David Edward Boettiger

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Emeritus Professor of Microbiology
Department: Microbiology

Contact information
211 Johnson Pavilion
3610 Hamilton Walk
Philadelphia, PA 19104
Office: (215) 898-8792
Fax: (215) 898-9557
Education:
B.A. (Chemistry)
Earlham College, 1964.
Ph.D. (Molecular Biology)
University of Wisconsin, 1972.
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Description of Research Expertise

Research Interests
Regulation of cell adhesion and adhesion-mediated signaling.

Key words: Cell adhesion, Integrin, FAK, src, fibronectin, bond strength, mechanochemical signaling.

Description of Research
Cells in solid tissues can receive signals from their environment in the form of soluble factors that interact with cellular receptors or through cell surface receptors that also function to adhere the cell to adjacent cells or to extracellular matrix. The two types of receptors are distinguished by the mechanical component of the adhesion receptors. My laboratory focuses on the adhesion receptors and particularly on the elements that distinguish adhesion receptors from other signaling receptors. There are two major issues:

1. How is the binding of ligand regulated?
2. How is the mechanical component of the signal detected and transmitted in the cell?

In order to properly address the issues of the control of ligand binding, it is necessary to be able to measure this binding. The problem is more complex than it would initially appear because this binding takes place in the space between the receptor-bearing cell and the ligand bearing surface, or the contact zone. The binding process is influenced by the separation between the surfaces, diffusion of receptor and ligand on their respective surfaces, and clustering of receptor and ligand. Measurement of this binding requires the development of new methods. My laboratory has developed two methods, one is dependent of specific chemical cross-linking and the other is based on the application of a hydrodynamic shear (flow) gradient to measure the relative force required to break the bonds. In this later case the force required to break the bonds and free the cell from a surface is directly proportional to the number of bound adhesion receptors. These tools provide a method to examine the ligand binding in the context of a cell binding to an extracellular matrix (or potentially to another cell) in a context that is closer to its functional use in cell adhesion. Using our approach and other mechanical approaches to the analysis of binding in the contact zone, it has been found that the binding association constant is 4-5 orders of magnitude less that expected based on the binding of soluble ligands and implies that only a small proportion of the available receptors in the contact zone are actually bound. This analysis provides a basis for analysis of the roles for intracellular signaling processes in the control of cell adhesion. Currently, we are analyzing the role(s) for abl, src, and FAK in this process.

The classic model for transmitting signals from the binding of ligand to cell surface receptors is receptor dimerization. This is based primarily on the analysis of the receptor tyrosine kinases. Integrins also cluster during cell adhesion and thus could use a similar mechanism. However, it is difficult to see how this form of signaling would be sensitive to the mechanical components of the cell's environment. We have found that there are two separable signaling components. Clustering of integrins induced the phosphorylation of FAK Y861, but tethering to the substrate was required for induction of FAK Y397 phosphorylation. The Y397 phosphorylation is the site that is most critical for downstream signaling. Because attachment of the integrin to the substrate was required for this signaling, it implies a mechanical component to the process. The problem is to analyze how tethering leads to the phosphorylation of FAK Y397. We expect that this will provide insight into the mechanism by which mechanical aspects of the cell environment can be sensed and responded to by the cell.

Rotation Projects for 2006-2007
1. Role of ILK (integrin-linked kinase) in the regulation of a5b1 integrin-mediated adhesion to fibronectin. The approach will use RNAi to suppress ILK expression and measure the effect on binding and activation of a5b1. It is currently controversial whether the kinase activity or adaptor activity mediates the process. In Drosophila deletion of ILK-homolog results in detachment of muscle cells at their initial contraction (in the embryo) suggesting that it plays an essential mechanical role in the process.

2. Development of a system for quantitative analysis of cadherin-mediated adhesion. Currently the analysis of cadherin-mediated adhesion is semi-quantitative at best. Quantitation will provide a basis for a more comprehensive analysis of the regulation of cadherin binding and the mechanical properties of the bond (i.e. how it functions in cell adhesion). The approach will use purified recombinant E cadherin and E cadherin transfected mouse L cells in combination with the spinning disc apparatus developed here.

3. The binding of cell adhesion receptors to their ligands takes place in the space between cell and substrate. Because both the receptor and ligand are constrained through their attachment to surfaces the efficiency of the binding reactions (basically Kon) is reduced. Current data suggests that this reduction is often 4-5 orders of magnitude. A portion of this loss of binding is probably attributable to the separation between the cell and substrate mediated by the negatively charged glycocalyx on the cell surface. If the separation exceeds the length of the receptor and ligand length no bonds will form. Experiments that manipulate the cell's glycosylation and measure the consequences on the number of a5b1 bonds that form and the adhesion strength will be performed.

Lab personnel:
Marina Guvakova, PhD - Research Assistant Professor
Laura Lynch, PhD - Research Specialist
Qi Shi - Research Specialist

Selected Publications

Shi., Q. and Boettiger, D: A novel mode for integrin-mediated signaling: Tethering is required for the phosphorylation of FAK Y397. Mol.Biol.Cell Oct 2003.

Miller, T, and Boettiger, D: Control of intracellular signaling by modulation of the fibronectin conformation at the cell-materials interface. Langmuir 19: 1730-37, 2003.

Garcia, A.J., Schwarzbauer, J, and Boettiger, D.: Distinct activation states of a5b1 integrin show differential binding to RGD and synergy domains of fibronectin. Biochemistry 41: 9063-69, 2002.

Datta, A. and Boettiger, D.: Phosphorylation of b3 integrin controls ligand binding strength. J.Biol.Chem. 277: 3943-49, 2002.

Garcia, A.J., Huber,F., and Boettiger, D.: Force required to break a5b1 integrin-fibronectin bonds in intact adherent cells is sensitive to integrin activated state. J. Biol.Chem. 273: 10988-93, 1998.

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Last updated: 03/30/2010
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