scholarly journals Diffusion of the disordered E-cadherin tail on β-catenin

2021 ◽  
Author(s):  
Felix Wiggers ◽  
Samuel Wohl ◽  
Artem Dubovetskyi ◽  
Gabriel Rosenblum ◽  
Wenwei Zheng ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Single Molecule ◽  
Protein Interactions ◽  
Energy Landscape ◽  
Specific Protein ◽  
Adhesion Protein ◽  
High Affinity ◽  
The Stability ◽  
Binding Interfaces ◽  
E Cadherin

AbstractSpecific protein interactions typically require well-shaped binding interfaces. Here, we report a cunning exception. The disordered tail of the cell-adhesion protein E-cadherin dynamically samples a large surface area of the proto-oncogene β-catenin. Single-molecule experiments and molecular simulations resolve these motions with high resolution in space and time. Contacts break and form within hundreds of microseconds without dissociation of the complex. A few persistent interactions provide specificity whereas unspecific contacts boost affinity. The energy landscape of this complex is rugged with many small barriers (3 – 4 kBT) and reconciles specificity, high affinity, and extreme disorder. Given the roles of β-catenin in cell-adhesion, signalling, and cancer, this Velcro-like design has the potential to tune the stability of the complex without requiring dissociation.

scholarly journals Mapping transmembrane binding partners for E-cadherin ectodomains

2020 ◽  
Author(s):  
Omer Shafraz ◽  
Bin Xie ◽  
Soichiro Yamada ◽  
Sanjeevi Sivasankar
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Transmembrane Protein ◽  
Binding Assays ◽  
Binding Interactions ◽  
Adhesion Protein ◽  
Bait Protein ◽  
Binding Partners ◽  
Extracellular Region ◽  
E Cadherin

ABSTRACTWe combine proximity labeling and single molecule binding assays, to discover novel transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a bait protein with TurboID biotin ligase, and identify proximal proteins that are biotin-tagged on both their extracellular and intracellular regions. We then test direct binding interactions between the proximal proteins and the bait, using single molecule Atomic Force Microscope binding assays. Using this approach, we identify novel binding partners for the extracellular region of E-cadherin, an essential cell-cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-α2β1, and the receptor tyrosine kinase ligand ephrin-B1, all directly interact with E-cadherin ectodomains. Our discovery of previously unknown heterophilic E-cadherin binding interactions, suggest the existence of novel cadherin cross-talk in epithelial cells.

scholarly journals Mapping transmembrane binding partners for E-cadherin ectodomains

2020 ◽  
Vol 117 (49) ◽  
pp. 31157-31165
Author(s):  
Omer Shafraz ◽  
Bin Xie ◽  
Soichiro Yamada ◽  
Sanjeevi Sivasankar
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Transmembrane Protein ◽  
Binding Assays ◽  
Adhesion Protein ◽  
Bait Protein ◽  
Binding Partners ◽  
Extracellular Region ◽  
Focal Adhesion Protein ◽  
E Cadherin

We combine proximity labeling and single molecule binding assays to discover transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a “bait” protein with BioID biotin ligase and identify proximal proteins that are biotin tagged on both their extracellular and intracellular regions. We then test direct binding interactions between proximal proteins and the bait, using single molecule atomic force microscope binding assays. Using this approach, we identify binding partners for the extracellular region of E-cadherin, an essential cell–cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-α2β1, the receptor tyrosine kinase ligand ephrin-B1, and the classical cadherin P-cadherin, all directly interact with E-cadherin ectodomains. Our data shows that combining extracellular and cytoplasmic proximal tagging with a biophysical binding assay increases the precision with which transmembrane ectodomain interactors can be identified.

scholarly journals Diffusion of a disordered protein on its folded ligand

2021 ◽  
Vol 118 (37) ◽  
pp. e2106690118
Author(s):  
Felix Wiggers ◽  
Samuel Wohl ◽  
Artem Dubovetskyi ◽  
Gabriel Rosenblum ◽  
Wenwei Zheng ◽  
...  
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Energy Landscape ◽  
Binding Kinetics ◽  
Disordered Proteins ◽  
Adhesion Protein ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
High Affinity

Intrinsically disordered proteins often form dynamic complexes with their ligands. Yet, the speed and amplitude of these motions are hidden in classical binding kinetics. Here, we directly measure the dynamics in an exceptionally mobile, high-affinity complex. We show that the disordered tail of the cell adhesion protein E-cadherin dynamically samples a large surface area of the protooncogene β-catenin. Single-molecule experiments and molecular simulations resolve these motions with high resolution in space and time. Contacts break and form within hundreds of microseconds without a dissociation of the complex. The energy landscape of this complex is rugged with many small barriers (3 to 4 kBT) and reconciles specificity, high affinity, and extreme disorder. A few persistent contacts provide specificity, whereas unspecific interactions boost affinity.

Regulation of the cell-cell adhesion protein, E-cadherin, in dog and human thyrocytes in vitro.

