as in microgravity: cell proliferation was enhanced whereas cell growth markers were depleted. These results also demonstrated delocalisation of auxin signalling in the root tip despite the fact that levitation of the seedling as a whole does not prevent the sedimentation of statoliths in the root cells. Conclusions In our model system, we found that diamagnetic levitation led to changes that are very similar to those caused by real- [e.g. on board the International Space Station (ISS)] or mechanically-simulated microgravity [e.g. using a Random Positioning Machine (RPM)]. These changes decoupled meristematic cell proliferation from ribosome biogenesis, and altered auxin polar transport.

Background Previous experiments have shown that the reduced gravity aboard the International Space Station (ISS) causes important alterations in Drosophila gene expression. These changes were shown to be intimately linked to environmental space-flight related constraints. Results Here, we use an array of different techniques for ground-based simulation of microgravity effects to assess the effect of suboptimal environmental conditions on the gene expression of Drosophila in reduced gravity. A global and integrative analysis, using “gene expression dynamics inspector” (GEDI) self-organizing maps, reveals different degrees in the responses of the transcriptome when using different environmental conditions or microgravity/hypergravity simulation devices. Although the genes that are affected are different in each simulation technique, we find that the same gene ontology groups, including at least one large multigene family related with behavior, stress response or organogenesis, are over represented in each case. Conclusions These results suggest that the transcriptome as a whole can be finely tuned to gravity force. In optimum environmental conditions, the alteration of gravity has only mild effects on gene expression but when environmental conditions are far from optimal, the gene expression must be tuned greatly and effects become more robust, probably linked to the lack of experience of organisms exposed to evolutionary novel environments such as a gravitational free one.

Background Many biological systems respond to the presence or absence of gravity. Since experiments performed in space are expensive and can only be undertaken infrequently, Earth-based simulation techniques are used to investigate the biological response to weightlessness. A high gradient magnetic field can be used to levitate a biological organism so that its net weight is zero. Results We have used a superconducting magnet to assess the effect of diamagnetic levitation on the fruit fly D. melanogaster in levitation experiments that proceeded for up to 22 consecutive days. We have compared the results with those of similar experiments performed in another paradigm for microgravity simulation, the Random Positioning Machine (RPM). We observed a delay in the development of the fruit flies from embryo to adult. Microarray analysis indicated changes in overall gene expression of imagoes that developed from larvae under diamagnetic levitation, and also under simulated hypergravity conditions. Significant changes were observed in the expression of immune-, stress-, and temperature-response genes. For example, several heat shock proteins were affected. We also found that a strong magnetic field, of 16.5 Tesla, had a significant effect on the expression of these genes, independent of the effects associated with magnetically-induced levitation and hypergravity. Conclusions Diamagnetic levitation can be used to simulate an altered effective gravity environment in which gene expression is tuned differentially in diverse Drosophila melanogaster populations including those of different age and gender. Exposure to the magnetic field per se induced similar, but weaker, changes in gene expression.

