Hoenger Lab
University of Colorado at Boulder
Department of Molecular, Cellular and Developmental Biology

Structural and Functional Investigations
into Cytoskeletal Assemblies by
Cryo Electron Microscopy
and 3D Image Analysis

 
Research:: Microtubules

The cytoskeletal system of eukaryotic and prokaryotic cells is composed of three types of structures, microtubules, intermediate filaments, and actin.

Microtubules have a variety of functions in the cell including: scaffolding to give cell shape, they serve as roadways for intracellular transport of mitochondria and other vesicles, and are the core components of mitotic spindles, centrosomes, basal bodies, and cilia and flagella. Microtubules dynamics drive cellular processes such as chromosome segregation and cell division

Microtubules polymerize from tubulin dimers (alpha and beta subunits) and in nature are often 13 protofilaments. They are about 25 nanometers wide and shaped like a hollow tube.

In an effort to elucidate the structure and describe the function and the interaction of molecular motors and microtubules, our laboratory examines these molecules using cryo electron micorscopy and three dimensional image reconstruction.

microtubules
 microtubules

 

Selected Publications:

 

Structural rearrangements in tubulin following microtubule formation (pdf)


EMBO Rep. 2005 March; 6(3): 227–232.
Published online 2005 February 25.

Angelika Krebs, Kenneth N Goldie, and Andreas Hoenger

Abstract

Microtubules are essential cytoskeletal structures that mediate several dynamic processes in a cell. To shed light on the structural processes relating to microtubule formation and dynamic instability, we investigated microtubules composed of 15 protofilaments using cryo-electron microscopy, helical image reconstruction and computational modelling. Analysis of the configuration of the αβ-tubulin heterodimer shows distinct structural differences in both subunits, and illustrates that the tubulin subunits have different roles in the microtubule lattice. Our modelling data suggest that after GTP hydrolysis microtubules, adopt a conformational state somewhere between a straight protofilament conformation—as found in zinc-induced tubulin sheets—and an outward curved conformation—as found in tubulin–stathmin complexes. The tendency towards a curved conformation seems to be mediated mostly by β-tubulin, whereas α-tubulin resembles a state more related to the straight structure. Our data suggest a possible explanation of dynamic instability of microtubules, and for nucleotide-sensitive microtubule-binding properties of microtubule-associated proteins and molecular motors.

3-D Reconstructions from Ice-Embedded and Negatively Stained Biomacromolecular Assemblies:
A Critical Comparison

Journal of Structural Biology, Volume 117, Number 2, September 1996 , pp. 99-116(18)

Hoenger A.; Aebi U.

Abstract

Three-dimensional (3-D) image reconstruction of electron microscopy (EM) data offers a powerful tool in structural biology that provides important information about the structure and function of individual biomacromolecules, their oligomers, and supramolecular assemblies. Forming an important link between atomic-resolution X-ray or NMR data of individual proteins and their supramolecular complexes, 3-D EM data allow the combining of structural information from all sources to assess protein function at atomic detail. Within the last several years, a range of image reconstruction methods were introduced aiming to determine 3-D structures of biomacromolecular assemblies to intermediate resolution (i.e., <20 A). To achieve a faithful representation of the specimen under investigation its preparation for EM is of critical importance. Whereas earlier 3-D reconstructions were predominantly obtained with negatively stained specimens, more recently the potential of cryofixation/ice embedding has definitely become widely recognized. Imaging the biomacromolecule itself rather than a heavy metal cast surrounding it, by freezing the sample in its physiological buffer environment, cryofixation/ice embedding was found to reduce adsorption artifacts and to reproduce certain structural details more accurately and faithfully. Most importantly, in addition to mapping out the overall size and shape of biomacromolecules, cryo-EM enables us to look "inside" biomacromolecules and image their secondary structure elements or even atomic detail. Despite these obvious advantages of ice embedding over negative staining, however, both preparation methods have their own strengths and limitations which should be carefully evaluated based on the particular structural and biological question asked.

Polarity of 2-D and 3-D Maps of Tubulin Sheets and Motor-decorated Sheets

Journal of Molecular Biology, Volume 263, Number 2, October 1996 , pp. 114-119(6)

Hoenger A.; Milligan R.A.

Abstract

As there has been considerable disagreement about the polarity of two-dimensional and three-dimensional maps of tubulin and tubulin - motor complexes, we have re-investigated this issue by analyzing images of tubulin sheets and motor-decorated sheets found at the distal ends of microtubules nucleated from centrosomes and sperm tails. Our results are unambiguous and define the relationship of the structural features in the maps to the microtubule plus and minus ends. Possible reasons for previous mis-assignments of the polarity are discussed briefly.
 

University of Colorado, MCDB, 347 UCB, Boulder, CO 80309-0347