Summer Research
* Student(s): Bryden Considine , Kateri Whitebean
Advisor: Herman Lehman
Title: Sf9 Cells as a Model for Signal Transduction
Abstract:
Cells in culture present the ability to explore signal transduction, the transfer and conversion of information signals into the cell. Sf9 cells, derived from the pupal ovary of Spondoptera frugiperda, have been maintained in cell culture for the past two decades, however, little is known about signal transduction in these cells. The Sf9 cells were isolated during the early pupal stage (1). Pupal growth and development is highly dependent on steroid hormone titers during this early stage. Ecdysone, a pro-hormone that is transformed by mitochondria into the active hormone 20-hydroxyecdysone, has been found to be an important steroid in insect growth and development. Because Sf9 cells were isolated during the early pupal stage when steroid hormone titers begin to increase, we observed effects in cell growth and morphology in cultured Sf9 cells treated with the steroid hormone 20-hydroxyecdysone.
We cultured Sf9 cells in Grace's Supplemented medium with Fetal Bovine serum, Penicillin-Streptomysin solution, and Serum-free media (SFM). We incorporated 20-hydroxyecdyone at a titer of 1 X 10^-6 M. The cells were incubated for different periods of time: 24 hours, 48 hours, and 72 hours. We used various fluorescent stains to detect specific organelles. Rhodamine-Phalloidin was used to visualize actin filaments, 4, 6-Diamidino-2-Phenylindole Dihydrochloride Hydrate (DAPI) was used to visualize nuclei, and Monoclonal Anti-a-Tubulin FITC Conjugate Goat Anti-Mouse Immunoglobulin was used to visualize microtubules. The cells were viewed and recorded using fluorescent microscopy.
We observed that Sf9 cells with and without the addition of the steroid hormone 20-hydroxyecdysone. Sf9 cells without the addition of 20-hydroxyecdysone developed microtubule containing tail-like extensions, whereas the Sf9 cells with the addition of 20-hydroxyecdysone had fewer of these extensions. We also observed differences in cell growth and density. Cells were originally plated at equal densities of 1 X 10^5 cells per milliliter. After 72 hours the Sf9 cells treated with 20-hydroxyecdysone were confluent having >1000 cells per field of view, whereas the cells not treated with the hormone had on average 530 cells per field of view. In addition we observed that cells treated with 20-hydroxyecdysone did not adhere to the slide as well as those not treated. 20-hydroxyecdysone may have caused a change in a protein specific to cell adhesion. By incorporating a steroid hormone into the growing Sf9 cells we have begun to develop a model of steroid hormone signal transduction.
1 Bollenbacher, W. E., Smith S. L., Goodman, W., and Gilbert, L. I. (1981). Ecdysteriod Titer during Larval - Pupal - Adult Development of the Tobacco Hornworm Manduca sexta. Gen. Comp. Endocrinol. 44, 302-306.
BC's research supported by the Camille and Henry Dreyfus Foundation. KW's research supported by NSF/STEP.