[EN] Gravity, a constant component of the terrestrial environment, affects numerous functions of living beings. Plant growth and development are vital functions which are founded on cellular processes such as cell proliferation and growth that occur in meristematic tissues, where "meristematic competence" consists of the strict coupling between the proliferative state of the cell and its growing capability. In previous studies, carried out in the International Space Station (ISS) and in a microgravity simulator on ground, our group has shown that the absence of gravity is capable of decoupling cell growth and proliferation in the root meristem. In order to consolidate and extend these previous studies, we have used a new device for simulation of altered gravity, based on the physical phenomenon of magnetic levitation and we have approached for the first time the effects of hypergravity on these biological processes, using both the Magnetic Levitator and the Large Diameter Centrifuge (LDC). Studies on the cell proliferation rate, nucleolar ultrastructure and the levels of nucleolar proteins, performed on samples grown under microgravity (0g*), confirmed the decoupling of cell proliferation and cell growth. Expression of the cyclin B1 gene, a marker of the G2/M transition in the cell cycle, evaluated by GUS staining on transgenic plants, decreased in 0g* despite the increase of the cell proliferation rate. Regarding hypergravity, moderate alterations recorded in these conditions in the two devices utilized indicate that the stress produced is weaker than under microgravity. Otherwise, since the sole influence of a high magnetic field was capable of altering plant growth and development, the effects of altered gravity detected in the Magnetic Levitator could have been somewhat masked by the magnetic field effects., In conditions of simulated microgravity, as well as under the influence of a high magnetic field, inhibition of the auxin polar transport was observed, according to the GUS staining pattern of the root of transgenic seedlings in which it was possible to visualize the expression of an auxin responsive gene. As a consequence, auxin was accumulated in the root tip and this could explain the higher proliferation rate in root meristematic cells in these conditions. However, hypergravity did not alter the auxin polar transport. With the purpose of testing whether cells not specialized in gravity perception respond to gravity alteration or not, and also for allowing the use of molecular techniques highly demanding in terms of biomass, we have used solid in vitro cell cultures, as a homogeneous proliferating cellular material. Changes detected either in gene expression and in the protein pattern at the level of the whole genome (studies based on microarrays and in bi-dimensional protein gels), or in specific genes (qPCR) and proteins (Westernblot), as well as the ultrastructural parameters of the nucleolus of cultured cells, reveal alterations in cell growth and proliferation which can be compared to those found in seedlings, indicating that at least a significant part of the response to altered gravity does not depend on specialized cells containing mechanoreceptors. On cell cultures we verified the alteration of the relative proportion of cell cycle phases (and probably also of the duration of the cycle), the change in expression of numerous genes acting as regulators in cell cycle checkpoints of phase transitions, and the deregulation of ribosome biogenesis by means of the alteration of nucleolar structure and of transcriptional and post-translational changes of key proteins of this process, such as nucleolin and fibrillarin. The intensity of alterations caused by the gravitational stress could be weakened by improving the seedling culture conditions with an increase of, [ES] La gravedad es un componente constante del medio ambiente en la Tierra que condiciona numerosas funciones de los seres vivos. El crecimiento y el desarrollo de las plantas son funciones vitales que se fundamentan en procesos celulares como la proliferación y el crecimiento celular que ocurren en los tejidos meristemáticos, donde la "competencia meristemática" consiste en el estricto acoplamiento entre el estado proliferativo de las células y su capacidad de crecimiento. En estudios previos, realizados en la Estación Espacial Internacional (ISS) y en un simulador de microgravedad en Tierra, nuestro grupo ha mostrado que la ausencia de gravedad es capaz de desacoplar el crecimiento y la proliferación celular en el meristemo de la raíz. Para consolidar, extender y ampliar estos estudios previos hemos utilizado un nuevo dispositivo simulador de la gravedad alterada basado en el fenómeno físico de la levitación magnética y hemos abordado por primera vez los efectos de la hipergravedad sobre estos procesos biológicos, tanto en el Levitador magnético como en la Centrífuga de Gran Diámetro (LDC). Los estudios sobre la tasa de proliferación celular, la ultraestructura nucleolar y los niveles de proteínas nucleolares, realizados sobre muestras en microgravedad (0g*), confirmaron el desacople entre la proliferación y el crecimiento celular. La expresión de la ciclina B1, gen marcador de la transición G2/M del ciclo celular, evaluada mediante la tinción GUS en plantas transgénicas, disminuyó en 0g* a pesar del aumento en la tasa de división celular. En cuanto a la hipergravedad, las moderadas alteraciones detectadas en estas condiciones en cualquiera de los dos dispositivos utilizados indican que produce un estrés menos intenso para las plantas que la microgravedad. Por otra parte, la influencia de un campo magnético de alta intensidad alteró por sí sola el crecimiento y desarrollo de la planta, pudiendo enmascarar los efectos de la alteración de la gravedad detectados en, En condiciones de microgravedad simulada, y también por efecto de un campo magnético elevado, se observó la inhibición del transporte polar de auxinas según el patrón de tinción GUS de la raíz de plántulas transgénicas en las que puede visualizarse la expresión de un gen inducible por estas hormonas. La consecuencia fue la acumulación de auxinas en la punta de la raíz, que podría ser responsable de la mayor proliferación celular en el meristemo radicular en estas condiciones. Sin embargo, la hipergravedad no alteró el transporte polar de auxinas. Con objeto de comprobar si existe respuesta a la alteración de la gravedad por parte de células no especializadas en su percepción, y también para permitir la realización de técnicas moleculares que demandan más biomasa, hemos utilizado cultivos celulares sólidos como un material celular homogéneo en proliferación. Los cambios detectados en la expresión génica y en el patrón de proteínas a nivel de genoma completo (estudios basados en microarrays y en geles bidimensionales de proteínas) o de genes (PCRq) y proteínas específicas (Western-blot), así como los parámetros ultraestructurales del nucleolo en los cultivos, revelan alteraciones del crecimiento y la proliferación celular comparables a las encontradas en plántulas, indicando que al menos una parte significativa de la respuesta a la gravedad alterada es independiente de las células especializadas que contienen mecanoreceptores. En los cultivos celulares se verificó la alteración de las proporciones de las distintas fases del ciclo celular (y probablemente de la duración del ciclo), el cambio en la expresión de numerosos genes reguladores que intervienen en los puntos de chequeo (checkpoints) de las transiciones entre fases, y también la desregulación de la biogénesis de los ribosomas, mediante la alteración de la estructura nucleolar y los cambios transcripcionales y post-traduccionales en proteínas claves del proceso, como nucleolina y fibrilarina. La intensidad de la

In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre–rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes.Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

