Modification of the neck-linker of KIF18A alters Microtubule subpopulation preference.

Kinesins support many diverse cellular processes, including facilitating cell division through mechanical regulation of the mitotic spindle. However, how kinesin activity is controlled to facilitate this process is not well understood. Interestingly, posttranslational modifications have been identified within the enzymatic region of all 45 mammalian kinesins, but the significance of these modifications has gone largely unexplored. Given the critical role of the enzymatic region in facilitating nucleotide and microtubule binding, it may serve as a primary site for kinesin regulation. Consistent with this idea, a phosphomimetic mutation at S357 in the neck-linker of KIF18A alters the localization of KIF18A within the spindle from kinetochore microtubules to nonkinetochore microtubules at the periphery of the spindle. Changes in localization of KIF18A-S357D are accompanied by defects in mitotic spindle positioning and the ability to promote mitotic progression. This altered localization pattern is mimicked by a shortened neck-linker mutant, suggesting that KIF18A-S357D may cause the motor to adopt a shortened neck-linker-like state that decreases KIF18A accumulation at the plus-ends of kinetochore microtubules. These findings demonstrate that posttranslational modifications in the enzymatic region of kinesins could be important for biasing their localization to particular microtubule subpopulations.

Glucose-stimulated KIF5B-driven microtubule sliding organizes microtubule networks in pancreatic beta cells.

In pancreatic islet beta cells, molecular motors use cytoskeletal polymers microtubules as tracks for intracellular transport of insulin secretory granules. Beta-cell microtubule network has a complex architecture and is non-directional, which provide insulin granules at the cell periphery for rapid secretion response, yet to avoid over-secretion and subsequent hypoglycemia. We have previously characterized a peripheral sub-membrane microtubule array, which is critical for withdrawal of excessive insulin granules from the secretion sites. Microtubules in beta cells originate at the Golgi in the cell interior, and how the peripheral array is formed is unknown. Using real-time imaging and photo-kinetics approaches in clonal mouse pancreatic beta cells MIN6, we now demonstrate that kinesin KIF5B, a motor protein with a capacity to transport microtubules as cargos, slides existing microtubules to the cell periphery and aligns them to each other along the plasma membrane. Moreover, like many physiological beta-cell features, microtubule sliding is facilitated by a high glucose stimulus. These new data, together with our previous report that in high glucose sub-membrane MT array is destabilized to allow for robust secretion, indicate that MT sliding is another integral part of glucose-triggered microtubule remodeling, likely replacing destabilized peripheral microtubules to prevent their loss over time and beta-cell malfunction.

Tau, microtubule dynamics, and axonal transport: New paradigms for neurodegenerative disease.

The etiology of Tauopathies, a diverse class of neurodegenerative diseases associated with the Microtubule Associated Protein (MAP) Tau, is usually described by a common mechanism in which Tau dysfunction results in the loss of axonal microtubule stability. Here, we reexamine and build upon the canonical disease model to encompass other Tau functions. In addition to regulating microtubule dynamics, Tau acts as a modulator of motor proteins, a signaling hub, and a scaffolding protein. This diverse array of functions is related to the dynamic nature of Tau isoform expression, post-translational modification (PTM), and conformational flexibility. Thus, there is no single mechanism that can describe Tau dysfunction. The effects of specific pathogenic mutations or aberrant PTMs need to be examined on all of the various functions of Tau in order to understand the unique etiology of each disease state.

Modification of the Neck Linker of KIF18A Alters Microtubule Subpopulation Preference.

Kinesins support many diverse cellular processes, including facilitating cell division through mechanical regulation of the mitotic spindle. However, how kinesin activity is controlled to facilitate this process is not well understood. Interestingly, post-translational modifications have been identified within the enzymatic region of all 45 mammalian kinesins, but the significance of these modifications has gone largely unexplored. Given the critical role of the enzymatic region in facilitating nucleotide and microtubule binding, it may serve as a primary site for kinesin regulation. Consistent with this idea, a phosphomimetic mutation at S357 in the neck-linker of KIF18A alters the localization of KIF18A within the spindle from kinetochore microtubules to peripheral microtubules. Changes in localization of KIF18A-S357D are accompanied by defects in mitotic spindle positioning and the ability to promote mitotic progression. This altered localization pattern is mimicked by a shortened neck-linker mutant, suggesting that KIF18A-S357D may cause the motor to adopt a shortened neck-linker like state that prevents KIF18A from accumulating at the plus-ends of kinetochore microtubules. These findings demonstrate that post-translational modifications in the enzymatic region of kinesins could be important for biasing their localization to particular microtubule subpopulations.

The N-terminal disease-associated R5L Tau mutation increases microtubule shrinkage rate due to disruption of microtubule-bound Tau patches.

Regulation of the neuronal microtubule cytoskeleton is achieved through the coordination of microtubule-associated proteins (MAPs). MAP-Tau, the most abundant MAP in the axon, functions to modulate motor motility, participate in signaling cascades, as well as directly mediate microtubule dynamics. Tau misregulation is associated with a class of neurodegenerative diseases, known as tauopathies, including progressive supranuclear palsy, Pick's disease, and Alzheimer's disease. Many disease-associated mutations in Tau are found in the C-terminal microtubule-binding domain. These mutations decrease microtubule-binding affinity and are proposed to reduce microtubule stability, leading to disease. N-terminal disease-associated mutations also exist, but the mechanistic details of their downstream effects are not as clear. Here, we investigate the effect of the progressive supranuclear palsy-associated N-terminal R5L mutation on Tau-mediated microtubule dynamics using an in vitro reconstituted system. We show that the R5L mutation does not alter Tau interactions with tubulin by fluorescence correlation spectroscopy. Using total internal reflection fluorescence microscopy, we determined that the R5L mutation has no effect on microtubule growth rate, catastrophe frequency, or rescue frequency. Rather, the R5L mutation increases microtubule shrinkage rate. We determine this is due to disruption of Tau patches, larger order Tau complexes known to form on the GDP-microtubule lattice. Altogether, these results provide insight into the role of Tau patches in mediating microtubule dynamics and suggesting a novel mechanism by which mutations in the N-terminal projection domain reduce microtubule stability.

The pathogenic R5L mutation disrupts formation of Tau complexes on the microtubule by altering local N-terminal structure.

The microtubule-associated protein (MAP) Tau is an intrinsically disordered protein (IDP) primarily expressed in axons, where it functions to regulate microtubule dynamics, modulate motor protein motility, and participate in signaling cascades. Tau misregulation and point mutations are linked to neurodegenerative diseases, including progressive supranuclear palsy (PSP), Pick's disease, and Alzheimer's disease. Many disease-associated mutations in Tau occur in the C-terminal microtubule-binding domain of the protein. Effects of C-terminal mutations in Tau have led to the widely accepted disease-state theory that missense mutations in Tau reduce microtubule-binding affinity or increase Tau propensity to aggregate. Here, we investigate the effect of an N-terminal arginine to leucine mutation at position 5 in Tau (R5L), associated with PSP, on Tau-microtubule interactions using an in vitro reconstituted system. Contrary to the canonical disease-state theory, we determine that the R5L mutation does not reduce Tau affinity for the microtubule using total internal reflection fluorescence microscopy. Rather, the R5L mutation decreases the ability of Tau to form larger-order complexes, or Tau patches, at high concentrations of Tau. Using NMR, we show that the R5L mutation results in a local structural change that reduces interactions of the projection domain in the presence of microtubules. Altogether, these results challenge both the current paradigm of how mutations in Tau lead to disease and the role of the projection domain in modulating Tau behavior on the microtubule surface.