Cryo electron tomography of vitrified fibroblasts: Microtubule plus ends in situ

Roman I. Koning*, Sandra Zovko, Montserrat Barcena, Gert T. Oostergetel, Henk K. Koerten, Niels Galjart, Abraham J. Koster, A. Mieke Mommaas

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

40 Citaten (Scopus)

Samenvatting

Mouse embryonic fibroblasts (MEFs) are cells that have highly suitable biophysical properties for cellular cryo electron tomography. MEFs can be grown directly on carbon supported by EM grids. They stretch out and grow thinner than 500 mn over major parts of the cell, attaining a minimal thickness of 50 nm at their cortex. This facilitates direct cryo-fixation by plunge-freezing and high resolution cryo electron tomography. Both by direct cryo electron microscopy projection imaging and cryo electron tomography of vitrified MEFs we visualized a variety of cellular structures like ribosomes, vesicles, mitochondria, rough endoplasmatic reticulum, actin filaments, intermediate filaments and microtubules.

MEFs are primary cells that closely resemble native tissue and are highly motile. Therefore, they are attractive for studying cytoskeletal elements. Here we report on structural investigations of microtubule plus ends. We were able to visualize single frayed protofilaments at the microtubule plus end in vitrified fibroblasts using cryo electron tomography. Furthermore, it appeared that MEFs contain densities inside their microtubules, although 2.5-3.5 times less than in neuronal cells [Garvalov, B.K., Zuber, B., Bouchet-Marquis, C., Kudryashev, M., Gruska, M., Beek, M., Leis, A., Frischknecht, F., Bradke, F., Baumeister, W., Dubochet, J., and Cyrklaff, M. 2006. Luminal particles within cellular microtubules. J. Cell Biol. 174, 759-765]. Projection imaging of cellular microtubule plus ends showed that 40% was frayed, which is two times more than expected when compared to microtubule growth and shrinkage rates in MEFs. This suggests that frayed ends might be stabilized in the cell cortex. (C) 2007 Elsevier Inc. All rights reserved.

Originele taal-2English
Pagina's (van-tot)459-468
Aantal pagina's10
TijdschriftJournal of Structural Biology
Volume161
Nummer van het tijdschrift3
DOI's
StatusPublished - mrt-2008
Evenement4th International Conference on Electron Tomography - , Canada
Duur: 5-nov-20068-nov-2006

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