No abstract

No abstract

No abstract

No abstract

The microtubule associated protein (MAP) Tau, primarily expressed in neurons, is known to have a variety of functions such as regulation of microtubule dynamics, modulation of kinesin motor motility and participation in signaling cascades. Research on the microtubule binding behavior of Tau reveals that Tau binds to the microtubule surface in an equilibrium between static and diffusive states. These functional states have been shown to be important in regulating kinesin motility during axonal transport. However, the structural relationship between the states has not been characterized. Therefore, using Total Internal Reflection Fluorescence (TIRF) microscopy, we developed a threecolor imaging assay to study the structural changes underlying Tau's dynamic behavior while bound to the microtubule surface using single molecule Fluorescence Resonance Energy Transfer (smFRET). Additionally, Alternating Laser Excitation (ALEX) is used to distinguish between single labeled populations and low FRET efficiency states. We have generated three 3RS-Tau FRET constructs to measure distances between distinct locations within Tau, between the N and C termini (N-C), between the microtubule binding repeats and the C terminal (3-C), and between the N terminal and the microtubule binding repeats (N-3). Initial studies indicate 3RS-Tau possesses distinct N and C termini interactions that allow for static versus diffusive binding. The examination of additional interactions will define overall structural changes in Tau on the microtubule surface. The smFRET-ALEX approach we have developed has applications for studying differences in other highly dynamic MAPs as well.