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Holger Schmitz


 

 

  • Dr. Holger Schmitz
  • Thomas-Mann-Str. 17
  • 51429 Bergisch Gladbach
  • Germany
  • phone: +49 2204 984442
  • HSchm5@freenet.de

I was a post-doc in this lab from 1993-1997. Insect flight muscle is one of the best ordered muscles in nature, a fact that makes it an ideal specimen for studying the structure of crossbridges in different states. By crossbridges, we mean the enzymatic heads of the protein myosin which acts as a “motor” molecule to produce force during muscle contraction. We have been studying the structure of these crossbridges in different biochemical states that can be produced using non-hydrolyzable nucleotide analogs such as AMPPNP. Myosin cannot cleave AMPPNP to produce energy during contraction. However, addtion of AMPPNP to a rigor buffer in which striated muscle fibers are suspended will cause a decrease in the affintiy of myosin for actin. This change mimics the effect of ATP in contracting muscle which causes detachment of crossbridges at the end of the power stroke. We are investigating this effect using electron tomography to obtain 3-D images of the crossbridges. The picture shown at the right illustrates the effect of adding AMPPNP in concert with ethylene glycol on the strtucture. The crossbridges change in both attachment angle and redistribute to different actin binding sites. Specific markers on the thin filament allow us to determine which crossbridges are binding to actin specifically, in which position they could become force bearing crossbridges if the nucleotide was withdrawn, and which attach non-specifically, in which position they must detach and later reattach to a different site on actin in order to become force bearing. These crossbridges that bind actin specifically we call target zone crossbridges. These reconstructions have allowed to verify for the first time that myosin crossbridges can attach to actin in an angle other than the rigor angle.


PUBLICATIONS

  • H. Schmitz, C. Lucaveche, M. K. Reedy ; K. A. Taylor. Oblique section 3-D reconstruction of relaxed insect flight muscle reveals the crossbridge lattice in helical registration. Biophys. J. 67, 1620-1633 (1994).
  • H. Schmitz, Mary C. Reedy, Michael K. Reedy, Richard T. Tregear, Hanspeter Winkler, Kenneth A. Taylor. Electron tomography of Insect Flight Muscle in Rigor and AMPPNP at 23oC. J. Mol. Biol. 264, 279-301 (1996).
  • H. Schmitz, M. C. Reedy, M. K. Reedy, R. T. Tregear, H. Winkler, K. A. Taylor. Tomographic 3-D Reconstruction of Insect Flight Muscle Partially Relaxed by AMPPNP and Ethylene Glycol. J. Cell Biol. 139(3), 695-707 (1997).
  • Kenneth A. Taylor, Holger Schmitz, Mary C. Reedy, Yale E. Goldman, Clara Franzini-Armstrong, Hiro Sasaki, Richard T. Tregear, Kate Poole, Carmen Lucaveche, Robert J. Edwards, Li Fan Chen, Hanspeter Winkler, and Michael K. Reedy. Tomographic 3-D reconstruction of quick frozen, Ca++-activated contracting insect flight muscle. Cell 99, 421-431 (1999).
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Debbie Kelly

  • Assistant Professor
  • Virginia Tech Carilion Research Institute (VTCRI)
  • 2 Riverside Circle
  • Roanoke, VA 24016

I was a graduate student in Prof. Taylor’s laboratory from 1999-2003. My project was to develop methodology to assemble focal adhesion complexes on a lipid monolayer and determine their structures. I accomplished this by using a synthetic peptide corresponding in sequence to the cytoplasmic domain of the Β1-integrin but which had an additional 4 histidines just N-terminal to the first histidine of the native sequence. I then bound this peptide to a derivatized lipid that uses Ni++ to form a complex with the 5 histidines, a so-called his-tag. This integrin peptide was then used to bind α-actinin to the monolayer which then formed 2-D arrays that were isomorphous with monolayer arrays formed using α-actinin alone. By binding a gold cluster label to the cytoplasmic domain, I was able to identify the integrin binding site on α-actinin. I then added the vinculin domain 1, vinculin-D1 to the α-actinin and made a ternary complex that was also isomorphous with the arrays of α-actinin alone. I then obtained a 3-D reconstructed image of the vinculin-D1 domain plus α-actinin and the integrin peptide and identified the vinculin-D1 from a difference map. Before I left at the end of 2003, I was working on a quaternary complex that contained the talin FERM domain, vinculin-D1, the Β1-integrin cytoplasmic domain and α-actinin.

