Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2012

Thesis Director

Michael A. Menze

Thesis Committee Member

Gary A. Bulla

Thesis Committee Member

Nilay Chakraborty

Thesis Committee Member

Britto P. Nathan

Abstract

In anhydrobiotic animals a reoccurring strategy to survive water stress involves the accumulation of low molecular weight solutes (e.g., trehalose) and highly hydrophilic macromolecules [e.g., late embryogenesis abundant (LEA) proteins]. We utilized two different water-stress models (spin-drying, and hyperosmotic dehydration) to investigate the impact of transgenic expression of a trehalose transporter [BT003466 (TRET)] and of a group 1 LEA protein [ACX81198 variant (LEA1)] on membrane integrity in cell lines from Spodoptera frugiperda (Sf-21) and Drosophila melanogaster (Kc167), respectively. Kc167 cells were challenged with sucrose or trehalose concentrations ranging from 0 to 200 mM supplemented in standard culture medium. Significant inhibition of proliferation occurred at all sugar concentrations tested after both 24 and 48 h as compared to unstressed controls (n =3 p < 0.05). Kc167-LEA1 cells showed significant improvement in tolerance over wild-type Kc167 cells in hyperosmotic sucrose medium containing 50 mM and 200 mM sucrose after 24 hand at all concentrations tested (50 mM, 100 mM, and 200 mM) after 48 h of incubation (n = 3, p < 0.05). When Kc167 wild-type and LEA1 expressing cells were incubated in hyperosmotic trehalose medium, Kc167-LEA1 cells showed significant amelioration of hyperosmotic stress at 100 and 200 mM trehalose after 24 and 48 h as compared to control Kc167 cells (n = 3, p < 0.05).

To over-express trehalose transporters a nucleotide sequence [BT003466 (TRET)] from Drosophila melanogaster was stably transfected into Sf-21 cells (Sf-21-TRET). The TRET transporter protein shows high homology to trehalose transporters from other insects including the anhydrobiotic insect Polypedilum vanderplanki and it is targeted to the plasma membrane of both Kcl67 and Sf-21 cells. To investigate the effects of severe desiccation on wild-type Sf-21 and Sf-21-TRET cells spin drying was employed. Sf-21 and Sf-21-TRET cells were either pre-incubated with 400 mM trehalose for 3h and then spun in 1.8 M trehalose containing buffer or not pre-incubated and then spun in 0.2 M trehalose containing buffer. Spin-drying caused 100% loss of membrane integrity in control Sf-21 cells regardless of the condition tested, whereas membrane integrity in Sf- 21-TRET cells preincubated with trehalose and dried in 1.8 M trehalose buffer was comparable to non-dried control cells 48 h after rehydration (n = 3). The population of Sf-21-TRET cells not preincubated with trehalose and dried in 0.2 M trehalose buffer showed a 17.5% reduction in membrane integrity as compared to non-dried control cells 48 h after rehydration (n = 2). Interestingly, trehalose accumulation after 3 h of incubation in either 400 mM or 800 mM trehalose supplemented media was the same between Sf-21 and Sf-21-TRET cells. Trehalose accumulation resulted in concentrations of 68.82 mM ± 16.84 (400 mM) and 174.56 mM ± 25.32 (800 mM) intracellular trehalose in wild-type Sf-21 cells and 60.71 mM ± 9.47 (400 mM) and 179.19 mM ± 24.03 (800 mM) in Sf-21-TRET expressing cells (n = 3 ± S.E.). These results suggest that wild-type Sf-21 cells contain an endogenous trehalose transporter and that the over-expression of trehalose transporters alone has an effect during the drying and/or rehydration process. The over-expression of trehalose transporters and LEA proteins appear to aid in maintenance of membrane integrity during water stress.

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