Tau differentially regulates the transport of early endosomes and lysosomes.

Microtubule-associated proteins (MAPs) modulate the motility of kinesin and dynein along microtubules to control the transport of vesicles and organelles. The neuronal MAP tau inhibits kinesin-dependent transport. Phosphorylation of tau at Tyr-18 by fyn kinase results in weakened inhibition of kinesin-1. We examined the motility of early endosomes and lysosomes in cells expressing wild-type (WT) tau and phosphomimetic Y18E tau. We quantified the effects on motility as a function of the tau expression level. Lysosome motility is strongly inhibited by tau. Y18E tau preferentially inhibits lysosomes in the cell periphery, while centrally located lysosomes are less affected. Early endosomes are more sensitive to tau than lysosomes and are inhibited by both WT and Y18E tau. Our results show that different cargoes have disparate responses to tau, likely governed by the types of kinesin motors driving their transport. In support of this model, kinesin-1 and -3 are strongly inhibited by tau while kinesin-2 and dynein are less affected. In contrast to kinesin-1, we find that kinesin-3 is strongly inhibited by phosphorylated tau.

Polyglutamylation of tubulin's C-terminal tail controls pausing and motility of kinesin-3 family member KIF1A.

The kinesin-3 family member KIF1A plays a critical role in site-specific neuronal cargo delivery during axonal transport. KIF1A cargo is mislocalized in many neurodegenerative diseases, indicating that KIF1A's highly efficient, superprocessive motility along axonal microtubules needs to be tightly regulated. One potential regulatory mechanism may be through posttranslational modifications (PTMs) of axonal microtubules. These PTMs often occur on the C-terminal tails of the microtubule tracks, act as molecular "traffic signals" helping to direct kinesin motor cargo delivery, and include C-terminal tail polyglutamylation important for KIF1A cargo transport. KIF1A initially interacts with microtubule C-terminal tails through its K-loop, a positively charged surface loop of the KIF1A motor domain. However, the role of the K-loop in KIF1A motility and response to perturbations in C-terminal tail polyglutamylation is underexplored. Using single-molecule imaging, we present evidence that KIF1A pauses on different microtubule lattice structures, linking multiple processive segments together and contributing to KIF1A's characteristic superprocessive run length. Furthermore, modifications of the KIF1A K-loop or tubulin C-terminal tail polyglutamylation reduced KIF1A pausing and overall run length. These results suggest a new mechanism to regulate KIF1A motility via pauses mediated by K-loop/polyglutamylated C-terminal tail interactions, providing further insight into KIF1A's role in axonal transport.

KIF18A's neck linker permits navigation of microtubule-bound obstacles within the mitotic spindle.

KIF18A (kinesin-8) is required for mammalian mitotic chromosome alignment. KIF18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that KIF18A's relatively long neck linker is required for the motor's accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of KIF18A display a deficiency in accumulation at the ends of K-fibers at the center of the spindle. Depletion of K-fiber-binding proteins reduces the KIF18A sNL localization defect, whereas their overexpression reduces wild-type KIF18A's ability to accumulate on this same K-fiber subset. Furthermore, single-molecule assays indicate that KIF18A sNL motors are less proficient in navigating microtubules coated with microtubule-associated proteins. Taken together, these results support a model in which KIF18A's neck linker length permits efficient navigation of obstacles to reach K-fiber ends during mitosis.

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.