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Vol. 19, Issue 7, 3138-3146, July 2008

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Microtubule Plus-End Conformations and Dynamics in the Periphery of Interphase Mouse Fibroblasts

Sandra Zovko^*, Jan Pieter Abrahams, Abraham J. Koster^*, Niels Galjart, and A. Mieke Mommaas^*

*Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands; Department of Biophysical Structural Chemistry, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands; Department of Cell Biology and Genetics, Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands

Submitted August 15, 2007; Revised April 21, 2008; Accepted May 2, 2008
Monitoring Editor: Erika L. Holzbaur

The plus ends of microtubules (MTs) alternate between phases of growth, pause, and shrinkage, a process called "dynamic instability." Cryo-EM of in vitro–assembled MTs indicates that the dynamic state of the plus end corresponds with a particular MT plus-end conformation. Frayed ("ram's horn like"), blunt, and sheet conformations are associated with shrinking, pausing, and elongating plus ends, respectively. A number of new conformations have recently been found in situ but their dynamic states remained to be confirmed. Here, we investigated the dynamics of MT plus ends in the peripheral area of interphase mouse fibroblasts (3T3s) using electron microscopical and tomographical analysis of cryo-fixed, freeze-substituted, and flat-embedded sections. We identified nine morphologically distinct plus-end conformations. The frequency of these conformations correlates with their proximity to the cell border, indicating that the dynamic status of a plus end is influenced by features present in the periphery. Shifting dynamic instability toward depolymerization with nocodazole enabled us to address the dynamic status of these conformations. We suggest a new transition path from growth to shrinkage via the so-called sheet-frayed and flared ends, and we present a kinetic model that describes the chronology of events taking place in nocodazole-induced MT depolymerization.

Address correspondence to: Sandra Zovko (zovko.sandra{at}gmail.com).

Abbreviations used: EB1, end binding protein 1; EM, electron microscopy; ET, electron tomography; GDP, guanosine 5'-diphosphate; GTP, guanosine 5'-triphosphate; kMT, kinetochore microtubule; LM, light microscopy; MT, microtubule; +TIPS, plus-end tracking proteins.