The model to the left illustrates the components of a focal adhesion.

PRESENTATIONS

  • Deborah F. Kelly, Dianne W. Taylor, Constantina Bakolitsa, Andrey A. Bobkov, Laurie Bankston, Robert C. Liddington and Kenneth A Taylor. Structure of the α-actinin-vinculin head domain complex determined by cryo-electron microscopy J. Mol. Biol., 357(2), 562-573 (2006).
  • Debbie Kelly, Dianne W. Taylor, Kenneth A. Taylor. Formation of the α1-integrin-β-actinin-F-actin ternary complex on a lipid monolayer. Mol. Biol. Cell 11(Suppl), 551a (2000).
  • Debbie Kelly, Dianne W. Taylor and Kenneth A. Taylor. Examining the α-actinin-β1-integrin structural relationship using cryo-electron microscopy. Biophys. J. 82(1), 384a (2002).
  • K. A. Taylor, D. F. Kelly, D. W. Taylor, C. Bakolitsa, A. Bobkov, R. C. Liddington. Structure of the α-Actinin-Vinculin Head Domain Complex Determined by Cryo-Electron Microscopy. Proceedings of the 45th Annual Meeting of the American Society of Cell Biology. page 54 (poster 340).

PUBLICATIONS

  • Deborah F. Kelly and Kenneth A. Taylor. Identification of the α1-integrin binding site on β-actinin by cryo-electron microscopy. J. Struct. Biol. 149(3), 290-302 (2005).
  • Deborah F. Kelly, Dianne W. Taylor, Constantina Bakolitsa, Andrey A. Bobkov, Laurie Bankston, Robert C. Liddington and Kenneth A Taylor. Structure of the α-actinin-vinculin head domain complex determined by cryo-electron microscopy J. Mol. Biol., 357(2), 562-573 (2006).
  • Dianne W. Taylor, Deborah F. Kelly, Anchi Cheng and Kenneth A Taylor. On the freezing of lipid monolayer specimens for cryoelectron microscopy. J. Struct. Biol., 160(3), 305-312 (2007) http://dx.doi.org/10.1016/j.jsb.2007.04.011.
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Jun Liu

  • Dr. Jun Liu (Assistant Professor)
  • The University of Texas – Houston Medical School
  • Department of Pathology & Laboratory Medicine
  • 6431 Fannin, MSB 2.228
  • Houston, TX 77030
  • Web page
  • E-Mail:Jun.Liu.1@uth.tmc.edu

I was a post-doc in the lab from 1998 through March of 2006. I worked on 3D reconstruction of chicken smooth muscle α-actinin by cryo-electron microscopy. A 3D atomic model of α-actinin was builted and refined against the 3D EM density map with 2.0nm resolution. To demonstrate how the α-actinin is cross-linked with F-actin, a tilted series of F-actin bundles was collected on Philips CM300-FEG electron microscope from -70 degree to +70 degree. Electron tomography was applied to generate the 3D structure of F-actin bundles. This project is an important step to demostrate the 3D structure of α-actinin and its structural relationship with F-actin. Other structures I have worked on are insect flight muscle, the IOS structure, full length smooth muscle myosin, the inhibited conformation of myosin-V, and the the envelope spikes of SIV and HIV.

Here are some models of the α-actinin structure I worked with:

Bipolar
act4bipolar_fa1.pdb: 1st actin filament of the crosslink
act4bipolar_fa2.pdb: 2nd actin filament of the crosslink
act4bipolar.pdb: Bipolar alpha-actinin model
act4bipolar_titin.pdb: Titin Z-repeat bound to EF34

Polar

act4polar_fa1.pdb: 1st actin filament of the crosslink
act4polar_fa2.pdb: 2nd actin filament of the crosslink
act4polar.pdb: Polar alpha-actinin model


