The project's long-term objective is to elucidate, at the molecular level, how the motor protein kinesin transports vesicles from a neuron's cell body to its axon tip, a distance of as much as 1 m, in only 4 days. The velocity-force relation of one kinesin motor is well understood when the motor is in solution and the viscous work load on it is small. However, within a cell, the viscoelastic drag force opposing the motor is at least 100 times greater, which makes it likely that 2-5 motors are required to pull a single vesicle. Quantitative velocity-force curves have been obtained for 1 kinesin but not for the 2, 3, or more active motors actually needed to pull a single load in vivo, and qualitative data on the effect of multiple motors are contradictory. We hypothesize that 2 or more motors pulling a single cargo will share the load equally. As a result, if the velocity-force curve for 1 kinesin is v1 (F), the velocity-force function v2(F) for two kinesins will be v1 (F/2), v3(F)= v1(F/3), etc.
Our specific aim, then, is to measure velocity-force curves for 1,2, and 3 kinesins over a physiologically realistic force range, 0-20 pN, which will directly test our hypothesis. We will use speckled microtubules to provide numerous fiduciary marks along the length of each microtubule. This will greatly improve the spatial precision of tracking over classical gliding assays, which use uniformly labeled microtubules. Also, the temporal precision will be 0.1s or better. We expect the number of motors to change approximately once per second. If the gliding microtubule can be tracked for 5-10s, the pattern of velocity changes will reveal the number of active motors. Force will be generated by viscous drag and by magnetic beads bound to the gliding microtubule. The exceptional length of some neurons places exceptionally stringent demands on their vesicle transport systems and suggests that some neuronal disease may originate in a transport system failure. Mutations in microtubule motor proteins have been shown to cause disease phenotypes in Drosophila as well as degenerative diseases of the human nervous system. A more quantitative analysis of multiple motor mechanics will expedite the identification of degenerative diseases associated with defective transport and facilitate rational intervention. ? ?