Genome-wide transcriptional profiling shows that reducing gravity levels during Drosophila metamorphosis in the International Space Station (ISS) causes important alterations in gene expression: a large set of differentially expressed genes (DEGs) are observed compared to 1g controls. However, the preparation procedures for spaceflight and the nonideal environmental conditions on board the ISS subject the organisms to additional environmental stresses that demonstrably affect gene expression. Simulated microgravity experiments performed on the ground, under ideal conditions for the flies, using the random position machine (RPM), show much more subtle effects on gene expression. However, when the ground experiments are repeated under conditions designed to reproduce the additional environmental stresses imposed by spaceflight procedures, 79% of the DEGs detected in the ISS are reproduced by the RPM experiment. Gene ontology analysis of them shows they are genes that affect respiratory activity, developmental processes and stress-related changes. Here, we analyse the effects of microgravity on gene expression in relation to the environmental stresses imposed by spaceflight. Analysis using 'gene expression dynamics inspector' (GEDI) self-organizing maps reveals a subtle response of the transcriptome to microgravity. Remarkably, hypergravity simulation induces similar response of the transcriptome, but in the opposite direction, i.e. the genes promoted under microgravity are usually suppressed under hypergravity. These results suggest that the transcriptome is finely tuned to normal gravity and that microgravity, together with environmental constraints associated with space experiments, can have profound effects on gene expression. © 2010 Blackwell Publishing Ltd.

Seeds of Arabidopsis thaliana were sent to space and germinated in orbit. Seedlings grew for 4 d and were then fixed in-flight with paraformaldehyde. The experiment was replicated on the ground in a Random Positioning Machine, an effective simulator of microgravity. In addition, samples from a different space experiment, processed in a similar way but fixed in glutaraldehyde, including a control flight experiment in a 1g centrifuge, were also used. In all cases, comparisons were performed with ground controls at 1g. Seedlings grown in microgravity were significantly longer than the ground 1g controls. The cortical root meristematic cells were analyzed to investigate the alterations in cell proliferation and cell growth. Proliferation rate was quantified by counting the number of cells per millimeter in the specific cell files, and was found to be higher in microgravity-grown samples than in the control 1g. Cell growth was appraised through the rate of ribosome biogenesis, assessed by morphological and morphometrical parameters of the nucleolus and by the levels of the nucleolar protein nucleolin. All these parameters showed a depletion of the rate of ribosome production in microgravity-grown samples versus samples grown at 1g. The results show that growth in microgravity induces alterations in essential cellular functions. Cell growth and proliferation, which are strictly associated functions under normal ground conditions, appeared divergent after gravity modification; proliferation was enhanced, whereas growth was depleted. We suggest that the cause of these changes could be an alteration in the cell cycle regulation, at the levels of checkpoints regulating cell cycle progression, leading to a shortened G2 period. © 2009 Elsevier GmbH.

Some years ago Prof. Marco and his team discovered that Drosophila show noticeably different behaviour as well as accelerated aging in Space (1-4). It was also proven that by simulating these conditions on Earth, it was possible to replicate these phenomena (2,5,6). In order to demonstrate how closely connected the enhanced motility and the accelerated aging are, five Drosophila strains have been selected, each behaving differentially under altered gravity conditions. Three of these strains have been selected some years ago and reselected over the last six months according to their gravitropic response in a vertically-oriented ten-exit maze for 25 generations. The behaviour of flies at 1g resembles an exaggerated escape response to gravity, a neutral behaviour to gravity and a negative (attractive) behaviour to gravity, respectively. The other two strains have been selected over several decades according to the longevity of their parents to experimentally produce a short and a long-life strain. The five selected strains are scored against a gravitropic index, median survival and median motility in 1g conditions to evaluate their usability as microgravity research tools. Preliminary experiments have been performed at ESTEC/DESC altered gravity facilities (hypergravity and simulated microgravity). The establishment of these strains will provide science with an exciting new tool in Space Biology and their future utilization on space missions or long term stays in the International Space Station is promising.

The study of the modifications induced by altered gravity in functions of plant cells is a valuable tool for the objective of the survival of terrestrial organisms in conditions different from those of the Earth. We have used the system “cell proliferation–ribosome biogenesis”, two inter-related essential cellular processes, with the purpose of studying these modifications. Arabidopsis seedlings belonging to a transformed line containing the reporter gene GUS under the control of the promoter of the cyclin gene CYCB1, a cell cycle regulator, were grown in a Random Positioning Machine, a device known to accurately simulate microgravity. Samples were taken at 2, 4 and 8 days after germination and subjected to biometrical analysis and cellular morphometrical, ultrastructural and immunocytochemical studies in order to know the rates of cell proliferation and ribosome biogenesis, plus the estimation of the expression of the cyclin gene, as an indication of the state of cell cycle regulation. Our results show that cells divide more in simulated microgravity in a Random Positioning Machine than in control gravity, but the cell cycle appears significantly altered as early as 2 days after germination. Furthermore, higher proliferation is not accompanied by an increase in ribosome synthesis, as is the rule on Earth, but the functional markers of this process appear depleted in simulated microgravity-grown samples. Therefore, the alteration of the gravitational environmental conditions results in a considerable stress for plant cells, including those not specialized in gravity perception