PUBLICATIONS

  • Jun Liu, Thomas Wendt, Dianne W. Taylor and Kenneth A. Taylor. Refined model of the 10S conformation of smooth muscle myosin by cryoEM 3-D image reconstruction. J. Mol. Biol. 329(5), 963-972 (2003).
  • Ping Zhu, Elena Chertova, Julian Bess, Jr., Jeffrey D. Lifson, Larry O. Arthur, Jun Liu, Kenneth A. Taylor, and Kenneth H. Roux. Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions. Proc. Natl. Acad. Sci. 100(26), 15812-15817 (2003).
  • Jun Liu, Dianne W. Taylor and Kenneth A. Taylor. A 3-D reconstruction of smooth muscle α-actinin by cryoEM reveals two different conformations at the actin binding region. J. Mol. Biol. 338(1), 115-125 (2004).
  • Jun Liu, Mary C. Reedy, Yale E. Goldman, Clara Franzini-Armstrong, Hiroyuki Sasaki, Richard T. Tregear, Carmen Lucaveche, Hanspeter Winkler, Bruce A. J. Baumann, John M. Squire, Thomas C. Irving, Michael K. Reedy, and Kenneth A. Taylor. Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges. J. Struct. Biol. 147(3) 268-282 (2004).
  • Jun Liu, Dianne W. Taylor and Kenneth A. Taylor. A 3-D reconstruction of smooth muscle alpha-actinin by cryoEM reveals two different conformations at the actin binding region. J. Mol. Biol. 338(1), 115-125 (2004).
  • Ping Zhu, Jun Liu, Julian Bess Jr., Elena Chertova, Jeffrey D. Lifson, Henry Gris, Gilad Ofek, Kenneth A. Taylor, and Kenneth H. Roux. Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 441, 847-852 (2006).
  • Jun Liu, Dianne W. Taylor, Elena Krementsova, Kathy Trybus & Kenneth A. Taylor. 3-D structure of the myosin V inhibited state by cryoelectron tomography. Nature 442, 208-211 (2006).
  • Jun Liu, Shenping Wu, Mary C. Reedy, Hanspeter Winkler, Carmen Lucaveche, Yifan Cheng, Michael K. Reedy, and Kenneth A. Taylor. Electron tomography of swollen rigor fibers of insect flight muscle reveals a short and variably angled S2 domain. J. Mol. Biol. 362, 844-860 (2006).
  • K. A. Taylor, J. Liu & H. Winkler. Localization and classification of repetitive structures in electron tomograms of paracrystalline assemblies. In: Electron Tomography: Methods for Three-dimensional Visualization of Structures in the Cell, 2nd edition, Joachim Frank, Ed. Springer-Verlag. pp 417-439 (2006).
  • Hanspeter Winkler, Jun Liu, Kenneth A. Taylor, Ping Zhu, Kenneth H. Roux. Electron tomography of macromolecular assemblies. In: Proceedings of the 4th IEEE International Symposium on Biomedical Imaging: From Nano to Macro, pg 240-243 (2007).
  • Wei Dai, Qingmei Jia, Eric Bortz, Sanket Shah, Jun Liu, Ivo Atanasov, Kenneth A. Taylor, Ren Sun & Z. Hong Zhou. Unique Structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy. J. Struct. Biol. 161, 428-438 (2008).
  • Feng Ye, Jun Liu, Hanspeter Winkler and Kenneth A. Taylor. Integrin αIIb β3 in a membrane environment remains the same height after Mn2+ activation when observed by cryo-electron tomography. J. Mol. Biol. 378(5), 976-986 (2008).
  • Cheri M. Hampton, Jun Liu, Dianne W. Taylor, David J. DeRosier and Kenneth A. Taylor. The 3D structure of villin as a unique F-actin cross-linker. Structure, 16(12), 1882-1891 (2008).
  • Hanspeter Winkler, Ping Zhu, Jun Liu, Feng Ye, Kenneth H. Roux, and Kenneth A. Taylor. Tomographic subvolume alignment and subvolume classification applied to myosinV and SIV envelope spikes. J. Struct. Biol., 165(2), 64-77 (2009).
  • Shenping Wu, Jun Liu, Mary C. Reedy, Hanspeter Winkler , Michael K. Reedy & Kenneth A. Taylor. Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle. J. Struct. Biol. 168, 485-502 (2009). PMCID: PMC2805068.
  • Shenping Wu, Jun Liu, Mary C. Reedy, Richard T. Tregear, Hanspeter Winkler,  Clara Franzini-Armstrong, Hiroyuki Sasaki, Carmen Lucaveche, Yale E. Goldman, Michael K. Reedy, and Kenneth A. Taylor. Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions. PLoS ONE 5(9): e12643 (2010)
  • Pradeep Luther, Hanspeter Winkler, Kenneth Taylor, Maria-Elena Zoghbi, Roger Craig, Raul Padron, John Squire and Jun Liu. Direct visualization of the binding of myosin binding protein C to actin filaments in intact muscle (PNAS, in press) PMCID: PMC313626
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Feng Ye

