Debbie Kelly

Dept. Cell Biology Harvard Medical School 240 Longwood Ave. Boston, MA 02115

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.