Direct observation of individual tubulin dimers binding to growing microtubules

Keith J. Mickolajczyk, Elisabeth A. Geyer, Tae Kim, Luke M. Rice, William O. Hancock

Research output: Contribution to journalArticlepeer-review

Abstract

The biochemical basis of microtubule growth has remained elusive for over thirty years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make the first direct, single-molecule measurements of tubulin on- and off-rates. We detect two populations of transient dwell times, and determine via binding-interface mutants that they are separated by the formation of inter-protofilament bonds. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data, and furthermore predict plus-end tapering. Overall, we provide the most direct and complete quantification of how microtubules grow to date. SIGNIFICANCE Microtubule polymerization dynamics are fundamental to cell migration and cell division, where they are targets for chemotherapy drugs. Despite significant progress, the precise structural and biochemical events occurring at growing microtubule tips are not well defined, and better understanding is necessary for discriminating mechanisms of microtubule dynamics regulation in cells. Here, we visualize individual tubulin subunits reversibly and irreversibly interacting with dynamic microtubule tips, and thereby directly measure tubulin on- and off-rates. By analyzing plus-tip residence times of wild-type and mutant tubulin, we characterize the relative contributions of longitudinal (along protofilaments) and lateral (between protofilaments) bond energies to microtubule growth. This work provides insights into microtubule tip structure and potential modes of microtubule dynamics regulation.

Original languageEnglish (US)
JournalUnknown Journal
DOIs
StatePublished - Sep 14 2018

Keywords

  • Interferometric scattering microscopy
  • microtubule dynamics
  • single-molecule kinetics

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

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