Nanomechanics of Tubulin Extraction from Microtubules and Adhesin Catch-Bond Rupture

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

Cell shape and structure are supported by a filamentous network composed in part by microtubules, dynamic assemblies of building blocks known as tubulin dimers, that are constantly remodeled according to the cell’s needs. These assemblies play important roles in cell division and organization and serve as “tracks” for the transport of cargo. The assemblies can be disassembled by cellular machinery that extracts a tubulin dimer from the microtubule, but the force required to do so and the overall stability of the microtubule is unknown. Understanding this process will provide insight in how mutations lead to microtubule defects that have physiological consequences, and to understanding how cancer drugs like Taxol exert their effects. This project’s technology development will facilitate better characterization of compounds such as Taxol by enabling direct measurement of the degree of stabilization at the level of a single tubulin dimer. The interdisciplinary nature of this project will provide an excellent training opportunity for high-school, undergraduate, and graduate students and will be integrated into a course, with a hands-on lab component. This project will establish a single-molecule force-spectroscopy assay to extract tubulin from a microtubule (MT) by pulling on the C-terminal tail of β tubulin. Advanced atomic force microscopy (AFM) studies will characterize the nanomechanical properties of tubulin extraction using a variety of pulling protocols and modified AFM cantilevers for improved force precision and temporal resolution. Analysis of the resulting data will yield the energetics and forces that stabilize tubulin within the MT lattice. It will also yield insight into the mechanism of severing enzymes, which act against the force needed to extract tubulin. Severing enzymes are molecular motors that extract tubulin dimers from MTs and are an important means to modulate MT dynamics. The mechanism of severing enzymes remains unknown. Related hexameric molecular motors exert a maximum force of ~20–30 pN. Yet, coarse-grained simulations predict a much higher force (~400 pN) needed to extract a tubulin dimer. This project will resolve this dichotomy by directly measuring the force needed to extract an individual tubulin dimer from a MT, a long-sought goal. To facilitate this assay development, the mechanical properties of several adhesin-ligand pairs will be characterized as a means to optimize their use as short, genetically encoded handles and evaluate their catch bond behavior. This project will thereby accelerate AFM-based force-spectroscopy studies of diverse biological systems including membrane proteins and nucleic acids. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.