UC Santa Barbara

Microtubules are an essential component of the cytoskeleton that provide structural integrity and facilitate important functions in cells. In this work, I will explore several aspects of microtubule mechanics and function using a wide range of biophysical techniques. I am particularly interested in the effects of stabilizing agents, including small molecule inhibitors of microtubule dynamics (such as taxol or epothilone), and microtubule-binding proteins, such as tau. First, using advanced spectral analytical techniques, we studied the effects of tau proteins on the mechanical properties of single microtubule (MT) filaments under controlled in vitro conditions. We found that the short forms of 3-repeat (3R) and 4-repeat (4R) wild-type (WT) tau proteins reduce the stiffness of taxol-assembled MTs compared to a no tau control, despite the fact that the microtubule diameter is known to increase when these tau proteins bind. In contrast, single point tau mutants P301L, R406W and ΔN296, which are all linked to devastating neurological diseases, do not have significant effects on microtubule stiffness compared to a no tau control, raising interesting questions about possible mechanical origins of tauopathy diseases.

Next, we examined the effects of tau proteins on in vitro transport properties of kinesin-bound cargoes on microtubules. In comparison to the no tau control, kinesin-bound quantum dot cargoes had lower velocity and shorter run lengths in the presence of tau when moving on taxol-assembled microtubules. Most of the single point tau mutants that we tested showed similar slowing of cargo translocation, with speed reductions similar to those observed with WT tau. One exception was the 4R short R406W mutant tau, in which the velocities of kinesin-bound cargoes were significantly lower than in WT case. The disease mechanism for mutant has been particularly puzzling, so our results may suggest a more prominent role in disruption of axonal transport than was previously appreciated.

Next, we investigated the effects of microtubule-stabilizing chemicals epothilone-A and -B on 1) microtubule mechanics, 2) the transport properties of kinesins and 3) the ability of WT tau proteins modify the stiffness of microtubules assembled with epothilone. We found that microtubules assembled in the presence of epothilone-A or -B were less stiff compared to taxol-assembled MTs, and the addition of WT tau further reduced the stiffness. However, the differences in stiffness/persistence length between epothilone-assembled microtubules and taxol-assembled microtubules diminished after the addition of WT tau. Kinesin translocation speed was sensitive to the type of stabilizing chemical, and the changes in velocity in the presence of tau were also depended on the assembly condition. Epothiolone and taxol compunds are both frequently used in cancer chemotherapies, and our results shed light on the possible molecular mechanisms of neurological side effects (such as debilitating nerve pain) when these agents are used.

Lastly, we looked at the effects of tau on intracellular trafficking in COS-7 cells. We found that the microtubule network in the cells were dramatically modified after the introduction of tau via transient transfection, with substantial bundling, aggregation and membrane-association in the presence of tau. However, there were no obvious differences in the microtubule network structures between cells that were transfected with mutant tau and wild-type tau. Interestingly, despite the major modification of the cytoskeletal structures in the presence of tau, the measured velocities of lysosomes in directed transport in cells that were transfected with tau were not significantly different compared to the lysosome velocities in cells with no tau.

Taken together, studies provide important new insights into how tau proteins and other small molecule stabilizers modulate the mechanical and functional properties of cytoskeletal microtubules and how misregulations in tau may be related to the development of neurodegeneration and dementia diseases.