  • Department of Medicine, UCSD
  • Leichtag Building Room 149-M
  • 9500 Gilman Drive, Mail Code 0726
  • La Jolla, CA 92093-0726
  • phone: 858-822-6496
  • feye@ucsd.edu

Structure of Integrins in the Membrane

I was a Ph.D. student in Prof. Taylor’s laboratory from 2002-2005. My project was to obtain a 3-D image of an integrin in both the active and inactive states while the protein was embedded in a lipid bilayer, its normal state in the cell. I isolated and purified the α-IIb Β3 integrin in a triton solubilized form from platelets, removed any active integrins by chromatography, and reincorporated the inactive integrins into small unilamellar vesicles. I collected tilt series of these integrins-vesicles in both the inactive state as isolated from the cells and also in an activated state using Mn++ and obtained 3-D images using electron tomography. These specimens were preserved in the frozen hydrated state. Subvolumes of individual integrins were extracted, aligned and classified to produce 3-D images with improved signal-to-noise ratio so that the molecular conformation could be elucidated. Publication of this work is currently in progress.


PUBLICATIONS

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Ann Imber


 

 

My role in the lab is sample preparation. Sample preparation is an important aspect of molecular biology. Whether the sample of interest is extracted from a natural source or cloned into an expression vector, sample preparation usually starts with the development of a purification protocol. Biotechnology such as polyhistidine and GST tags can be added to cloned DNA sequences to create fusion proteins. Fusion tags can be cleaved off using proteases with specific recognition sequences, or else retained in the final sample to create proteins capable of adhering to desired surfaces. Fusion proteins can be purified by affinity chromatography or based on their molecular weight, solubility, or overall charge. Purified samples can be further evaluated and combined with other samples to study protein interactions. An example of a purification scheme is as follows:

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Cheri Hampton

  • Howard Hughes Medical Institute
  • Columbia University Medical Center
  • P&S Black Building 2-221
  • 650 W 168th Street
  • New York, NY 10032
  • Curriculum Vitae

Structure of F-actin bundles with α-actinin and villin.

My research interests pertain to the cytoskeleton, its morphology and regulation. The cytoskeleton is a highly adaptive scaffold of proteins that respond to both internal and external stimuli. The cytoskeleton thus functions as both a structure and a signaling complex. My work in the Taylor lab has centered on the structural analysis of macromolecular assemblies of cytoskeletal proteins on lipid monolayers. In this manner we can create 2D paracrystalline arrays of F-actin cross-linked by actin-bundling proteins such as α-actinin and villin. With these arrays we have pushed the image analysis envelope beyond simple spatial averaging techniques into the realm of single-particle methods with 3D volume alignment and correspondence analysis.

α-Actinin is a modular protein belonging to the spectrin superfamliy that cross-links and bundles actin filaments in both muscle and non-muscle cells. α-Actinin cross-links F-actin to form large (> 1μm) rafts on the lipid monolayer. In order to improve the stochastic signal-to-noise ratio of the cross-link motifs, we first aligned all motifs to the common actin filament and followed with classification of the left- and right-side cross-links independently. The resulting left and right class averages could then be pasted together down the central, aligned actin filament to recreate a class average for every motif. With the improved detail observed in these averages we could perform quantitative measurements of cross-link length and build comparative molecular models to show the observed variation in the length and the angle of the α-actinin cross-links. Unique to this study are the numbers of α-actinin molecules bound to successive crossovers on the same actin filament. These monofilament-bound α-actinins may resemble the form of α-actinin involved in actin-membrane attachments in focal adhesions. These results suggest that α-actinin is not simply a rigid spacer between actin filaments, but rather a flexible cross-linking, scaffolding, and anchoring protein. We suggest these properties of apha-actinin may contribute to tension sensing in actin bundles.

Atomic model of gelsolin segments docked into the average density of villin (center) cross-linking two actin filaments.

