Research at the Cooke Lab

Motor proteins perform roles in numerous biological processes, including: muscle contraction, cell division and the movement of intracellular organelles. We study the mechanism of action of two classes of motor proteins, the myosin family and the kinesin family, which share a structural homology. One long-range goal of our laboratory is to understand the molecular mechanisms by which these two classes of motor proteins generate force. 

The interaction of two proteins, actin and myosin, produces the force of muscle contraction and is also involved in the motility of all eukaryotic cells. We have used spectroscopic probes, both paramagnetic and fluorescent, to measure the orientation and conformation of both actin and myosin in active muscle fibers. 

Kinesin motors are smaller than myosin but generate forces and displacements that are similar. To monitor conformational changes in this motor we have placed paramagnetic probes on active elements. This work, carried out in collaboration with the laboratory of Dr. Ron Vale, led to the first molecular model of kinesin motility. 

Another goal of the lab is to determine how muscle fibers adapt to intense activity and fatigue. Using muscle fibers that are permeable, we can simulate the intracellular conditions that occur in fatigued muscle. These studies have shown that the accumulation of a number of metabolic products in the interior of the muscle cell can account for some of the fatigue felt by the long distance runner, but it does not explain all of it.  A subunit of myosin becomes phosphorylated during heavy use.  Our recent results suggest that myosin phosphorylation works synergistically with metabolite accumulation to inhibit fiber shortening velocity.   

The relevance of our research is to explain the complex behavior of muscles (skeletal, smooth and cardiac), as well as the motility of non muscle cells, in terms of the interactions of the motor proteins. Our results will help develop more rational methods to manipulate muscle contraction and cell motility for medical purposes.

Our laboratory has recently pursued a new area, the energetics of resting muscle, see Stewart et al, references #162. We have found a new mechanism for generating thermogenesis in skeletal muscle fibers. The metabolic activity of resting skeletal muscle is of interest, because it plays a significant role in the whole body resting energy expenditure. Muscle metabolism is involved in cold induced thermogenesis, in consumption of calories from excess food intake and is a major regulator of blood sugar levels. The mechanism of muscle thermogenesis and its regulation remain an active area of investigation.

We started from the premise that thermogenesis was linked to an old observation that myosin in a test tube had a much greater activity than it has in living fibers. No one had previously found an in vitro system that replicates the in vivo activity. We employed quantitative fluorescence spectroscopy to measure single nucleotide turnovers in relaxed skinned fibers, and finally we observed the elusive inhibited myosin. We called the new state of myosin with the highly inhibited ATPase activity the “Super Relaxed State”. In the Super Relaxed State it takes about 250 seconds for myosin to turnover an ATP, as opposed to ~30 seconds in the normal relaxed state. We propose that the inhibition of myosin ATPase activity in skeletal fibers results from incorporation into a thick filament structure previously identified using electron microscopy by Craig, Padron and co-workers. By analogy with another motor, active myosin is akin to a car racing down the road. Myosin in the normal relaxed state is similar to a car stopped at a traffic light with the motor idling, and the counterpart of the super relaxed state is a car parked beside the road with the motor off. 

We propose that myosin, well known as a motor protein, has an alter ego. Alterations in the ATP turnover rate by myosin in resting muscle is one of the mechanisms that produce thermogenesis in muscle. Manipulations of factors that affect the population of myosin in the super relaxed state would directly affect muscle metabolism and thus whole body energy expenditure. Our work suggests reasonable targets for therapies to alter whole body thermogenesis. Such interventions could treat health problems such as diabetes or obesity. 

We have recently extended these results to vertebrate cardiac muscle, where we also observe myosin with a very slow ATP turnover rate. We propose that in cardiac muscle this state plays a role in lowering the metabolic rate, especially in times of stress such as ischemia.