BACKGROUND
gravity is a universal force and affects everything in nature.
gravitational effects on biological events and functions, including the mechanism of gravity sensation in cells, remain unclear.
previous spaceflight experiments have reported the irregular formation of cytoskeletal fibers in spaceflown experimental cells.
lewis et al.
observed that the microtubule filaments extended from a poorly defined centrosome in human lymphocytes   <cit> .
gruener and hughes-fulford reported that actin reorganization responded to the gravity level and showed abnormal assembly of actin stress fibers  <cit> .
in the field of space biology, to examine the effects of microgravity on biological events in ground-based research, clinostat is often used to simulate the microgravity condition.
a three-dimensional  clinostat is an apparatus that nullifies the effect of gravity, and it has been used in substitution studies on microgravity effects  <cit> .
in a study on xenopus myocytes, 3d clinorotation caused disorganization, condensation, and irregular arrangement of the actin filaments  <cit> .
the rho family of gtpases plays an important role in controlling the organization and remodeling of the actin-based cytoskeleton, and these gtpases act as molecular switches that are active in the gtp-bound state and inactive in the unbound state  <cit> .
the activation of rho is catalyzed by the dbl family of guanine nucleotide exchange factors   <cit> , and activated rho proteins subsequently induce the formation of actin stress fibers in mammalian cells  <cit> .
the dbl family comprises a number of proteins with variable modular structures, expression patterns, and cellular functions  <cit> .
leukemia-associated rhogef  is one of the rhoa-selective rhogefs that are directly regulated by activated gα12/ <dig> proteins and play a key role in oncogenic transformation induced by g protein-coupled receptors  <cit> .
in this study, we found that the activation of the small gtpase rho was influenced by the simulated microgravity generated by the 3d clinostat.
furthermore, the full-length cdna of bovine larg was identified, and its gene expression was downregulated under clinorotation.
this downregulation subsequently resulted in the repression of rhoa activation.
these results indicated that the disorganization of the actin fibers was caused by the inhibition of rho activation by 3d clinorotation.
RESULTS
formation of actin filaments under clinorotation
disorganization of the actin cytoskeleton has been demonstrated in previous studies on spaceflight and ground-based simulation of microgravity.
we determined whether the disorganization of the actin cytoskeleton is observed in the bovine brain microvascular endothelial  cells cultured in the simulated microgravity condition.
we first observed the f-actin filament by rhodamine-phalloidin staining.
bbme cells are adherent cells and normally show actin stress fibers  under the stationary condition as shown in figure 1a.
after exposure to clinorotation for  <dig> h, the cores of the actin filaments formed clusters, which are indicated by arrows in figure 1b, and stress fibers were distinctly observed in the control cells.
paxillins , which are cytoskeletal proteins involved in actin-membrane attachment, were also observed more distinctly in the control cells than in the clinorotated cells.
these observations clearly indicated that clinorotation caused the disorganization of the actin cytoskeleton.
reverse transcription polymerase chain reaction-based differential display, cloning, and sequencing of bovine larg
in order to study the effect of altered gravity on gene expression in the cells, reverse transcription polymerase chain reaction -based differential display, which is an mrna fingerprinting technique, was performed.
rt-pcr was performed using the total rna isolated from the clinorotated and stationary control cells as templates and all the  <dig> primer sets listed in the additional files .
therefore, a total of  <dig> pcrs were run, and each contained a different combination of 5' and 3' primers.
from the polyacrylamide gel electrophoresis images, we identified  <dig> dna segments that showed different expression levels under clinorotation .
the dna sequences of some segments could be determined, and homology searches were performed using the basic local alignment search tool  program from the dna data bank of japan , shizuoka.
although the function of the identified segments was unknown, two segments showed high homology to human rhogef or the activated leukocyte cell adhesion molecule.
one of the segments that showed differential expression levels in the mrna fingerprinting demonstrated substantial similarity to the 3' region of human larg mrna, which is one of the rhogefs.
we performed several cloning experiments to obtain longer cdnas corresponding to this segment.
full-length bovine larg cdna, including the open reading frame , was obtained using the rt-pcr, 5' rapid amplification of cdna ends , and 3' race methods.
the resulting assembled cdna comprised  <dig>  nucleotides including a  <dig> -bp orf corresponding to a predicted  <dig>  amino acid  polypeptide similar to that of human larg.
the predicted protein showed the greatest similarity to human and mouse larg proteins .
the molecular weight of the protein was estimated as  <dig>  kda; this is the same as that of the human and mouse larg proteins.