The polar actin bundles of the microvillus are tightly cross-linked by two proteins, villin and fimbrin. Villin is an F-actin nucleating, cross-linking, severing, and capping protein within the gelsolin superfamliy. Villin is unique in this group by its ability to cross-link filaments, which is due to an additional small headpiece domain. Villin shares high sequence homology to gelsolin, which has no known cross-linking ability. In order to define the villin cross-linking structure we have used electron tomography of 2-D rafts of F-actin cross-linked with villin on a lipid monolayer to generate 3-D volumes of F-actin: villin cross-links. These rafts are > or = to 1μm across and consist of polar arrays of F-actin spaced ~126 angstroms apart with villin cross-links occurring approximately once per actin crossover. More than 6,000 paired F-actin crossover repeats with villin protein bound between them were selected as single particles, aligned, and classified by correspondence analysis to produce class averages. Docking of the homologous gelsolin domain structures plus the villin head piece structure into the average density reveals the invariant localization on the actin N-terminus which is quite distinct from that of other actin-binding proteins, such as cofilin, profillin, DNase I, or gelsolin domains G1 and G2. This is the first glimpse of the entire structure of villin in a cross-linking role. Up until now it has been assumed that villin interacts with F-actin in a similar fashion to its close homolog, gelsolin. This study shows this assumption to be wrong and instead lends concrete evidence to the notion that there can be different modes of interaction with actin among highly homologous actin-binding proteins.

Movie showing side-by-side comparison of alpha-actinin cross-link motifs before (left, raw image) and after (right, averaged image) left-right classification technique.

Villin homology model fit to villin actin rafts.


PUBLICATIONS

  • Cheri M Hampton and Kenneth A Taylor. α-Actinin-2.AfCS-Nature Molecule Pages (2006). (doi:10.1038/mp.a000195.01).
  • Cheri M Hampton and Kenneth A Taylor. α-Actinin-3. AfCS-Nature Molecule Pages (2006). (doi:10.1038/mp.a000196.01).
  • Cheri M Hampton and Kenneth A Taylor. α-Actinin-4. AfCS-Nature Molecule Pages (2006). (doi:10.1038/mp.a000197.01).
  • Cheri M Hampton, Dianne W Taylor, Kenneth A Taylor. Novel structures for α-actinin: F-actin interactions and their implications for actin-membrane attachment and tension sensing in the cytoskeleton. J. Mol. Biol. 368, 92-104 (2007) http://dx.doi.org/10.1016/j.jmb.2007.01.071
  • Cheri M. Hampton, Jun Liu, Dianne W. Taylor, David J. DeRosier and Kenneth A. Taylor. The 3D structure of villin as a unique F-actin cross-linker. Structure, 16(12), 1882-1891 (2008).

POSTERS AND PRESENTATIONS

  • Gordon Research Conference: Signaling by Adhesion Receptors, 2004 The Structural Regulation of Calcium-Sensitive α-Actinin-1.
  • Gordon Research Conference: Three Dimensional Electron Microscopy, 2005 Structures of 2D Rafts of F-actin and Bundling Proteins on Lipid Monolayers.
  • American Society for Cell Biology, December 10-14, 2005. San Francisco, CA Quantification of Structural Morphology of α-Actinin Cross-linking Ability.
  • American Society for Cell Biology, December 9-13, 2006. San Diego, CA Villin interacts with F-actin in a manner distinct from gelsolin. Quantification of Structural Morphology of α-Actinin Cross-Linking Flexibility.
  • Gordon Research Conference: Three-Dimensional Electron Microscopy, 2007. F-actin: Villin Crosslinks.
  • Invited Speaker: Gordon Research Conference. Three Dimensional Electron Microscopy, 2007. “Structure of the Villin-Actin Cross-link using Single-Particle Analysis of 3D Volumes.”

PUBLISHED ABSTRACTS

  • Hampton, C. M., Taylor, D. W. and Taylor, K. A. (2005). Quantification of Structural Morphology of α-Actinin Cross-linking Ability. Mol. Biol. Cell 16 (suppl), L96. (Late Abstracts, The American Society for Cell Biology 45th Annual Meeting).
  • Hampton, C. M., Taylor, D.W., Ouyang, G., DeRosier, D. J. and Taylor, K. A. (2006). Villin Interacts with F-actin in a Manner Distinct from Gelsolin. Mol. Biol. Cell 17 (suppl), L34 (Monday). (Late Abstracts, The American Society for Cell Biology 46th Annual Meeting).
  • Hampton, C. M., Taylor, D.W., Ouyang, G., DeRosier, D. J. and Taylor, K. A. (2007). Villin Interacts with F-actin in a Manner Distinct from Gelsolin as Determined by Electron Tomography. (Microscopy and Microanalysis Proceedings 2007).
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