In the GENE experiment performed during an 11-day Soyuz Mission to the International Space Station (ISS), we intended to determine if microgravity affects Drosophila metamorphosis processes. Control experiments were performed including a 1g ground control parallel to the ISS flight samples and a Random Position Machine microgravity simulated control. A preliminary analysis of the results indicates that five hundred to one thousand genes change their expression profiles depending on the cut-off levels selected. Especially affected among them are the mitochondrial ones (an example with the respiratory chain is presented). We show here that there is a synergic effect of the constraints introduced to meet the requirements of the space experiment (mainly, a cold step and the use of hermetically closed Type-I containers). The cold transport step to the launch site was introduced to slow down the pupal development. The hermetically closed Type I containers were required to ensure the containment of the fixative (acetone) in the experiment. As shown here, the oxygen concentration inside the container was not optimal but fully compatible with pupal development. It is highly likely that such combined environmental effects will become a common finding in these types of studies as they become more complicated and extensive. They could open the way to understand how the gene expression patterns and the actual phenotypes can adjust to the environment. These findings indicate the importance of a vigorous ground based program in support of real microgravity experiments. Only then we can utilize the ISS in order to understand the consequences of the modified environment in outer space on living organisms

En el presente trabajo se han realizado nuevos avances en el conocimiento de la organización funcional del proceso de biogénesis de ribosomas en el nucleolo de células proliferantes de la especie vegetal modelo Arabidopsis thaliana, mediante la caracterización de una proteína que desempeña un papel clave en varias etapas del proceso, la proteína homóloga de la nucleolina. Además, se ha utilizado este modelo biológico y, en concreto, el sistema funcional proliferación/ciclo celular-crecimiento celular/biogénesis de ribosomas para identificar alteraciones inducidas por el ambiente de microgravedad, real o simulada, en plántulas germinadas y crecidas en estas condiciones. A partir de la existencia en A. thaliana de dos genes de proteínas homólogas a la nucleolina AtNUC-L1 y AtNUC-L2, de los que sólo AtNUC-L1 se expresa en condiciones normales, se localizó la distribución de la proteína en el nucleolo a nivel ultraestructural. En un mutante incapaz de expresar AtNUC-L1 se observó la expresión de AtNUC-L2, cuyo producto también se localizó en el nucleolo, sugiriendo que AtNUC-L2 complementa la función de AtNUC-L1. Sin embargo, la complementación no es total porque en los mutantes Atnuc-L1 se observó una profunda desorganización estructural del nucleolo, con la aparición de estructuras que manifiestan errores en el procesamiento del pre-rRNA. Además, se detectaron desequilibrios en la transcripción del rDNA y en el procesamiento temprano del transcrito. Por otra parte, las alteraciones celulares inducidas por la ausencia de gravedad en el nucleolo de células proliferantes de plantas, se investigaron en el experimento “ROOT”, realizado en la Estación Espacial Internacional durante la “Misión Cervantes”, el cual consistió en la germinación de semillas de Arabidopsis y crecimiento en el espacio durante cuatro días; se realizaron controles en Tierra (1 g) y en microgravedad simulada, en una Random Positioning Machine (RPM), en las mismas condiciones. Más adelante se llevó a c

From the autumn of 2001 till spring of 2005 a series of six flights to the International Space Station, ISS, were conducted using the Russian Soyuz manned launcher. These flights initially known as ‘taxi-missions’, were characterized by the participation and co-funding from both the European Space Agency, ESA, and the five national delegations from France, Italy, Belgium, Spain, and the Netherlands. The national participation was reflected both in the flight of a cosmonaut/astronaut, originating from the country co-sponsoring the flight as well as in the origin of the majority of experiments and other activities carried out during these missions. In these six Soyuz missions: Andromède (October 2001), Marco Polo (April 2002), Odissea (October 2002), Cervantes (October 2003), DELTA (April 2004) and Eneide (April 2005), some more than one hundred experiments were carried out. These experiments covered the areas of basic and applied research and technology in biology, human physiology, fluid and plasma physics, material science and Earth observation. Also a significant number of education activities were part of these missions. This paper gives a complete overview of these missions, of all science, education and related activities performed. The perspectives of these activities in the light of the space exploration programs in the XXI century and some of the uncertainties and paradoxes are discussed

