74.2 Friday, Jan. 6 Protective Mechanisms Against Water Stress Evaluated in Insect Cells ANDERSON, J.M.*; HARDER, A.M.; HAND, S.C.; TONER, M.; CHAKRABORTY, N.; MENZE, M.A.; Eastern Illinois University, Dept. Biol. Sci., Charleston, IL; Eastern Illinois University, Dept. Biol. Sci., Charleston, IL; Louisiana State University, Dept. Biol. Sci., Baton Roughe, LA; Center for Engineering in Medicine Harvard Medical School, Boston, MA; Center for Engineering in Medicine Harvard Medical School, Boston, MA; Eastern Illinois University, Dept. Biol. Sci., Charleston, IL email@example.com
We utilized three different water-stress models (controlled-rate freezing, spin-drying, and osmotic dehydration) to investigate the impact of intracellular trehalose or transgenic expression of a group 1 LEA protein (ACX81198 variant) on membrane integrity in cell lines from Spodoptera frugiperda (Sf-21) and Drosophila melanogaster (Kc167), respectively. Neither species is tolerant to severe desiccation. Kc167 cells were challenged with 100mM sucrose in standard culture medium. Expression of LEA1 protein increased the number of cells with intact membranes by 25±3.5% (n=3,±SE) after 48h of incubation compared to controls. To facilitate trehalose uptake a nucleotide sequence (trehalose transporter BT003466) was stably transfected into Sf-21 cells (Sf-21-BT). Cells were incubated for 3h at 27 °C in a buffer containing 200mM trehalose. After incubation 43±3.7% (n=5,±SE) of control Sf-21 cells showed compromised membranes while Sf-21-BT cells maintained higher numbers of cells with intact membranes [76±5.4%(n=5,±SE, P<0.05)]. However, Sf-21 and Sf-21-BT cells frozen in this buffer at 1°C•min-1 showed no significant difference in membrane integrity after thawing (60±3.3% control vs. 60±2.8% Sf-21-RHO; n=3;±SE). After incubation in culture media with 400mM trehalose for 3h, severe water stress was applied in the form of spin-drying. Spin-drying caused 100% loss of membrane integrity in control Sf-21 cells, whereas membrane integrity in Sf-21-BT cells 48h after rehydration was comparable to non-dried control cells. Trehalose and LEA proteins appear to aid in maintenance of membrane integrity during water stress. (NSF-IOS-0920254, CFR-EIU-2010).