{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/99209"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/99209","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Force-dependent changes in alpha-catenin conformation","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Barrick, Samantha Kirstin Tieszen"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Leckband, Deborah E.","Gruebele, Martin","Selvin, Paul R.","Gennis, Robert B."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018-03-13T15:25:14Z","date_published":"2018-03-13T15:25:14Z","updated_at":"2026-07-22T22:24:37Z","subjects":["Alpha-catenin","Cell adhesion","Protein biophysics"],"languages":["en"],"rights":["Copyright 2017 Samantha Barrick"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/99209","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Leckband, Deborah E.","Gruebele, Martin","Selvin, Paul R.","Gennis, Robert B."]},{"key":"dc:creator","label":"Author","values":["Barrick, Samantha Kirstin Tieszen"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2018-03-13T15:25:14Z","2020-03-14T09:15:16Z","2017-11-28","2017-12"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Alpha-catenin","Cell adhesion","Protein biophysics"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2017 Samantha Barrick"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/99209"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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). 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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). 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