In an experiment conducted in the “Cervantes” Spanish Soyuz Mission, a 10-day flight to the International Space Station, Arabidopsis seeds were germinated, seedlings grew for 4 days at 22ºC, and they were fixed in flight with paraformaldehyde. A ground 1 g control experiment was replicated, and an additional experiment in simulated microgravity, using a Random Positioning Machine, was performed in the same conditions. Structural, morphometric and immunocytochemical data were compared. Glutaraldehyde-fixed seedlings similarly grown in the Space Shuttle (STS-84 Mission) were also used for ultrastructural and morphometric studies. Seedlings grown for 4 days in real or simulated weightlessness showed a longer size than the ground 1 g control. Root meristematic cells showed an enhanced proliferating rate, but ribosome biogenesis was reduced, as inferred from the nucleolar size and from the levels of the nucleolar protein nucleolin. This could be the consequence of the acceleration of the cell cycle, with shortening of its phases. Weightlessness induces stress in the plant, influencing cellular processes decisive for development and morphogenesis. This stress may endanger the plant and would require the action of compensating specialized defence mechanisms

Nucleolin is a major highly phosphorylated nucleolar protein involved in the regulation of r-chromatin condensation/expansion and rDNA transcription as well as in rRNA processing. The nucleolar protein homologous to the mammalian nucleolin and to the onion nucleolin-like protein NopA100, was detected in the nuclear soluble protein fraction, and in the nuclear matrix fractionfrom Lepidium sativum root meristematic cells, using the selective silver staining method and the cross-reaction with the anti-NopA100 antibody. In 2-DE Western blots of both nuclear fractions, the nucleolin-like protein was revealed as a smear on the level of 90 kDa extending through a certain range of pI. In both extracts obtained from seedlings germinated and grown under slow clinorotation, the extension of the pI range was shorter and the molecular weight diapason was thinner than in the 1 g control; moreover, in the nuclear matrix fraction, the spread of the pI range was separated into two clusters. The results obtained could indicate a lower phosphorylation of the protein, suggesting a decrease in the activity of L. sativum nucleolin-like protein under clinorotation

Human exploration of outer space will eventually take place. In preparation for this endeavour, it is important to establish the nature of the biological response to a prolonged exposure to the space environment. In one of the recent Soyuz Missions to serve the International Space Station (ISS), the Spanish Soyuz mission in October 2003, we exposed four groups of Drosophila male imagoes to microgravity during the almost eleven days of the Cervantes mission to study their motility behaviour. The groups were three of young flies and one of mature flies, In previous space experiments, we have shown that when imagoes are exposed to microgravity they markedly change their behaviour by increasing their motility, especially if subjected to these conditions immediately after hatching. The constraints of the current Soyuz flights made it impossible to study the early post-hatching period. A low temperature cold transport was incorporated as a possible way out of this constraint. It turned out that on top of the space flight effects, the cold treatment by itself, modifies the motility behaviour of the flies. Although the four groups increased their motility, the young flies did it in a much lower extent than the mature flies that had not been exposed to the low temperature during transportation. Nevertheless, the flies flown in the ISS are still more active than the parallel ground controls. As a consequence of the lower motility stimulation in this experiment, a likely consequence of the cold transport step, no effects on the life spans of the flown flies were detected. Together with previous results, this study confirms that high levels of motility behaviour are necessary to produce significant decreases in fly longevity.

The European Soyuz missions have been one of the main routes for conducting scientific experiments onboard the International Space Station, which is currently in the construction phase. A relatively large number of life and physical sciences experiments as well as technology demonstrations have been carried out during these missions. Included among these experiments are the Gene experiment during the Spanish “Cervantes” Soyuz mission and the ICE-1st experiment during the Dutch “Delta” mission. In both experiments, full genome microarray analyses were carried out on RNA extracted from whole animals recovered from the flight. These experiments indicated relatively large scale changes in gene expression levels in response to spaceflight for two popular model systems, Drosophila melanogaster (Gene) and Caenorabditis elegans (ICE-1st). Here we report a comparative analysis of results from these two experiments. Finding orthologous genes between the fruit fly and the nematode was far from straightforward, reducing the number of genes that we could compare to roughly 20% of the full comparative genome. Within this sub-set of the data (2286 genes), only six genes were found to display identical changes between species (decreased) while 1809 genes displayed no change in either species. Future experiments using ground simulation techniques will allow producing a better, more comprehensive picture of the putative set of genes affected in multicellular organisms by changes in gravity and getting a deeper understanding of how animals respond and adapt to spaceflight.

The nucleolus is the transcription site of rRNA genes as well as the site of processing and initial packaging of their transcripts. The plant nucleolin homologue NopA100 is involved in the regulation of r-chromatin condensation/expansion and rDNA transcription as well as in rRNA processing. We have investigated with immunogold electron microscopy the location of nucleolar DNA and NopA100 in cress root meristematic cells grown under slow horizontal clinorotation, reproducing an important feature of microgravity, namely the absence of an orienting action of a gravity vector, compared to control conditions. We demonstrate redistribution of both rDNA and NopA100 in nucleolar subcomponents induced by clinorotation. Ribosomal DNA concentrated predominantly in fibrillar centers in the form of condensed r-chromatin inclusions and internal non condensed fibrils, redistributing from the dense fibrillar component and the transition zone between fibrillar centers and the dense fibrillar component, recognized as the loci of rDNA transcription. The content of NopA100 was much higher in the inner space of fibrillar centers and reduced in the dense fibrillar component as compared to the control. Based on these data, an effect of slow horizontal clinorotation in lowering the level of rDNA transcription as well as rRNA processing is suggested. © 2005 COSPAR. Published by Elsevier Ltd. All rights reserved.

