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广东快乐十分奖金:Direct observation of individual tubulin dimers binding to growing microtubules
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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 plus-tip kinetics. 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 insight into microtubule tip structure and potential modes of microtubule dynamics regulation.
The biochemical basis of microtubule growth has remained elusive for over 30 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 direct, single-molecule measurements of tubulin association and dissociation rates. We detect two populations of transient dwell times and determine via binding-interface mutants that they are distinguished by the formation of one interprotofilament bond. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data and, furthermore, predicts plus-end tapering. Overall, we provide the most direct and complete experimental quantification of how microtubules grow to date.
?1E.A.G. and T.K. contributed equally to this work.
- ?2To whom correspondence may be addressed. Email: or .
Author contributions: K.J.M., L.M.R., and W.O.H. designed research; K.J.M., E.A.G., and T.K. performed research; E.A.G. contributed new reagents/analytic tools; K.J.M. built microscope for studies; T.K. carried out simulations; K.J.M. and T.K. analyzed data; and K.J.M., L.M.R., and W.O.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
See Commentary on page 7163.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1815823116/-/DCSupplemental.
Published under the PNAS license.