therefore, we concluded that this protein was bovine larg.
the alignments of the bovine, human, and mouse protein sequences are shown in figure  <dig> .
no other protein sequence in genbank, except the bovine larg and translated bovine larg expressed sequence tags, showed a higher similarity to the human and mouse larg proteins.
these facts strongly indicate that the cloned bovine larg cdna corresponds to a bonafide bovine orthologue of the human gene.
motif analysis of the bovine larg protein sequence revealed the presence of four conserved functional domains, namely, the closely juxtaposed dbl homology  and pleckstrin homology  domains, the pdz domain and the regulator of g protein signaling  domain.
as expected, the pdz and ph domains of the bovine, human, and mouse proteins are highly conserved, and they showed the highest degree of sequence conservation.
the pdz domains of these proteins are identical, whereas the ph domains differ by a single conservative amino acid substitution.
it is important to note the remarkably high degree of conservation of the entire n-terminal part of the larg protein encompassing the pdz domain.
within the first  <dig> amino acid residues, substitutions were observed in several amino acids, indicating an overall identity of 98%.
in comparison, the regions between the pdz and rgs domains and the rgs and dh domains as well as the entire c-terminal domains are substantially more variable.
for example, the region between the ph domain and the c terminal of bovine larg has an aa identity of  <dig> % and  <dig> % with those of human and mouse larg, respectively.
real-time quantitative pcr
from the mrna fingerprinting results, we predicted that the larg gene expression was downregulated under clinorotation.
to confirm the change in the larg gene expression under clinorotation, real-time quantitative pcr was performed.
as shown in figure  <dig>  the level of larg gene expression in the clinorotated cells decreased to  <dig>  ±  <dig> % of that in the stationary control cells.
this result indicated that under clinorotation, the larg gene expression was downregulated at the mrna level, and it suggested that rho activation would be influenced by changes in the gravity level.
western blot analysis and rho activation assay
western blot was performed to examine the amount of larg and rhoa proteins.
as shown in figure  <dig>  the expression level of immunoreactive larg densitometrically decreased to  <dig> %; however, the rhoa expression level increased to 138% under clinorotation.
furthermore, the amount of activated rhoa was estimated using the g-lisa rhoa activation assay kit .
as shown in figure  <dig>  the level of active rhoa in the clinorotated cells was  <dig> % of that in the stationary control cells.
these results indicated that the amounts of the larg proteins and active rhoa decreased under simulated microgravity.
discussion
previous reports demonstrated that the effects of microgravity on biological events vary.
the microgravity environment has been reported to cause reduction in the secretion of growth hormones  <cit> , blunting of the response to mitogen activation in t cells  <cit> , retardation of osteoblast growth, and disorganization of cytoskeletal formation  <cit> .
cytoskeletal formation is highly sensitive to alteration in gravity.
it was reported that in cells, the actin microfilament system constitutes the gravity-sensitive cell component  <cit>  and that microgravity affects cytoskeletal formation  <cit> .
this disorganization is caused by differences in the gravity level between the space  and ground  environments.
in ground-based studies, a 3d clinostat is commonly used to simulate microgravity conditions.
it has been reported that the results obtained using clinorotation were similar to those obtained in spaceflight experiments, suggesting that clinorotation is effective in simulating microgravity  <cit> .
however, the mechanism by which the gravity signal is converted to an intercellular signal is unclear.
it is well known that the small g proteins of the rho family regulate the remodeling of actin fibers  <cit> .
therefore, we hypothesized that the influence of microgravity on the rho signaling pathway caused the disorganization of actin, and we examined the differences in gene expression between the static and clinorotated cells by using the mrna fingerprinting method.
we found that several gene fragments responded to the altered gravity.
subsequently, we identified a part of the larg fragment that was downregulated under clinorotation.
the larg gene has been identified in some vertebrates  <cit> ; however, this is the first study that has identified the entire bovine larg gene.
motif analysis of the bovine larg protein sequence demonstrated the presence of four conserved functional domains – the dh, ph, pdz, and rgs domains.
these four domains displayed a high degree of sequence conservation in the bovine and human/mouse proteins.
our western blotting result showed that the total amount of rho protein increased in the clinorotated cells, although the disorganization of actin fibers was observed.
these results indicated that clinorotation affects not only rho expression but also rho activation.
the real-time quantitative pcr and western blotting results demonstrated that the larg expression significantly decreased in the clinorotated cells .