Cell proliferation is a crucial cellular process which influences development. In plants, meristems are formed by actively proliferating cells, in which the main expression of proliferation is the existence of a cell division cycle. Many cell activities are influenced by the cell proliferation status and cell cycle progression, among them ribosome biogenesis, which is morphologically expressed as the nucleolus. The connection is established through nucleolar proteins, which regulate the synthesis and processing of preribosomal precursors and, at the same time, are targets of various cell cycle regulators, such as certain kinases. Nucleolin is one of these nucleolar proteins, whose level increases with cell proliferation and depends on the cell cycle stages. Not only the levels, but also other important features of the protein, such as its distribution in situ in the nucleolus, its phosphorylation and its physiological degradation, depend on these parameters. Furthermore, since the nucleolar structure is highly sensitive to functional variations, distinct nucleolar structures, regarding the nucleolar size and the distribution of nucleolar subcomponents, have been defined for each period of the cell cycle, using synchronized cells. In addition to increasing our knowledge of cellular physiology, these relationships can be used to mark the proliferative state of the cell and the periods of cell cycle.

The importance of preservation of samples during space experiments is discussed. Preservation is important because of the atypical long storage times before and after the experiment without surveillance by the investigators. The preservation requirements are highly variable, depending on the quality of the samples and the objectives of the research. Dedicated procedures should be developed for every case and biological model system.

Seeds of Arabidopsis thaliana were sent to the International Space Station in the “Cervantes Mission” (Spanish Soyuz Mission). Seed germination was initiated in flight by supplying culture medium. Seedlings were grown for 4 days at 22 °C, and growth was stopped by the addition of paraformaldehyde fixative. Once back on the ground, samples were immediately processed for microscopy. A ground control experiment was simultaneously replicated. Glutaraldehyde-fixed root cells from seedlings grown in the Biorack on board of the Space Shuttle (STS-84 Mission) in similar conditions were also ultrastructurally examined. The length of seedlings grown at 1g was conspicuously shorter than parallel samples grown under microgravity. We examined the morphology of the root meristematic cells, with a focus on their nucleoli in the cortex and stele. In general, root cortical cells proliferate at a higher rate and their nucleoli are more active than those of stele cells. While the stele showed longer cells with larger nucleoli in the flight samples, cortical cells from space-grown seedlings were shorter, more numerous and more densely packed than ground controls. However, nucleoli were smaller and less active in fast proliferating flight cells than in the ground controls. This reduced level of ribosome synthesis in the flight samples is probably the result of an accelerated cell cycle. An altered rate of cell proliferation may be detrimental for the plant and could be the reason for the reported smaller size of older space-grown seedlings. Finally, two-dimensional protein electrophoresis showed noticeable differences between space samples and ground controls

