Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biology

Supervisor

Staples, James F.

Abstract

The electron transport system complexes form supercomplexes (SCs) within mitochondrial membranes, perhaps increasing respiratory capacity or reducing reactive oxygen species production. My project aimed to determine the abundance, composition, and stability of SCs in a hibernator. Hibernators have dynamic metabolisms that change greatly during the winter. I isolated mitochondria from rats and thirteen-lined ground squirrels (TLGS) in different hibernation states and measured mitochondrial respiration. I extracted mitochondrial proteins using two detergents of different strengths, and quantified SC abundance using 2-dimensional gel electrophoresis and immunoblotting. Rats had fewer SCs than TLGS. SCs are dynamic in hibernation and the complex III composition of different SCs differed between hibernation states and seasons. There was no correlation between SC abundance and mitochondrial respiration. The stability of SCs differed: torpor SCs were most stable. This is the first report of SC changes during hibernation, and the first to demonstrate their dynamics on a short timescale.

Summary for Lay Audience

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) hibernate throughout the winter, using only stored fat as fuel. Hibernation consists of two steady states: torpor and interbout euthermia (IBE). In torpor, squirrels are inactive, body temperature is around 4℃, and metabolic rate is low. Torpor lasts for 6-20 days, then squirrels arouse to IBE and return to a warm body temperature and high metabolic rate for 6-12 hours, before entering another bout of torpor. This cycle repeats until spring.

Ground squirrel physiology changes drastically between these two states, especially within mitochondria. Mitochondria convert energy derived from food to ATP, a form useable by all cells. Mitochondria generate ATP using, in part, the electron transport system (ETS). The ETS is a system of five different enzyme complexes that generate an electrochemical potential and use it to produce ATP. We previously thought that ETS complexes existed separately, but recent research shows that they physically associate with one another, forming supercomplexes (SCs).

Given that metabolic rate and temperature change drastically in ground squirrels during hibernation, I expected that some characteristics of SCs would change as well. I also hypothesized that there would be differences between the SCs of ground squirrels and rats that do not hibernate. To answer these questions, I isolated mitochondria from several tissues of ground squirrels in different hibernation states, and quantified SCs. Within ground squirrel mitochondria, SCs exist in the form of “respirasomes” (CI/CIII/CIV), and smaller SCs (CIII/CIV). I found that ETS complex III associations within SCs were dynamic, and the percent of CIII associated with each SC type differed between torpor and IBE, and between winter and summer. I also found that ground squirrels had more supercomplexes than rats did, and that SC stability differed among hibernation conditions: SCs in torpid animals were the most stable.

This is the first report of SC changes during hibernation, and of differences between a hibernator and a non-hibernator. These findings will contribute another piece to the puzzle of understanding how hibernators are able to alter metabolism to survive extreme conditions.

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