Porter room B031A
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Explore Andy Hoenger's areas of research and more in Vivo
Ph.D., University of Basel, 1993
Structural and functional investigations into cytoskeletal assemblies by cryo electron microscopy and 3D image analysis.
My function at MCDB is two-fold. First, I took over the directorship of the IVEM Lab from Prof. JR McIntosh, and secondly I will continue my personal research that focuses on cryo-electron microscopy (cryo-EM) based three-dimensional (3-D) reconstruction of large macromolecular assemblies and cellular structures, whenever possible within the in vivo context of an intact cell. These two responsibilities match well together and as such there will be a strong interaction between the two units. So far structural biology was often confined to in vitro approaches, thereby reducing a complex biological system to a very limited problem. While this will certainly still be the case for some time for generating atomic-resolution data (X-ray, electron crystallography or NMR spectroscopy), cryo-electron microscopy in combination with new (tomographic) 3D reconstruction approaches clearly has a perspective to investigate cellular structures in vivo, not necessarily at atomic resolution, but at 2-3 nm detail which will allow to recognize single protein domains within the context of larger macromolecular assemblies and cellular organelles, directly in the cell. With my background on microtubule structures and associated proteins such as MAPS and molecular motors of the Dynein and Kinesin families, a correlative microscopy approach will be very useful for high-resolution studies on various dynamic aspects in cells such as cell division (e.g. Cancer research), and intracellular transport processes (e.g. in axons, neuronal tissues, Neuroscience). Apart from focussing on microtubule structures alone, I am planning to further investigate the regulation of the entire cytoskeleton, and in particular the functional connections between microtubules, actin and intermediate filament structures in an intact cell.
Preserving the cellular architecture will be a key-preparative issue, while handling 3D data of large cellular structures will be the key-interpretative issue for the future. This requires developing procedures to handle large specimens and applying methods for cryo-EM based 3-D reconstructions, which are beyond the conventional averaging methods. To date the large majority of (cryo-) EM derived 3-D data are derived from approaches, which attempt to average large numbers of (seemingly) identical image elements and therefore either rely on the accuracy of various kinds of symmetries expected to exist within a macromolecular assembly (e.g. icosahedral viruses, helical filaments or 2-D crystals), or at least expect individual particles to be structurally identical (so called single particle averaging). However, the biological default state of the vast majority of structures from protein complexes to cellular structures is usually far from this requirement. To this end we are moving towards tomographic 3-D data acquisition, which has been demonstrated by various pioneering groups (Ellisman, Baumeister, McEwen, etc.) to be an extremely promising technique. In principle tomography allows reconstructing in 3-D any object that is suitable to be visualized in an EM. Once an initial 3-D dataset exists averaging may still be useful to further refine pre-reconstructed particles and complexes at various conformational states. On my previous post at EMBL I have just now succeeded to collect funds for the purchase of one of the newest generation cryo-EM, which uses liquid-helium technology for a better specimen conservation, and a revolutionary new tilt-stage design for enhanced stability. Both features are ideal to optimise tomography-based 3-D data acquisition. Ranging in resolution typically from 1-3 nm, tomography is not the method to generate data at atomic detail, but yields 3-D scaffolds, which can be used to dock individual X-ray or NMR-data for structural and functional interpretations at near-atomic detail. When going in the other direction on the resolution scale, tomographical EM data allows an advanced interpretation of light-microscopy and laser-confocal 3-D data, and therefore constitutes a very useful bridge to link structural details from cells to atoms. Based on these perspectives I would like to formulate our (maybe slightly over-ambitious) aim for a distant future of being able to reconstruct highly complex cellular structures (e.g. an entire spindle, nucleus, axon and synapse, etc.) at near atomic detail, using tomography 3-D data as a basis to combine all required information from various sources.
Fig. 2: Correlative aspects of light- and electron microscopy with high-resolution data. This figure also illustrates the main EM techniques applied in our lab. Background: cryo-EM image of microtubules. Insets from left to right: fluorescence LM (mitotic spindle), surface shadowing (tubulin sheet), helical 3-D reconstruction of a motor-microtubule complex, molecular docking of X-ray data.
Cryo-electron tomography on vitrified sections: A critical analysis of benefits and limitations for structural cell biology.
Bouchet-Marquis, C and Hoenger, A Micron, 42(2):152-62. 2011
Cryo-electron tomography of microtubule-kinesin motor complexes.
Cope, J, Gilbert, S, Rayment, I, Mastronarde, D, and Hoenger, A J Struct Biol, 170(2):257-65. 2010
Lattice structure of cytoplasmic microtubules in a cultured Mammalian cell.
McIntosh, JR, Morphew, MK, Grissom, PM, Gilbert, SP, and Hoenger, A J Mol Biol, 394(2):177-82. 2009
The Schizosaccharomyces pombe EB1 homolog Mal3p binds and stabilizes the microtubule lattice seam.
Sandblad, L, Busch, KE, Tittmann, P, Gross, H, Brunner, D, and Hoenger, A Cell, 127(7):1415-24. 2006
A structural model for monastrol inhibition of dimeric kinesin Eg5.
Krzysiak, TC, Wendt, T, Sproul, LR, Tittmann, P, Gross, H, Gilbert, SP, and Hoenger, A EMBO J, 25(10):2263-73. 2006