Cell 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. / A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2003. / March 27, 2003. / Intracellular and Extracellular Scaffolding, Adhesion / Includes bibliographical references. / Kenneth A. Taylor, Professor Directing Dissertation; Thomas M. Roberts, Outside Committee Member; Michael Blaber, Committee Member; Kenneth H. Roux, Committee Member; Thomas C. S. Keller, III, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_181568 |
| Contributors | Kelly, Deborah F. (authoraut), Taylor, Kenneth A. (professor directing dissertation), Roberts, Thomas M. (outside committee member), Blaber, Michael (committee member), Roux, Kenneth H. (committee member), Keller, Thomas C. S. (committee member), Program in Molecular Biophysics (degree granting department), Florida State University (degree granting institution) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource, computer, application/pdf |
| Rights | This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them. |
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