2026-09-21T21:15:27Z https://www.ideals.illinois.edu/oai-pmh

oai:www.ideals.illinois.edu:2142/99209 2023-07-11 col_2142_5131 col_2142_14789 com_2142_5130 com_2142_14788 com_2142_8903

Leckband, Deborah E. Leckband, Deborah E. Gruebele, Martin Selvin, Paul R. Gennis, Robert B. Barrick, Samantha Kirstin Tieszen 2018-03-13T15:25:14Z 2018-03-13T15:25:14Z 2020-03-14T09:15:16Z 2017-11-28 2017-12 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. Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2019-12-01 The student, Samantha Barrick, accepted the attached license on 2017-11-28 at 11:40. The student, Samantha Barrick, submitted this Dissertation for approval on 2017-11-28 at 11:52. This Dissertation was approved for publication on 2017-11-28 at 13:50. DSpace SAF Submission Ingestion Package generated from Vireo submission #11765 on 2018-03-13 at 09:55:55 Made available in DSpace on 2018-03-13T15:25:14Z (GMT). No. of bitstreams: 3 BARRICK-DISSERTATION-2017.pdf: 5006510 bytes, checksum: d33ac5a2d6069c8731bfb164ce6bca64 (MD5) LICENSE.txt: 4213 bytes, checksum: d644efaf4a606de27afb13c0d83647d9 (MD5) PROQUEST_LICENSE.txt: 4559 bytes, checksum: 2e0ef541a7842cc45bccc693e2ed85c1 (MD5) Previous issue date: 2017-11-28 Embargo set by: Seth Robbins for item 105172 Lift date: 2020-03-13T15:25:40Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system Embargo set by: Seth Robbins for item 105172 Lift date: 2020-03-13T15:28:52Z Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system U of I Only Restriction Lifted for Item 105172 on 2020-03-14T09:15:16Z. application/pdf http://hdl.handle.net/2142/99209 en Copyright 2017 Samantha Barrick Alpha-catenin Cell adhesion Protein biophysics Force-dependent changes in alpha-catenin conformation text text Chemistry Chemistry University of Illinois at Urbana-Champaign Dissertation Ph.D.