ORCID

http://orcid.org/0000-0001-5253-5769

Date of Award

Winter 1-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Plant & Microbial Biosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Mechanical forces play critical roles throughout the lives of all organisms and, as such, diverse arrays of mechanotransduction systems have evolved to detect and initiate responses to force. Many mechanotransduction systems consist of mechanosensitive (MS) ion channels, membrane pores that open in response to sufficient mechanical force. My dissertation focuses on both the study and application of force-dependent conformational changes of MS channels. I tested whether charged pore-lining residues R326 and D327 of the Arabidopsis thaliana mitochondrial MS channel MSL1 function in inward rectification or gating kinetics. Mutating these residues showed no effect on MSL1 rectification; however, these residues are important for gating transitions and open state stability of MSL1. I also attempted to develop a genetically-encoded, fluorescence-based membrane tension sensor using the Mechanosensitive ion channel of Small conductance (MscS) from E. coli as a tension-sensing scaffold and circularly permuted GFP (mcpGFP) as the fluorescence reporter. While responses to tension by sensor candidates could not be ruled out, signal changes from mcpGFP that were not dependent on the tension-sensing scaffold dominated sensor responses to osmotic shock. Finally, I developed a detailed protocol for single-channel analysis of plant MS channels using patch-clamp electrophysiology in E. coli spheroplasts. This protocol provides users with detailed descriptions covering sample prep through data analysis of a typical experiment and includes equipment and set-up descriptions geared towards the inexperienced user. Thus my dissertation advances the study of plant mechanobiology by increasing knowledge about the function, study, and application of MS ion channels.

Language

English (en)

Chair and Committee

Elizabeth S. Haswell

Committee Members

Philip V. Bayly, Jianmin Cui, Ram Dixit, Joseph M. Jez,

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