Biology subjects

Reid, T. A.

Publications and source records attributed to Reid, T. A..

3 recordsLinked to original sources

bioRxiv2022

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

EB1 is a key cellular protein that delivers critical 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 tubulin hydrolysis state, recapitulated experimental EB1 tip tracking. To test this model, we used cell-free biophysical assays, as well as live-cell imaging, in combination with the chemotherapy drug Eribulin. We found that Eribulin blocked EB1 protofilament-edge binding, which led to a dramatic decrease in EB1 tip tracking on dynamic microtubules. We conclude that rapid EB1 binding to microtubule protofilament-edge sites increases the efficiency of EB1 tip tracking at the growing microtubule plus end.

cell biology↗

bioRxiv2020

Student-focused development of a next-generation centrifuge force microscope

Advanced biological molecule force probing methods such as atomic force microscopy and optical tweezers used to quantify forces at the single-molecule level are expensive and require extensive training and technical knowledge. However, the technologies underlying a centrifuge force microscope (CFM) are relatively straight forward, allowing for construction by labs with relatively low budgets and minimal training. Design ideas from previously constructed CFMs served as a guide in the development of this CFM. There were two primary goals: first, to develop an inexpensive, functional CFM using off-the-shelf and 3D printed parts; and second, to do so in the context of providing an educational experience for a broad range of students. The team included high school students and undergraduates from local high schools, the University of Minnesota, and other local higher education institutions. This project created an environment for student-focused development of the CFM that fostered active learning, individual ownership, as well as excellence in research. The instrument discussed herein represents a fully functional CFM designed and built by a postdoctoral researcher and a graduate student who together mentored several high school and undergraduate students. STATEMENT OF SIGNIFICANCEThe presented centrifuge force microscope (CFM) builds on features of existing designs specifically engineered for probing macromolecular force interactions at the single-molecule level. In the coming years, more versatile and modular CFM designs will be utilized in the force spectroscopy field, and the presented design is a step in that direction. In addition to constructing a functional instrument, true student ownership of the project design was equally an end goal. Students from high school through graduate school were included, and the project was structured so that everyone was seen as peers. This active learning project allowed students to acquire scientific concepts and techniques and apply them to real-life situations.

biophysics↗

bioRxiv2019

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends in cells

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a [~]6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the conversion of growing microtubule ends from a tapered into a blunt configuration resulted in reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

cell biology↗