[EN] In this worfc wc have studicd the phytico-chcmical fea t u r o of thc nuclcolar protctm NopAlOO and NopAM. which are majar protctns in thc soluble nuclear fraetton tn proliferating celK. although they al so display significan! levéis in thc most involuhlc fraclion (nuclear matrix). The lepéis of these proteins « e r e higher in thc 0 2 phasc (han in any other nhase of the cell cyc'le. Their nuclcolar diMnhutmn. studied by light and electrón microsopy. was uneven. mainly appcanng in thc fibnllar components of thc nucleolus. Western blot analysis with anu-NopAlOO antibody revealed a major band at thc Icvcl of 100 kDa and .IIMI ihe presence of a seríes of mmor and smallcr bands. which are thc rcsult of thc proteolytic maturation of NopAlOO. This fací has been cvidcnccd afler treatment with leupeptin. a general proteasc inhibitor: a rclationship between thc mercase in thc activity of nucleolus. the activity of NopAlOO. thc cell eyele progression, and the proteolytic maturation of the protein. has been postulated. Varíations of NopAlOO through thc cell eyele are accompamed by a structural reorganizatton of thc nucleolus. which is expressed as quantitative changes in thc rclati\c contríbution of thc difieren) nucleolar subcomponents to the uholc nuclcolar volumen Conscquenily, we have designcd a structural model of nucleolar organi/ation for cach onc of the intcrphasc períods of proliferating cclls KM tu res of NopAlOO show intcrcsiing analogics wiih thosc of mammalian nucieolin, on its p.in. \ o p A 6 4 appcars lo be a stabic fragment of thc NopAlOO. "Ruis, both protaiM should be ineluded in thc protein group known as "nucleolin-like" protcmv This fact, together with a dctailed analysis of thc amino acid vcquenecs of the rest of "nuclcolin-like" proteins in other organisms. confirms thc high evolutionary conservation of nucleolin and its homologous proteins. which is thc consequence of the highly relevan! functional roleplayed by this protein in thc life of the cell, [ES] En este trabajo se han analizado las propiedades (luco-químicas de las proteínas núcleo lares NopA 100 y NopA64. que son mayontanas en la fracción soluble de los núcleos de células en proliferación, aunque también presentan nivele* significanvos en la fracción mil iruoluble (matriz nuclear). Las proteínas mostraron un incremento en la fase G2 del ciclo celular. Mediante inmunocitoquimica a nivel de microscopía óptica y electrónica, se observó una divinhucion desigual dentro del nucléolo, apareciendo principalmente en los componentes fibnlarcs I n análisis mediante Wet$tm blot con anticuerpo anti-NopA 100 mostró una banda principal a 1ÍX) kDa junto con una serie de proteínas relacionadas con ella, de menor tamaño Este resultado sugiere que la NopA 100 sufre un proceso fisiológico de maduración proteolitica. lo que fue confirmado mediante un tratamiento con un inhibidor de proteasas general, la Icupeptina. Se observó una relación entre el incremento de la actividad del nucléolo y la actividad de la NopA 100, el progreso en el ciclo celular y la maduración de la proteina. Hemos demostrado que las variaciones de la NopA 100 durante la progresión del ciclo celular van acompañadas de una reorganización estructural del nucléolo, que es expresada en cambios cuantitativo* de la contribución relativa de los diferentes subcomponentes nuclcolarcs al volumen total del nucléolo. Coasiguientemcnic. hemos diseñado un modelo estructural de la organización nucleolar para cada uno de los períodos de la m1crfa.se El conjunto de sus propiedades identifica a la NopA UKi como una proteina homologa a la nueleolina de mamíferos > a la NopA64 como un fragmento estable de la NopA 100 I sic hecho, junto con el análisis detallado de las secuencias de aminoácidos del resto de "proteínas semejantes a nueleolina" en otros organismos, confirma la alta conservación evolutiva que presenta, la cual es la consecuencia del papel funcional extraordinariamente relevante de esta proteína en la vida de

