Rheometric studies on the in vitro polymerization of microtubules
Nelson, Rickey L.
McIntire, Larry V.
Master of Arts
Microtubules form weakly associated networks with many properties similar to networks of rigid rods. To study the in vitro polymerization of bovine brain tubulin, a Fluids Rheometer was used in the oscillatory mode. The maximum elastic modulus for microtubule networks in the standard PIPES buffer was observed to be proportional to the tubulin concentration over much of the investigated range. A maximum elastic modulus of 2 dynes/cm2 occurred at a tubulin concentration of 8 mg/ml. At even higher concentrations, a decrease in the maximum elastic modulus was observed. Other additions to the standard buffer primarily affected the interactions between microtubules as opposed to shifting the overall polymer-heterodimer equilibrium. 9% deuterium oxide increased the maximum elastic modulus by a factor of 4, while 25% glycerol led to approximately a doubling in the maximum elastic modulus when compared to microtubule networks in the standard buffer. Microtubule associated proteins (MAPs) were shown to inititate the formation of microtubules when added back to purified tubulin solutions. Even though more microtubules were formed as the MAPs were added, the elasticity decreased with increasing MAPs additions because shorter, more numerous microtubules were present. The interactions between bovine brain tubulin and rabbit muscle actin were also investigated. The resulting networks were found to be extremely strain sensitive but had elastic moduli considerably greater than the sum of the individual components. The MAPs fraction was found to cross-link actln filaments with the maximum interaction occurring at .3 mg MAPs/mg actln, corresponding to 1 mole of MAPs per 23 moles of actin. The extent of phosphorylation state of the MAPs also affected the interaction. At an actin concentration of 1 mg/ml, MAPs with a lower phosphate content would form a network with a maximum elastic modulus of 4 dynes/cm. However, at approximately the same actin and MAPs concentrations the maximum elastic modulus from MAPs with a higher phosphate content was 25% lower at 3 dynes/cm.