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Three-Dimensional Microtubule Behavior in Xenopus Egg Extracts Reveals Four Dynamic States and State-Dependent Elastic Properties

Philipp J. Keller ^*, Francesco Pampaloni ^*, Gianluca Lattanzi  and Ernst H. K. Stelzer ^*

^* Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany; and Department of Medical Biochemistry, Biology and Physics TIRES-Center and INFN, University of Bari, Bari, Italy

Correspondence: Address reprint requests to Philipp J. Keller or Ernst H. K. Stelzer, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117, Heidelberg, Germany. Tel.: 49-6221-3878123, Fax: 49-6221-387306. E-mail: keller{at}embl.de, stelzer{at}embl.de.

Although microtubules are key players in many cellular processes, very little is known about their dynamic and mechanical properties in physiological three-dimensional environments. The conventional model of microtubule dynamic instability postulates two dynamic microtubule states, growth and shrinkage. However, several studies have indicated that such a model does not provide a comprehensive quantitative and qualitative description of microtubule behavior. Using three-dimensional laser light-sheet fluorescence microscopy and a three-dimensional sample preparation in spacious Teflon cylinders, we measured microtubule dynamic instability and elasticity in interphase Xenopus laevis egg extracts. Our data are inconsistent with a two-state model of microtubule dynamic instability and favor an extended four-state model with two independent metastable pause states over a three-state model with a single pause state. Moreover, our data on kinetic state transitions rule out a simple GTP cap model as the driving force of microtubule stabilization in egg extracts on timescales of a few seconds or longer. We determined the three-dimensional elastic properties of microtubules as a function of both the contour length and the dynamic state. Our results indicate that pausing microtubules are less flexible than growing microtubules and suggest a growth-speed-dependent persistence length. These data might hint toward mechanisms that enable microtubules to efficiently perform multiple different tasks in the cell and suggest the development of a unified model of microtubule dynamics and microtubule mechanics.