Type of Document Dissertation Author Kelly, Deborah F. URN etd-09012003-024804 Title Visualizing Cell Adhesion Proteins Using Cryo-Electron Microscopy And 3D Reconstruction Techniques Degree Doctor of Philosophy Department Molecular Biophysics, Institute of Advisory Committee
Advisor Name Title Kenneth A. Taylor Committee Chair Kenneth H. Roux Committee Member Michael Blaber Committee Member Thomas C.S. Keller III Committee Member Thomas M. Roberts Committee Member Keywords
- Intracellular and Extracellular Scaffolding
Date of Defense 2003-06-01 Availability unrestricted AbstractCell adhesion assemblies occur at sites where cells either contact each other or components related to the extracellular matrix. They provide the structural integrity needed to support nonmigrating cells via a host of transmembraneous proteins. In addition to their structural role, these transmembrane receptors also establish a system of communicating with the cytoskeleton. One of the primary receptors found on the surface of stationary cells is the fibronectin receptor, also known as the a5b1-integrin. When bound to their extracellular ligand, fibronectin, these integrins assume an “ active” conformation. This external binding event is coordinated with a series of physical changes that are translated through the transmembrane portion of the receptor and passed along to its cytoplasmic domain. Inside the cell, these perturbations cause a series of structural changes that allow the F-actin cytoskeleton to be linked to the integrin’s cytoplasmic domain. Our interests lie in visualizing the macromolecular assemblies of the cytoskeletal components that support this linkage.
The method we used to observe these adhesive complexes is transmission electron microscopy (TEM).
We used the lipid monolayer crystallization technique for a dual purpose: 1) as a means of concentrating protein at the air:water interface, providing a crystallization surface; 2) to mimic the cytoplasmic leaflet, which contains a hydrophobic lipid layer on top of a bulk aqueous phase. Therefore, preparing an ordered EM specimen with these characteristics gives us a tool to study numerous biological systems.
In this project specifically, we synthesized the integrin cytoplasmic domain with a histidine (His)-tag at its N-terminus and bound it to a lipid monolayer containing a nickel-chelating group. This causes the integrin to assume an orientation that represents its native conformation at the cell membrane. We were able to produce EM specimens that contained this integrin domain along with other cytoskeletal proteins, such as talin, a-actinin, vinculin and F-actin. In particular, the b1-integrin:a-actinin and the b1-integrin:aactinin: vinculin samples formed ordered arrays that we used to make frozen-hydrated specimens. We collected EM images of untilted samples and calculated their averaged 2-D projections. Additionally, we produced a 3-D reconstruction of the b1-integrin:a-actinin:vinculin assembly by collecting images of samples tilted up to 70o. Using molecular modeling software, in combination with information deposited into various protein databases, we created atomic models of both the b1-integrin as well as the vinculin-head piece. The docking of these models into our 3-D Cryo-EM map was quantified and refined to produce an atomic model for our assembly. Overall, this combination of imaging and model building establishes a methodology of producing complexes of adhesive proteins whose spatial relationships are virtually unknown.
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