in mammalian cells, three rhogefs, namely, larg, p115-rhogef, and pdz-rhogef have been identified as rhoa-selective rhogefs  <cit> .
these rhogefs have close homologs and contain a regulator of g protein homology domains.
in our results, the level of the larg proteins showed a milder decrease  than that of active rhoa .
we suspected downregulated expression of not only the larg proteins but also the other rhogefs.
in consequence, the downregulation of these rhogefs does not induce rho activation and subsequently results in the disorganization of the actin fibers under simulated microgravity.
we demonstrated that clinorotation induces the inactivation of rho; however, the manner in which larg transcription is downregulated under simulated microgravity has yet to be elucidated.
furthermore, as shown in figure  <dig>  we assumed that changes in the level of gravity also affect the upstream regulation of rho, such as that via the heterotrimeric g proteins  <cit> , or the downstream regulation of rho via rho effectors such as p160rock   <cit> .
recently, it has been pointed out that numerous cytoskeletal and signaling molecules mediate the interaction between actin and the focal adhesion molecules involved in mechanosensitivity  <cit> .
in order to identify the exact molecule that converts the gravity signal into the intercellular signal, it is necessary that the upstream and downstream regulation of rho signaling, including regulation via heterotrimeric g proteins, rhogefs, and rho effectors such as rho kinase, be the focus of future investigations.
CONCLUSIONS
we concluded that the simulated microgravity generated by clinorotation affected rho activation, particularly, the larg gene expression, and this effect resulted in the disorganization of the actin fibers.
this conclusion supported the previous observations of spaceflight and ground-based experiments  <cit> .

 gravity is a universal force and affects everything in nature. gravitational effects on biological events and functions, including the mechanism of gravity sensation in cells, remain unclear. previous spaceflight experiments have reported the irregular formation of cytoskeletal fibers in spaceflown experimental cells. lewis et al. observed that the microtubule filaments extended from a poorly defined centrosome in human lymphocytes. gruener and hughes-fulford reported that actin reorganization responded to the gravity level and showed abnormal assembly of actin stress fibers. in the field of space biology, to examine the effects of microgravity on biological events in ground-based research, clinostat is often used to simulate the microgravity condition. a three-dimensional clinostat is an apparatus that nullifies the effect of gravity, and it has been used in substitution studies on microgravity effects. in a study on xenopus myocytes, 3d clinorotation caused disorganization, condensation, and irregular arrangement of the actin filaments. the rho family of gtpases plays an important role in controlling the organization and remodeling of the actin-based cytoskeleton, and these gtpases act as molecular switches that are active in the gtp-bound state and inactive in the unbound state. the activation of rho is catalyzed by the dbl family of guanine nucleotide exchange factors, and activated rho proteins subsequently induce the formation of actin stress fibers in mammalian cells. the dbl family comprises a number of proteins with variable modular structures, expression patterns, and cellular functions. leukemia-associated rhogef is one of the rhoa-selective rhogefs that are directly regulated by activated gα12/ proteins and play a key role in oncogenic transformation induced by g protein-coupled receptors. in this study, we found that the activation of the small gtpase rho was influenced by the simulated microgravity generated by the 3d clinostat. furthermore, the full-length cdna of bovine larg was identified, and its gene expression was downregulated under clinorotation. this downregulation subsequently resulted in the repression of rhoa activation. these results indicated that the disorganization of the actin fibers was caused by the inhibition of rho activation by 3d clinorotation.  formation of actin filaments under clinorotation disorganization of the actin cytoskeleton has been demonstrated in previous studies on spaceflight and ground-based simulation of microgravity. we determined whether the disorganization of the actin cytoskeleton is observed in the bovine brain microvascular endothelial cells cultured in the simulated microgravity condition. we first observed the f-actin filament by rhodamine-phalloidin staining. bbme cells are adherent cells and normally show actin stress fibers under the stationary condition as shown in figure 1a. after exposure to clinorotation for h, the cores of the actin filaments formed clusters, which are indicated by arrows in figure 1b, and stress fibers were distinctly observed in the control cells. paxillins, which are cytoskeletal proteins involved in actin-membrane attachment, were also observed more distinctly in the control cells than in the clinorotated cells. these observations clearly indicated that clinorotation caused the disorganization of the actin cytoskeleton. reverse transcription polymerase chain reaction-based differential display, cloning, and sequencing of bovine larg in order to study the effect of altered gravity on gene expression in the cells, reverse transcription polymerase chain reaction -based differential display, which is an mrna fingerprinting technique, was performed. rt-pcr was performed using the total rna isolated from