Endocrinology ◽  
1995 ◽  
Vol 136 (7) ◽  
pp. 3113-3119 ◽  
Author(s):  
G Brabant ◽  
C Hoang-Vu ◽  
J Behrends ◽  
Y Cetin ◽  
E Pötter ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Protein ◽  
Cell Adhesion Protein ◽  
E Cadherin ◽  
Cell Cell

scholarly journals Controlling protein function by fine-tuning conformational flexibility

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Sonja Schmid ◽  
Thorsten Hugel
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Protein Function ◽  
Specific Protein ◽  
Cellular Protein ◽  
Steady States ◽  
Fine Tuning ◽  
Cell Protein ◽  
Global Changes ◽  
Protein Regulation

In a living cell, protein function is regulated in several ways, including post-translational modifications (PTMs), protein-protein interaction, or by the global environment (e.g. crowding or phase separation). While site-specific PTMs act very locally on the protein, specific protein interactions typically affect larger (sub-)domains, and global changes affect the whole protein non-specifically. Herein, we directly observe protein regulation under three different degrees of localization, and present the effects on the Hsp90 chaperone system at the levels of conformational steady states, kinetics and protein function. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We disentangle specific and non-specific effects that control Hsp90’s ATPase function, which has remained a puzzle up to now. Lastly, we introduce a new mechanistic concept: functional stimulation through conformational confinement. Our results demonstrate how cellular protein regulation works by fine-tuning the conformational state space of proteins.

scholarly journals Early Events in Actin Cytoskeleton Dynamics and E-Cadherin-Mediated Cell-Cell Adhesion during Epithelial-Mesenchymal Transition

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 578 ◽  
Author(s):  
Irina Y. Zhitnyak ◽  
Svetlana N. Rubtsova ◽  
Nikita I. Litovka ◽  
Natalya A. Gloushankova
Keyword(s):  
Cell Adhesion ◽  
Epithelial Mesenchymal Transition ◽  
Immunofluorescent Staining ◽  
Actin Binding ◽  
Mesenchymal Transition ◽  
Actin Bundle ◽  
Cell Contacts ◽  
The Stability ◽  
E Cadherin ◽  
Cell Cell

Epithelial-mesenchymal transition (EMT) plays an important role in development and also in initiation of metastasis during cancer. Disruption of cell-cell contacts during EMT allowing cells to detach from and migrate away from their neighbors remains poorly understood. Using immunofluorescent staining and live-cell imaging, we analyzed early events during EMT induced by epidermal growth factor (EGF) in IAR-20 normal epithelial cells. Control cells demonstrated stable adherens junctions (AJs) and robust contact paralysis, whereas addition of EGF caused rapid dynamic changes at the cell-cell boundaries: fragmentation of the circumferential actin bundle, assembly of actin network in lamellipodia, and retrograde flow. Simultaneously, an actin-binding protein EPLIN was phosphorylated, which may have decreased the stability of the circumferential actin bundle. Addition of EGF caused gradual replacement of linear E-cadherin–based AJs with dynamic and unstable punctate AJs, which, unlike linear AJs, colocalized with the mechanosensitive protein zyxin, confirming generation of centripetal force at the sites of cell-cell contacts during EMT. Our data show that early EMT promotes heightened dynamics at the cell-cell boundaries—replacement of stable AJs and actin structures with dynamic ones—which results in overall weakening of cell-cell adhesion, thus priming the cells for front-rear polarization and eventual migration.

scholarly journals iTRVZ: liquid nano-platform for signal integration on the plasma membrane

2021 ◽  
Author(s):  
Taka Aki Tsunoyama ◽  
Christian Hoffmann ◽  
Bo Tang ◽  
Koichiro M Hirosawa ◽  
Yuri L Nemoto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Focal Adhesions ◽  
Specific Protein ◽  
Signalling Pathways ◽  
Signal Integration ◽  
Signalling Molecules ◽  
Raft Domains

Signalling is one of the most important functions of the cellular plasma membrane (PM). A variety of extracellular signalling molecules bind to their specific receptors in the PM, and the engaged receptors in turn trigger various cytoplasmic signalling cascades. These signalling pathways are intertwined and affect each other, in a process called crosstalk, which enables the cells to fine tune the overall signal. The crosstalk of different receptor signalling pathways has been examined quite extensively, but the platform responsible for signal integration has never been discovered. Here, using single-molecule imaging, we found a nanometer-scale (50-80 nm) liquid-like protein assembly on the PM cytoplasmic surface (at a density of ~2-μm apart from each other on average, with a lifetime of ~10 s), working as the signal transduction and integration platform for receptors, including GPI-anchored receptors (GPI-ARs), receptor-type tyrosine kinases (RTKs), and GPCRs. The platform consists of integrin, talin, RIAM, VASP, and zyxin, and is thus termed iTRVZ. These molecules are known as focal-adhesion constituents, but iTRVZ is distinct from focal adhesions, because iTRVZ exists on both the apical and basal PMs and lack vinculin. The iTRVZ formation is driven by specific protein-protein interactions, liquid-liquid phase separation, and interactions with actin filaments and raft domains via PI(4,5)P2. iTRVZ integrates and amplifies the GPI-AR and RTK signals in a strongly non-linear fashion, and thus works as an AND gate and noise filter. These findings greatly advance our understanding of the mechanism for crosstalk between signalling pathways.

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