Force-dependent changes in alpha-catenin conformation

Barrick, Samantha Kirstin Tieszen

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https://hdl.handle.net/2142/99209 Copy

Description

Title

Force-dependent changes in alpha-catenin conformation

Author(s)

Barrick, Samantha Kirstin Tieszen

Issue Date

2017-11-28

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

Leckband, Deborah E.

Doctoral Committee Chair(s)

Leckband, Deborah E.

Committee Member(s)

• Gruebele, Martin
• Selvin, Paul R.
• Gennis, Robert B.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Alpha-catenin
• Cell adhesion
• Protein biophysics

Abstract

Cadherin complexes are crucial intercellular adhesions that transduce force fluctuations at junctions to activate signals that reinforce stressed intercellular contacts. α-Catenin is an identified force sensor within cadherin complexes. Mechanical force regulates binding of α-catenin to the actin-binding protein vinculin and to the actin cytoskeleton itself. This regulation is accomplished through distinct conformational changes in the α-catenin central modulatory (M) domain and the C-terminal actin-binding domain (ABD), respectively. Force-induced unfurling of the M domain exposes a cryptic vinculin-binding site, resulting in increased vinculin binding under tension. Previous studies found that a cooperative network of salt bridges stabilizes the autoinhibited conformation of the M domain. Studies described in this dissertation established that disruption of salt bridges within the M domain triggers the activation (unfurling) of α-catenin to bind vinculin, both at equilibrium and under tension. These studies compared wild-type (WT) α-catenin to salt-bridge mutants designed to disrupt a key interaction within the salt-bridge network. Binding measurements quantified enhanced vinculin binding by a salt-bridge mutant, allowing the calculation of an equilibrium constant between the autoinhibited and active conformations of α-catenin. Equilibrium molecular dynamics (MD) simulations indicated that disrupting the salt-bridge network destabilizes the autoinhibited conformation of α-catenin. Imaging of live cells expressing a Fӧrster resonance energy transfer (FRET)-based α-catenin conformation sensor demonstrated that salt-bridge disruption promotes α-catenin unfurling under steady-state tension. Furthermore, a constant-force steered molecular dynamics (SMD) simulation of the M domain suggested the adoption of an intermediate conformation during force-induced activation, and identified a novel load-bearing salt bridge within this structure. The mechanism underlying tension-dependent strengthening of the α-catenin/actin linkage has not yet been established. MD simulations presented in this dissertation suggested force-induced conformational changes within the α-catenin ABD that increase the affinity for actin. Constant-force simulations of two α-catenin isoforms showed that force unfolds a short α-helix within the ABD while leaving the rest of the domain intact. Equilibrium MD simulations showed that a mutation designed to mimic this partially unfolded conformation resulted in exposure of a buried residue in the putative actin-binding site. These results suggest that tension-dependent conformational changes allosterically regulate actin binding by promoting a high-affinity conformation of the ABD. Single-molecule measurements of α-catenin unfolding by atomic force microscopy (AFM) investigated the mechanism of force-induced unfolding of the α-catenin M domain. The preliminary data presented in this dissertation demonstrated that the mechanical stability of α-catenin is too low for unfolding of the multiple independently folded domains within the M region to be consistently resolved by AFM. Nonetheless, the results were qualitatively consistent with other studies of α-catenin unfolding. The studies described in this dissertation provide molecular-level details of α-catenin-dependent reinforcement of stressed cell-cell adhesions. This reinforcement occurs through two distinct mechanisms: tension-dependent binding of vinculin at junctions, and force-induced enhancement of direct actin binding. These findings deepen our understanding of how force-dependent changes in the conformation of α-catenin transduce force at cell-cell junctions, which is critical for understanding diverse cellular processes such as maintenance of tissue integrity and embryonic development, as well as disease-related events such as cancer metastasis.

Graduation Semester

2017-12

Type of Resource

text

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Copyright and License Information

Copyright 2017 Samantha Barrick

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