the clinorotated and stationary control cells as templates and all the primer sets listed in the additional files. therefore, a total of pcrs were run, and each contained a different combination of 5' and 3' primers. from the polyacrylamide gel electrophoresis images, we identified dna segments that showed different expression levels under clinorotation. the dna sequences of some segments could be determined, and homology searches were performed using the basic local alignment search tool program from the dna data bank of japan, shizuoka. although the function of the identified segments was unknown, two segments showed high homology to human rhogef or the activated leukocyte cell adhesion molecule. one of the segments that showed differential expression levels in the mrna fingerprinting demonstrated substantial similarity to the 3' region of human larg mrna, which is one of the rhogefs. we performed several cloning experiments to obtain longer cdnas corresponding to this segment. full-length bovine larg cdna, including the open reading frame, was obtained using the rt-pcr, 5' rapid amplification of cdna ends, and 3' race methods. the resulting assembled cdna comprised nucleotides including a -bp orf corresponding to a predicted amino acid polypeptide similar to that of human larg. the predicted protein showed the greatest similarity to human and mouse larg proteins. the molecular weight of the protein was estimated as kda; this is the same as that of the human and mouse larg proteins. therefore, we concluded that this protein was bovine larg. the alignments of the bovine, human, and mouse protein sequences are shown in figure. no other protein sequence in genbank, except the bovine larg and translated bovine larg expressed sequence tags, showed a higher similarity to the human and mouse larg proteins. these facts strongly indicate that the cloned bovine larg cdna corresponds to a bonafide bovine orthologue of the human gene. motif analysis of the bovine larg protein sequence revealed the presence of four conserved functional domains, namely, the closely juxtaposed dbl homology and pleckstrin homology domains, the pdz domain and the regulator of g protein signaling domain. as expected, the pdz and ph domains of the bovine, human, and mouse proteins are highly conserved, and they showed the highest degree of sequence conservation. the pdz domains of these proteins are identical, whereas the ph domains differ by a single conservative amino acid substitution. it is important to note the remarkably high degree of conservation of the entire n-terminal part of the larg protein encompassing the pdz domain. within the first amino acid residues, substitutions were observed in several amino acids, indicating an overall identity of 98%. in comparison, the regions between the pdz and rgs domains and the rgs and dh domains as well as the entire c-terminal domains are substantially more variable. for example, the region between the ph domain and the c terminal of bovine larg has an aa identity of % and % with those of human and mouse larg, respectively. real-time quantitative pcr from the mrna fingerprinting results, we predicted that the larg gene expression was downregulated under clinorotation. to confirm the change in the larg gene expression under clinorotation, real-time quantitative pcr was performed. as shown in figure the level of larg gene expression in the clinorotated cells decreased to ± % of that in the stationary control cells. this result indicated that under clinorotation, the larg gene expression was downregulated at the mrna level, and it suggested that rho activation would be influenced by changes in the gravity level. western blot analysis and rho activation assay western blot was performed to examine the amount of larg and rhoa proteins. as shown in figure the expression level of immunoreactive larg densitometrically decreased to %; however, the rhoa expression level increased to 138% under clinorotation. furthermore, the amount of activated rhoa was estimated using the g-lisa rhoa activation assay kit. as shown in figure the level of active rhoa in the clinorotated cells was % of that in the stationary control cells. these results indicated that the amounts of the larg proteins and active rhoa decreased under simulated microgravity. discussion previous reports demonstrated that the effects of microgravity on biological events vary. the microgravity environment has been reported to cause reduction in the secretion of growth hormones, blunting of the response to mitogen activation in t cells, retardation of osteoblast growth, and disorganization of cytoskeletal formation. cytoskeletal formation is highly sensitive to alteration in gravity. it was reported that in cells, the actin microfilament system constitutes the gravity-sensitive cell component and that microgravity affects cytoskeletal formation. this disorganization is caused by differences in the gravity level between the space and ground environments. in ground-based studies, a 3d clinostat is commonly used to simulate microgravity conditions. it has been reported that the results obtained using clinorotation were similar to those obtained in spaceflight experiments, suggesting that clinorotation is effective in simulating microgravity. however, the mechanism by which the gravity signal is converted to an intercellular signal is unclear. it is well known that the small g proteins of the rho family regulate the remodeling of actin fibers. therefore, we hypothesized that the influence of microgravity on the rho signaling pathway caused the disorganization of actin, and we examined the