Single-molecule imaging of Tau dynamics on the microtubule surface.

The microtubule-associated protein Tau is primarily expressed in neurons and plays an integral role in the regulation of multiple functions within the axon. In the adult brain, the six Tau isoforms are expressed allowing for a complex system of control. Despite Tau's central role, the mechanisms by which Tau acts are not fully understood. We have used single-molecule total internal reflection fluorescence (TIRF) microscopy and the methods described in this chapter to further our knowledge of Tau's behavior and function. We have demonstrated that Tau's dynamic binding behavior allows for regulation of motor protein motility and microtubule dynamics in an isoform-specific manner. The continued use and refinement of the single-molecule techniques detailed here can only further our knowledge of Tau and other proteins integral to the maintenance of axonal transport.

Phosphoregulation of Tau modulates inhibition of kinesin-1 motility.

Microtubule-based axonal transport is tightly regulated by numerous pathways, ensuring appropriate delivery of specific organelle cargoes to selected subcellular domains. Highlighting the importance of this process, pathological evidence has linked alterations in these pathways to the pathogenesis of several neurodegenerative diseases. An important regulator of this system, the microtubule-associated protein Tau, has been shown to participate in signaling cascades, modulate microtubule dynamics, and preferentially inhibit kinesin-1 motility. However, the cellular means of regulating Tau's inhibition of kinesin-1 motility remains unknown. Tau is subject to various posttranslational modifications, including phosphorylation, but whether phosphorylation regulates Tau on the microtubule surface has not been addressed. It has been shown that tyrosine 18 phosphorylated Tau regulates inhibition of axonal transport in the disease state. Tyrosine 18 is both a disease- and nondisease-state modification and is therefore an attractive starting point for understanding control of Tau's inhibition of kinesin-1 motility. We show that pseudophosphorylation of tyrosine 18 reduces 3RS-Tau's inhibition of kinesin-1 motility. In addition, we show that introduction of negative charge at tyrosine 18 shifts Tau's previously described static-dynamic state binding equilibrium toward the dynamic state. We also present the first evidence of Tau's static-dynamic state equilibrium under physiological conditions.