[EN] In polytene cells, such as those of salivary glands from Chironomus larvae, DNA is amplified forming the polytene chromosomes, as a result of the lateral amplification of the sister chromatids that remain associated throughout successive rounds of replication. Ribosomal genes result, thus, amplified, and their activity gives rise to the formation of a giant nucleolus, which may be called Polytene Cell Nucleolus (PCN), measuring as much as 10-15 µm in diameter. We have used the advantages of such this biological model to study some basic aspects of PCN organization and to contribute to clarify pending problems on the functional organization of the nucleolus. At the ultrastructural level, the PCN is only formed by dense fibrillar component (DFC) and granular component (GC). Fibrillar centers (FCs) have not been identified in these nucleoli. Nevertheless, FCs are present in non-polytene nervous cells of the same individual. The absence of FCs in the PCN could be related to the levels and distribution of structural proteins in the nucleolus, which could be a specific feature of each cellular type. Alternatively, this absence could be the result of the high activity of the PCN. However, FCs did not appear indeed in the PCN after a treatment with cycloheximide (CHM) of the salivary glands, which inhibits nucleolar transcription. The treatment with this drug produced a reorganization or segregation of the structural components of PCN, so that the DFC appeared located in the zones nearer to the chromosome, and the GC in the periphery. By means of both confocal and electron microscope we have studied the organization of DNA within the PCN. Intranucleolar DNA appeared spread in the DFC, in the form of decondensed chromatin. The organization of DNA is highly dynamic, since, after CHM treatment, chromatin became condensed and retracted. The use of in situ hybridization with fluorescent markers (FISH) has made possible the specific localization of rDNA, whose distribution a, Furthermore, by means of an experiment of in vitro transcription, we have located the sites of the DFC where the transcription takes place. The in situ pattern of the nucleolar transcription also appeared organized in foci. These foci have the same functional significance as FCs in cells having them, so that the absence of FCs does not mean a change in the model of structural organization of rDNA and nucleolar transcription. Otherwise, immunodetection of fibrillarin and nucleolin, two proteins involved in pre-rRNA processing, has been carried out. Both proteins were located in the DFC, but, when colocalization under the confocal microscope was performed, we observed zones where only fibrillarin was located, zones where only nucleolin was present and zones containing the two proteins simultaneously. Although, ultrastructurally, the DFC appears as a homogenous component, its molecular composition and its functional organization demonstrate that it is heterogeneous. Treatment with the transcription inhibitor CHM, also caused the appearance in active transcription zones, in chromosomes as well as free in the nucleoplasm, of some spherical structures, fibrillar in nature, called droplets . Immunocytochemical experiments allowed us to locate in them nucleolar proteins such as fibrillarin and nucleolin, but they did not contain any signal of DNA. The origin of these structures could be a segregation and microfragmentation of DFC, leading to the formation of nucleoplasmic aggregates containing nucleolar proteins, which cannot be transported to the nucleolus when rRNA gene transcription was altered. Finally, we have explored the problem of the implication of nucleolus in cellular functions other than ribosome biosynthesis. In particular, we have studied the nucleolar localization of reverse transcriptase (rt), a protein having no relationship to ribosome biogenesis, by means of an antibody raised against a recombinant protein containing phylogenetically conserved motifs of t, [ES] En células politenizadas, como las de las glándulas salivales de larvas de Chironomus, el DNA está amplificado formando los cromosomas politénicos, como resultado de la amplificación lateral de las cromátidas hermanas que se mantienen unidas a lo largo de sucesivas rondas de replicación. Los genes ribosómicos resultan también amplificados y su actividad da lugar a la formación de un nucleolo gigante, el llamado Nucleolo de Células Politénicas (NCP), que llega a alcanzar un diámetro de 10-15 µm. Hemos utilizado las ventajas que ofrece este modelo biológico para estudiar algunos aspectos básicos de la organización del NCP, contribuyendo a esclarecer problemas pendientes sobre la organización funcional del nucleolo. A nivel ultraestructural, el NCP apareció compuesto únicamente por componente fibrilar denso (CFD) y componente granular (CG). No han sido identificados en estos nucleolos los centros fibrilares (CFs). Sin embargo, los CFs sí están presentes en los nucleolos de células nerviosas no politénicas del mismo individuo. La inexistencia de CFs en el NCP podría estar relacionada con los niveles y distribución de las proteínas estructurales del nucleolo, que podría ser una característica específica de tipo celular. Alternativamente, podría ser consecuencia de la elevada actividad del NCP. Sin embargo, los CFs tampoco aparecieron en el NCP tras el tratamiento con cicloheximida (CHM), que inhibe la transcripción nucleolar. El tratamiento con esta droga produjo una reorganización o segregación en los componentes estructurales del NCP, de modo que el CFD se sitúa en las zonas cercanas al cromosoma y el CG en la periferia. Mediante el microscopio confocal y al microscopio electrónico hemos estudiado la distribución del DNA en el NCP. El DNA intranucleolar se observó extendido en el CFD, en forma de cromatina descondensada. La organización del DNA es muy dinámica, ya que, tras el tratamiento con CHM, se produce una fuerte condensación y retracción de la cromatina nuc, implicadas en el procesamiento del pre-rRNA. Ambas proteínas se localizaron en el CFD, pero en la colocalización de ambas, observamos que hay zonas donde solamente se localizó la fibrilarina, zonas donde sólo se localizó la nucleolina y zonas donde se localizaron las dos proteínas simultáneamente. Aunque ultraestructuralmente el CFD aparece como un componente homogéneo, su composición molecular y su organización funcional demuestran que es heterogéneo. El tratamiento con CHM, inhibidor de la transcripción, provocó la aparición en zonas de transcripción activas, tanto en el cromosoma como libres en el nucleoplasma, de unas estructuras esféricas de naturaleza fibrilar, denominadas droplets. Los experimentos inmunocitoquímicos permitieron localizar en ellos proteínas nucleolares como la fibrilarina y la nucleolina, pero no apareció señal del DNA. El origen de estas estructuras podría ser una segregación y microfragmentación del CFD, formándose agregaciones nucleoplásmicas de las proteínas nucleolares, las cuales no pueden ser transportadas al nucleolo cuando la transcripción de los genes del rRNA está alterada. Finalmente, se ha abordado el problema de la implicación del nucleolo en otras funciones celulares distintas de la síntesis de ribosomas. En concreto, hemos estudiado la localización nucleolar de una proteína no relacionada con la biogénesis de ribosomas, la transcriptasa reversa (rt), mediante un anticuerpo obtenido contra una proteína recombinante que contiene motivos de esta enzima filogenéticamente conservados. La presencia de esta enzima está relacionada con la estrategia del mantenimiento de los telómeros en Dípteros Sorprendentemente, la transcriptasa reversa apareció localizada en el NCP, posiblemente en relación con los mecanismos reguladores de la actividad de la enzima. A partir de todos nuestros resultados podemos concluir que, a pesar de que el organizador nucleolar (NOR) se origina en el NCP como la adición lateral de múltiples copias del NOR origi

The International Space Station is currently in an advanced state of construction. The possibility of conducting in the Space environment longer term experiments than previously possible is becoming a reality, but there are several obstacles in this undertaking, some purely based on the availability of suitable hardware, some more technical. To eliminate some of them and make possible investigations using one of the more important model systems, Drosophila melanogaster, we have developed an automatic device to grow and carry out biological experiments in Space with Drosophila. The Experimental Support Equipment Item 1 (ESE–1) is an automated culture system for multigenerational experiments with insects developed for the ESA International Space Station facilities. The concept could be generalized for preparing culture units for small invertebrates.