differences in gene expression between the static and clinorotated cells by using the mrna fingerprinting method. we found that several gene fragments responded to the altered gravity. subsequently, we identified a part of the larg fragment that was downregulated under clinorotation. the larg gene has been identified in some vertebrates; however, this is the first study that has identified the entire bovine larg gene. motif analysis of the bovine larg protein sequence demonstrated the presence of four conserved functional domains – the dh, ph, pdz, and rgs domains. these four domains displayed a high degree of sequence conservation in the bovine and human/mouse proteins. our western blotting result showed that the total amount of rho protein increased in the clinorotated cells, although the disorganization of actin fibers was observed. these results indicated that clinorotation affects not only rho expression but also rho activation. the real-time quantitative pcr and western blotting results demonstrated that the larg expression significantly decreased in the clinorotated cells. in mammalian cells, three rhogefs, namely, larg, p115-rhogef, and pdz-rhogef have been identified as rhoa-selective rhogefs. these rhogefs have close homologs and contain a regulator of g protein homology domains. in our results, the level of the larg proteins showed a milder decrease than that of active rhoa. we suspected downregulated expression of not only the larg proteins but also the other rhogefs. in consequence, the downregulation of these rhogefs does not induce rho activation and subsequently results in the disorganization of the actin fibers under simulated microgravity. we demonstrated that clinorotation induces the inactivation of rho; however, the manner in which larg transcription is downregulated under simulated microgravity has yet to be elucidated. furthermore, as shown in figure we assumed that changes in the level of gravity also affect the upstream regulation of rho, such as that via the heterotrimeric g proteins, or the downstream regulation of rho via rho effectors such as p160rock. recently, it has been pointed out that numerous cytoskeletal and signaling molecules mediate the interaction between actin and the focal adhesion molecules involved in mechanosensitivity. in order to identify the exact molecule that converts the gravity signal into the intercellular signal, it is necessary that the upstream and downstream regulation of rho signaling, including regulation via heterotrimeric g proteins, rhogefs, and rho effectors such as rho kinase, be the focus of future investigations.  we concluded that the simulated microgravity generated by clinorotation affected rho activation, particularly, the larg gene expression, and this effect resulted in the disorganization of the actin fibers. this conclusion supported the previous observations of spaceflight and ground-based experiments. 
the irregular formation of cytoskeletal fibers in spaceflown experimental cells has been observed, but the disorganization process of fibers is still poorly understood. from the gene and protein expression analyses, we found that the expression level of leukemia-associated rho guanine nucleotide exchange factor, which activates rho, was downregulated under clinorotation. furthermore, the full-length cdna of bovine larg, a member of the rho guanine nucleotide exchange factor family, was identified, and its gene expression was observed to be downregulated under clinorotation. these results indicated that the disorganization of the actin fibers was caused by the inhibition of rho activation by 3d clinorotation. this downregulation subsequently resulted in the repression of rhoa activation. it is well known that the activation of the small gtpase rho leads to actin stress fibers assembly. clinorotation influences actin fiber remodeling and its related signaling pathways that involve the small gtpase rho. the activation of the small gtpase rho was influenced by simulated microgravity generated by a three-dimensional clinostat.
 the irregular formation of cytoskeletal fibers in spaceflown experimental cells has been observed, but the disorganization process of fibers is still poorly understood. it is well known that the activation of the small gtpase rho leads to actin stress fibers assembly. this study was performed to evaluate the effect of simulated microgravity on the activation of rho that is involved in actin fiber remodeling in cells.  clinorotation influences actin fiber remodeling and its related signaling pathways that involve the small gtpase rho. actin stress fiber remodeling was significantly inhibited to a greater extent in cells cultured under clinorotation than in static cultured cells. from the gene and protein expression analyses, we found that the expression level of leukemia-associated rho guanine nucleotide exchange factor, which activates rho, was downregulated under clinorotation. moreover, we identified the full-length larg cdna. the amount of gtp-bound rhoa, that is, the active form of rhoa, decreased under this condition.  the activation of the small gtpase rho was influenced by simulated microgravity generated by a three-dimensional clinostat. furthermore, the full-length cdna of bovine larg, a member of the rho guanine nucleotide exchange factor family, was identified, and its gene expression was observed to be downregulated under clinorotation. this downregulation subsequently resulted in the repression of rhoa activation. these results indicated that the disorganization of the actin fibers was caused by the inhibition of rho activation by 3d clinorotation. 