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Gravistimulation and Actin Cytoskeleton-Based Cell Function in Cultured Mammalian Cells.  Y. Chiu1,2, T.N. Thomas2, K. Fujiwara2.1Dept. of Biochemistry and Biophysics, and 2Center for Cardiovascular Research, University of Rochester, Rochester, NY

   Cells respond to a variety of mechanical stimuli such as fluid shear stress, stretch, pressure, and osmotic shock. Gravity is a constant and continuous form of mechanical force exerted to all animals and plants. While gravitropic responses of plants have been well characterized and amyloplast sedimentation has been placed to the most upstream in the cascade of events, gravity effects on mammalian cells is largely unknown. The goal of this study is to investigate both the cellular and molecular responses of cultured mammalian cells subjected to gravistimulation. When bovine aortic endothelial cells (BAEC) were grown up-side down and stained with rhodamine-phalloidin, transient development of lamellipodia and a reduction in the number and thickness of stress fibers were observed within 1hour. Surprisingly, the activity of small GTPase Rac, which regulates lamellipodia formation, was down-regulated in these cells. To further evaluate the potential signal transduction occurring in gravistimulated cells, phosphotyrosine proteins were extracted from cell lysates by immunopurification and analyzed by SDS-PAGE.  While dozens of proteins were purified, there were several proteins uniquely present or absent in up-side down cells compared with control cells, suggesting that the tyrosine phosphorylation level of some proteins was sensitive to the gravity vector change. To study effects of gravity on cell migration, scratch-injured BAEC monolayers were incubated either vertical (i.e. parallel to the direction of gravity) or up-side down. The wound closure was slower in both cases compared with the right-side up control cells. These observations indicate that mammalian cells respond to gravistimulation in many ways and suggest that one of the targets of gravistimulation is the actin-based cytoskeleton.

(Supported by NIH: HL69041 and AHA: 256265T.)