Rapid binding to protofilament edge sites facilitates tip tracking of EB1 at growing microtubule plus-ends.

EB1 is a key cellular protein that delivers regulatory molecules throughout the cell via the tip-tracking of growing microtubule plus-ends. Thus, it is important to understand the mechanism for how EB1 efficiently tracks growing microtubule plus-ends. It is widely accepted that EB1 binds with higher affinity to GTP-tubulin subunits at the growing microtubule tip, relative to GDP-tubulin along the microtubule length. However, it is unclear whether this difference in affinity alone is sufficient to explain the tip-tracking of EB1 at growing microtubule tips. Previously, we found that EB1 binds to exposed microtubule protofilament-edge sites at a ~70 fold faster rate than to closed-lattice sites, due to diffusional steric hindrance to binding. Thus, we asked whether rapid protofilament-edge binding could contribute to efficient EB1 tip tracking. A computational simulation with differential EB1 on-rates based on closed-lattice or protofilament-edge binding, and with EB1 off-rates that were dependent on the tubulin hydrolysis state, robustly recapitulated experimental EB1 tip tracking. To test this model, we used cell-free biophysical assays, as well as live-cell imaging, in combination with a Designed Ankyrin Repeat Protein (DARPin) that binds exclusively to protofilament-edge sites, and whose binding site partially overlaps with the EB1 binding site. We found that DARPin blocked EB1 protofilament-edge binding, which led to a decrease in EB1 tip tracking on dynamic microtubules. We conclude that rapid EB1 binding to microtubule protofilament-edge sites contributes to robust EB1 tip tracking at the growing microtubule plus-end.

Robust microtubule dynamics facilitate low-tension kinetochore detachment in metaphase.

During mitosis, sister chromatids are stretched apart at their centromeres via their attachment to oppositely oriented kinetochore microtubules. This stretching generates inwardly directed tension across the separated sister centromeres. The cell leverages this tension signal to detect and then correct potential errors in chromosome segregation, via a mechanical tension signaling pathway that detaches improperly attached kinetochores from their microtubules. However, the sequence of events leading up to these detachment events remains unknown. In this study, we used microfluidics to sustain and observe low-tension budding yeast metaphase spindles over multiple hours, allowing us to elucidate the tension history prior to a detachment event. We found that, under conditions in which kinetochore phosphorylation weakens low-tension kinetochore-microtubule connections, the mechanical forces produced via the dynamic growth and shortening of microtubules is required to efficiently facilitate detachment events. Our findings underscore the critical role of robust kinetochore microtubule dynamics in ensuring the fidelity of chromosome segregation during mitosis.

Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate dynamic instability.

Kinesin-14 molecular motors represent an essential class of proteins that bind microtubules and walk toward their minus-ends. Previous studies have described important roles for Kinesin-14 motors at microtubule minus-ends, but their role in regulating plus-end dynamics remains controversial. Kinesin-14 motors have been shown to bind the EB family of microtubule plus-end binding proteins, suggesting that these minus-end-directed motors could interact with growing microtubule plus-ends. In this work, we explored the role of minus-end-directed Kinesin-14 motor forces in controlling plus-end microtubule dynamics. In cells, a Kinesin-14 mutant with reduced affinity to EB proteins led to increased microtubule lengths. Cell-free biophysical microscopy assays were performed using Kinesin-14 motors and an EB family marker of growing microtubule plus-ends, Mal3, which revealed that when Kinesin-14 motors bound to Mal3 at growing microtubule plus-ends, the motors subsequently walked toward the minus-end, and Mal3 was pulled away from the growing microtubule tip. Strikingly, these interactions resulted in an approximately twofold decrease in the expected postinteraction microtubule lifetime. Furthermore, generic minus-end-directed tension forces, generated by tethering growing plus-ends to the coverslip using λ-DNA, led to an approximately sevenfold decrease in the expected postinteraction microtubule growth length. In contrast, the inhibition of Kinesin-14 minus-end-directed motility led to extended tip interactions and to an increase in the expected postinteraction microtubule lifetime, indicating that plus-ends were stabilized by nonmotile Kinesin-14 motors. Together, we find that Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate microtubule lengths in cells.

Oxidative stress pathogenically remodels the cardiac myocyte cytoskeleton via structural alterations to the microtubule lattice.

In the failing heart, the cardiac myocyte microtubule network is remodeled, which contributes to cellular contractile failure and patient death. However, the origins of this deleterious cytoskeletal reorganization are unknown. We now find that oxidative stress, a condition characteristic of heart failure, leads to cysteine oxidation of microtubules. Our electron and fluorescence microscopy experiments revealed regions of structural damage within the microtubule lattice that occurred at locations of oxidized tubulin. The incorporation of GTP-tubulin into these damaged, oxidized regions led to stabilized "hot spots" within the microtubule lattice, which suppressed the shortening of dynamic microtubules. Thus, oxidative stress may act inside of cardiac myocytes to facilitate a pathogenic shift from a sparse microtubule network into a dense, aligned network. Our results demonstrate how a disease condition characterized by oxidative stress can trigger a molecular oxidation event, which likely contributes to a toxic cellular-scale transformation of the cardiac myocyte microtubule network.