Probing surface protein patterning in biological systems using fluorescence nanoscopy

Agrawal, Utsav

Permalink

https://hdl.handle.net/2142/88273 Copy

Description

Title

Probing surface protein patterning in biological systems using fluorescence nanoscopy

Author(s)

Agrawal, Utsav

Issue Date

2015-07-10

Director of Research (if dissertation) or Advisor (if thesis)

Schroeder, Charles M.

Doctoral Committee Chair(s)

Schroeder, Charles M.

Committee Member(s)

• Kraft, Mary L.
• Rao, Christopher V.
• Kuhlman, Thomas E.

Department of Study

Chemical & Biomolecular Engr

Discipline

Chemical Engineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(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.

Graduation Semester

2015-8

Type of Resource

text

Permalink

http://hdl.handle.net/2142/88273

Copyright and License Information

Copyright 2015 Utsav Agrawal

Owning Collections