In Vivo Optical Trapping Applied to two cellular motors fighting

3/17/2013 Taekjip Ha

Optical traps were invented in 1987 and have been exquisite tools to measure nanometer--even Angstrom--changes as a function of force in biological systems. The trap acts like a spring made of light, where the light tends to hold a particle into the region which is brightest, with a force, F, equal to a spring constant times the distance from the center (F = -kx). When in water, which has a well-known viscosity, the spring constant is fixed and can be easily determine. However, inside of a cell, the retarding force is a function of the velocity (F=gv) because the cell is elastic in addition to being viscous. The cell’s media is, roughly speaking, like honey: push an object, let it go, and it keeps gliding, where the force slowing it down is a function of how fast it is going. So, the spring “constant” is not really a constant: it varies by time and can vary according to the frequency. It also varies with position, since the cell is spatially heterogeneous.

Fortunately, a theory to account for the viscoelastic effects has been developed. CPLC researchers led by Professor Selvin wanted to take this idea and make a real optical trap, to show that it worked, and to measure properties in a cell. They took two measurements. One was a regular optical trap, with a fixed laser. The particle moved with the usual Brownian force—in effect, at temperature T, the particle is moved around with a finite amount of energy ½ k_BT, where k_B is the Boltzmann’s constant. Second, the laser beam was oscillated in space at a multitude of frequencies, and the particle’s motion was observed. (The laser was shaken with a square-wave so it was shaken at many frequencies at once.) The important point is that by taking a ratio of the two, one can get out the spring constant as a function of frequency or time.

Having done this, they were interested in understanding how cargoes (“particles”) are moved around the cell. The cell is like a dense city, with roadways and trucks moving things about. They were interested in looking at a roadway called microtubules, with cargoes being driven by two molecular motors. One molecular motor is called kinesin, which walks on the roadway while carrying the cargo towards the exterior or cell membrane, and the other is dynein, which moves the cargo in the opposite direction, towards the cell’s interior, or the nucleus. (Both literally walk, or run, each having two “legs”!) These two motors frequently attached at the same time and act on the cargo at the same time. So how does the cargo decide which way to move? In fact, cargoes are frequently observed to go in a “saltatory” motion, frequently moving up-and-back with no obvious rhyme-or-reason.

Furthermore, no one had ever measured the “stall force” of these molecular motors inside of a cell. The stall force is the maximum force that a molecular motor can apply before it is stopped by the optical trap. The stall force, however, was known in vitro, that is, in water: it was 6 pN for kinesin, and 1 pN for dynein.

What we found is that, when the cargo is moving towards the exterior, kinesin’s stall force was equal to 6 pN minus 1 pN times the number of dyneins being carried{C}⁶{C}. This is an extremely surprising result. It implies that kinesin is pulling the cargo while dynein(s) is resisting the motion. The more dyneins, the greater the resistance. What this further implies, is that dynein is staying bound to the microtubule when going outward. This is a totally surprising result since dynein was thought to mostly walk only when moving towards the interior of the cell. In fact, we found when the cargo is moving towards the interior, the stall force is equal to a multiple of 1 pN. That is, apparently the kinesin hops off the microtubule, and the stall force is equal to the number of dyneins attached to the cargo. Although it is not definitive, these measurements imply that the motors are in a tug-of-war, with each side pulling away, and the final direction of movement depending on which side happens to win.

The important point is that by developing new physical techniques, we could measure the properties of molecular motors inside the cell, which was not possible before. Now we are planning on extending this work, to simultaneously look at fluorescence, and to tinker with the molecular motors to understand what about them contributes to the saltatory motion.