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Title:Probing surface protein patterning in biological systems using fluorescence nanoscopy
Author(s):Agrawal, Utsav
Director of Research:Schroeder, Charles M.
Doctoral Committee Chair(s):Schroeder, Charles M.
Doctoral Committee Member(s):Kraft, Mary L.; Rao, Christopher V.; Kuhlman, Thomas E.
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):fluorescence nanoscopy
stochastic optical reconstruction microscopy (STORM)
B. subtilis
human immunodeficiency virus (HIV)
chemotaxis
McpB
envelope proteins
clustering
Abstract:A complete understanding of biological substructures is often obscured by the diffraction limit of visible light in conventional fluorescence microscopy. Recently developed fluorescence nanoscopy techniques such as stochastic optical reconstruction microscopy (STORM) effectively break the diffraction limit (~250 nm) to enable imaging with an order of magnitude smaller than the spatial resolution. In this work, we applied fluorescence nanoscopy to study the patterning of proteins in biological surfaces at a high spatial resolution of 25 nm. We also developed new analytical methods to extract quantitative information on protein arrangement within biological substrates to elucidate key phenomenon such as signal amplification in bacteria during chemotaxis. In B. subtilis, we observed reorganization in arrays of McpB receptor proteins, key chemotactic receptors for asparagine sensing, upon exposure to saturating stimulant concentrations. More specifically, receptors formed large polar clusters in the absence of stimulant but shifted towards smaller and more dispersed lattices throughout the cell when stimulant was added. In a first-of-its-kind fluorescence nanoscopic study on B. subtilis, we measured cluster sizes and intra cluster density of these receptors. Our results support the existing theories on the role of cooperativity via receptor clustering in signal amplification to respond to wide range of external stimuli in bacteria. In second project, we studied the role of viral proteins on HIV-1 infectivity. In HIV-1, envelope proteins gp120 and gp41 are known to play a significant role in infectivity and their arrangement is indicative of internal viral structure at different stages of its life cycle. We ascertained the distribution of both the envelope proteins on surface of virions at a high resolution of ~25 nm using three-color fluorescence nanoscopy. Our work effectively paves the way for revealing new levels of organization of surface proteins at nanometer scale spatial resolution for a molecular view understanding of dynamics in biological systems.
Issue Date:2015-07-10
Type:Thesis
URI:http://hdl.handle.net/2142/88273
Rights Information:Copyright 2015 Utsav Agrawal
Date Available in IDEALS:2015-09-29
Date Deposited:August 201


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