My Summer Undergraduate Research Experience

Kinetics of Thyroid Hormone Receptor α Nuclear Import Xenopus laevis

Thyroid gland secretes two similar hormones (T 3 and T 4 ) that play a pivotal role in development and maturation. Thyroid gland secretes two similar hormones (T 3 and T 4 ) that play a pivotal role in development and maturation. The thyroid hormone receptor (TR) is a nuclear protein which, in response to T 3, promotes or represses gene transcription. The thyroid hormone receptor (TR) is a nuclear protein which, in response to T 3, promotes or represses gene transcription. Therefore, the mechanism regulating the subcellular distribution of TR is an extremely important topic. Therefore, the mechanism regulating the subcellular distribution of TR is an extremely important topic. Background Thyroid Gland

Nuclear Pore Complex Molecules that are ~50kDa can passively diffuse through the NPC Molecules that are ~50kDa can passively diffuse through the NPC Energy-independent process Energy-independent process Does not require NPC interaction Does not require NPC interaction Temperature-independent Temperature-independent Molecules can be actively transported through the NPC Molecules can be actively transported through the NPC Energy-dependent process Energy-dependent process Requires interaction with the NPC or import receptor Requires interaction with the NPC or import receptor Temperature-dependent Temperature-dependent Central Transporter Cytoplasm Nucleus

QUESTION QUESTION Why, if TR can enter the nucleus by an energy- independent process it has also evolved to follow a signal-mediated process. Why, if TR can enter the nucleus by an energy- independent process it has also evolved to follow a signal-mediated process. HYPOTHESIS: HYPOTHESIS: Signal-mediated import will occur more rapidly than passive diffusion. Signal-mediated import will occur more rapidly than passive diffusion.

Xenopus laevis Surgically remove Xenopus oocytes. Surgically remove Xenopus oocytes.

Vegetal Pole Animal Pole (where nucleus resides) Inject 35 S-TR Manually microinject radioactively labeled TR into the cytoplasm. Manually microinject radioactively labeled TR into the cytoplasm.

Block signal mediated transport of TR by keeping oocytes at 4 °C. Block signal mediated transport of TR by keeping oocytes at 4 °C. Compare the nuclear localization rate of TR with its rate in oocytes incubated at 20 °C. Compare the nuclear localization rate of TR with its rate in oocytes incubated at 20 °C. 4°C 20 °C

At various time points, manually dissect nuclei from “cytoplasms” (enucleated oocytes). At various time points, manually dissect nuclei from “cytoplasms” (enucleated oocytes). Separate proteins using SDS-polyacrylamide gel electrophoresis. Separate proteins using SDS-polyacrylamide gel electrophoresis. Detect radioactive TR by exposing the dried gel to X-ray film. Detect radioactive TR by exposing the dried gel to X-ray film. Nucleus Cytoplasm

Autoradiogram 35 S-TRα 4° Cyto 20° Cyto 20° Nuc 4° Nuc Experiment incubated for 2.5 hours. Oocytes incubated at 20° C have a noticeably greater amount of TR in the nucleus as compared to oocytes at 4° C.

General trend of increased nuclear localization with time. General trend of increased nuclear localization with time. Oocytes at 20°C consistently had a greater %Nuclear TR than 4°C. Oocytes at 20°C consistently had a greater %Nuclear TR than 4°C.

Benefits of Research Patience, Determination and Perseverance Patience, Determination and Perseverance Surrounded daily by people with the same interests Surrounded daily by people with the same interests Perfect my public speaking skills. Perfect my public speaking skills. Drastically different experience than classes Drastically different experience than classes Analyzing novel data vs. regurgitation of facts. Analyzing novel data vs. regurgitation of facts. Hands on Experience- learn by doing. Hands on Experience- learn by doing. Get to know Professors in the Biology Department Get to know Professors in the Biology Department Dr Allison and I are now best friends. Dr Allison and I are now best friends. Lab meeting twice a week with Dr. Shakes in the summer. Lab meeting twice a week with Dr. Shakes in the summer. Scientific ethics seminars with Dr. Heideman. Scientific ethics seminars with Dr. Heideman.

My Future Plans Next year I will attend medical school. Next year I will attend medical school. Applied to schools with excellent research programs. Applied to schools with excellent research programs. Hope to combine my affinity for research and medicine. Hope to combine my affinity for research and medicine. Continue molecular biology research pertaining to human disorders. Continue molecular biology research pertaining to human disorders. Get involved with clinical research. Get involved with clinical research.

Acknowledgements Dr Allison for her expert advice, time and incredible patience. Dr Allison for her expert advice, time and incredible patience. Funding from NSF and NIH for L.A.A. Funding from NSF and NIH for L.A.A. Supported by HHMI Summer Research Fellowship, HHMI Minor Research Grant, Minor Research Grant from the College of William and Mary. Supported by HHMI Summer Research Fellowship, HHMI Minor Research Grant, Minor Research Grant from the College of William and Mary.