Force-dependent changes in alpha-catenin conformation

Alpha-catenin;Cell adhesion;Protein biophysics

Barrick, Samantha Kirstin Tieszen

关键词: Alpha-catenin;    Cell adhesion;    Protein biophysics;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/99209/BARRICK-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

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.