My Summer Undergraduate My Summer Undergraduate Research ExperienceResearch Experience

Kinetics of Thyroid Kinetics of Thyroid Hormone ReceptorHormone Receptor αα Nuclear Import Nuclear Import

Xenopus laevis

Thyroid gland secretes two similar Thyroid gland secretes two similar hormones (Thormones (T33 and T and T44) that play a pivotal ) that play a pivotal role in development and maturation.role in development and maturation.

The thyroid hormone receptor (TR) is a The thyroid hormone receptor (TR) is a nuclear protein which, in response to nuclear protein which, in response to TT33, promotes or represses gene , promotes or represses gene transcription.transcription.

Therefore, the mechanism regulating Therefore, the mechanism regulating the subcellular distribution of TR is an the subcellular distribution of TR is an extremely important topic. extremely important topic.

BackgroundBackground

Thyroid Gland

Nuclear Pore ComplexNuclear Pore Complex Molecules that are ~50kDa can passively diffuse Molecules that are ~50kDa can passively diffuse

through the NPCthrough the NPC Energy-independent processEnergy-independent process Does not require NPC interactionDoes not require NPC interaction Temperature-independentTemperature-independent

Molecules can be actively transported through Molecules can be actively transported through the NPCthe NPC

Energy-dependent processEnergy-dependent process Requires interaction with the NPC or import receptorRequires interaction with the NPC or import receptor Temperature-dependentTemperature-dependent

Central Transporter

Cytoplasm

Nucleus

http://www.scripps.edu/~stoffler/proj/NPC/npc.html

QUESTIONQUESTION Why, if TR can enter the nucleus by an Why, if TR can enter the nucleus by an

energy-independent process it has also energy-independent process it has also evolved to follow a signal-mediated evolved to follow a signal-mediated process.process.

HYPOTHESIS:HYPOTHESIS: Signal-mediated import will occur more Signal-mediated import will occur more

rapidly than passive diffusion.rapidly than passive diffusion.

Xenopus laevisXenopus laevis

Surgically Surgically remove Xenopus remove Xenopus oocytes.oocytes.

Vegetal Pole

Animal Pole (where nucleus resides)

Inject 35S-TR

Manually Manually microinject microinject radioactively labeled radioactively labeled TR into the TR into the cytoplasm.cytoplasm.

Block signal mediated transport of Block signal mediated transport of TR by keeping oocytes at 4 °C.TR by keeping oocytes at 4 °C.

Compare the nuclear localization Compare the nuclear localization rate of TR with its rate in oocytes rate of TR with its rate in oocytes incubated at 20 °C.incubated at 20 °C.

4°C 20 °C

At various time At various time points, manually points, manually dissect nuclei from dissect nuclei from “cytoplasms” “cytoplasms” (enucleated oocytes).(enucleated oocytes).

Separate proteins Separate proteins using SDS-using SDS-polyacrylamide gel polyacrylamide gel electrophoresis.electrophoresis.

Detect radioactive Detect radioactive TR by exposing the TR by exposing the dried gel to X-ray dried gel to X-ray film.film.

NucleusCytoplasm

AutoradiogramAutoradiogram

3535S-S-TRTRαα

4°

Cyt

o

20° C

yto

20° Nuc

4° N

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

TRα Nuclear Import Kinetics

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 0.5 1 1.5 2 2.5 3 3.5

Time (hours)

%T

R N

ucl

ear

20°C

4°C

Benefits of ResearchBenefits of Research Patience, Determination and Perseverance Patience, Determination and Perseverance Surrounded daily by people with the same Surrounded daily by people with the same

interestsinterests Perfect my public speaking skills.Perfect my public speaking skills. Drastically different experience than classesDrastically 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 DepartmentGet 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 Lab meeting twice a week with Dr. Shakes in the

summer.summer. Scientific ethics seminars with Dr. Heideman.Scientific ethics seminars with Dr. Heideman.

My Future Plans My Future Plans

Next year I will attend medical Next year I will attend medical school.school. Applied to schools with excellent Applied to schools with excellent

research programs.research programs.

Hope to combine my affinity for Hope to combine my affinity for research and medicine.research and medicine. Continue molecular biology research Continue molecular biology research

pertaining to human disorders.pertaining to human disorders. Get involved with clinical research.Get involved with clinical research.

AcknowledgementsAcknowledgements

Dr Allison for her expert advice, time Dr Allison for her expert advice, time and incredible patience.and incredible patience.

Funding from NSF and NIH for Funding from NSF and NIH for L.A.A.L.A.A.

Supported by HHMI Summer Supported by HHMI Summer Research Fellowship, HHMI Minor Research Fellowship, HHMI Minor Research Grant, Minor Research Research Grant, Minor Research Grant from the College of William Grant from the College of William and Mary.and Mary.