Influence of simulated microgravity on the activation of the small GTPase Rho involved in cytoskeletal formation--molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor.
BACKGROUND
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.


RESULTS
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 (LARG), 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.


CONCLUSIONS
The activation of the small GTPase Rho was influenced by simulated microgravity generated by a three-dimensional (3D) clinostat. Furthermore, the full-length cDNA of bovine LARG, a member of the Rho guanine nucleotide exchange factor (GEF) 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.
Higashibata
Akira
A
Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency, Tsukuba Space Center, 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505, Japan. higashibata.akira@jaxa.jp
Imamizo-Sato
Mari
M
Seki
Masaya
M
Yamazaki
Takashi
T
Ishioka
Noriaki
N
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D022502:Stress Fibers / Q000378:metabolism; D014893:Weightlessness; D020741:rho GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology
D020662:Guanine Nucleotide Exchange Factors; D020741:rho GTP-Binding Proteins