Type of Document	Dissertation
Author	Hampton, Cheri M.
Author's Email Address	cheri@sb.fsu.edu
URN	etd-08252006-120004
Title	Reading Between the Filaments: Structural Characterization of Two Different F-Actin Cross-Linking Proteins by Electron Microscopy
Degree	Doctor of Philosophy
Department	Molecular Biophysics, Institute of
Advisory Committee	Advisor Name Title P. Bryant Chase Committee Chair Charles Ouimet Committee Member Hong Li Committee Member Kenneth A. Taylor Committee Member Piotr Fajer Committee Member
Keywords	Cytoskeleton Electron Tomography Actin Alpha-Actinin Villin Correspondence Analysis Lipid Monolayer Electron Microscopy
Date of Defense	2006-08-11
Availability	unrestricted
Abstract We use a lipid monolayer system to prepare paracrystalline rafts of F-actin with two different cross-linking proteins, α-actinin and villin. α-Actinin cross-links F-actin into relatively loose networks throughout cells, while villin forms dense, tight bundles of filaments in specialized structures known as microvilli. Our monolayer system allows us to examine each of these cross-linkers as they interact with F-actin in a simplified two-component system. In order to analyze the data from these arrays, we have hybridized methods from 2-D crystal analysis with a single-particle approach to refine strategies for correspondence analysis and classification of images. For the α-actinin:F-actin rafts, we use this strategy to characterize the highly variable cross-links as 2-D projection images. The villin:F-actin rafts require further refinement of the hybrid strategy by application to 3-D volumes from electron tomography. Both protein arrays yield unique insights to the architectural arrangement of cross-linking proteins between filaments. In the α-actinin:F-actin rafts we use correspondence analysis to demonstrate that otherwise polar arrays of F-actin can have insertions of filaments of reversed polarity within them, and that these polarity differences do not influence the cross-links. We developed a method for left-right independent classification of the α-actinin cross-links to recover the high variability in the cross-link angular distribution by increasing the signal-to-noise ratio of the class averages. These averages are combined to recreate the original cross-link as it appears in a process we call "mapping-back." Measurements reveal that the length of cross-links can vary. From the classification of cross-links we demonstrate and model the novel occurrence of α-actinin bound to successive cross-over repeats on the same actin filament which we have termed “monofilament” binding. We further illustrate that the length variation of these monofilament-bound α-actinins are quantized to 55 Ĺ, the distance between two adjacent actin monomers in the filament. The villin:F-actin rafts displayed homogeneous cross-linking. Docking of the homologous gelsolin atomic coordinates into the three-dimensional volumes reveals that villin does not interact with F-actin in the same manner as gelsolin. This data supports multiplicity of binding modes even in highly homologous proteins, and suggests a new mode of F-actin-binding for the villin protein.
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