scholarly journals Molecular mechanism of cell-cell adhesion mediated by cadherin-23

2017 ◽  
Author(s):  
G. S. Singaraju ◽  
A. Kumar ◽  
J. S. Samuel ◽  
A. Sagar ◽  
J. P. Hazra ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Point ◽  
Cell Junctions ◽  
Single Point Mutation ◽  
Molecular Architecture ◽  
Binding Interface ◽  
Extracellular Region ◽  
Classical Cadherins ◽  
Cadherin 23 ◽  
Cell Cell

AbstractAdherin-junctions are traditionally described by the homophilic-interactions of classical cadherin-proteins at the extracellular region. However, the role of long-chain non-classical cadherins like cadherin-23(Cdh23) is not explored as yet even though it is implicated in tissue-morphogenesis, cancer, and force-sensing in neuronal tissues. Here, we identified a novel antiparallel-binding interface of Cdh23 homodimer in solution by combining biophysical and computational methods, in-vitro cell-binding, and mutational modifications. The dimer consists of two electrostatic-based interfaces extended up to two terminal domains, atypical to classical-cadherins known so far, and forms the strongest interactions in cadherin-family as measured using single-molecule force-spectroscopy. We further identified single point-mutation, E78K, that completely disrupts this binding. Interestingly, the mutation, S77L, found in skin cancers falls within the binding interface of the antiparallel-dimer. Overall, we provide the molecular architecture of Cdh23 at the cell-cell junctions which are likely to have far-reaching applications in the fields of mechanobiology and cancer.

scholarly journals Cis-clustering of cadherin-23 controls the kinetics of cell-cell adhesion

2021 ◽  
Author(s):  
Sabyasachi Rakshit ◽  
Cheerneni Srini ◽  
Gayathri Singaraju ◽  
Nisha Arora ◽  
Sayan Das ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Single Molecule ◽  
Liquid Droplets ◽  
Extracellular Region ◽  
Cadherin 23 ◽  
Multicellular Organisms ◽  
Cis And Trans ◽  
Kinetics Of ◽  
Cis Interaction ◽  
Cell Cell

Abstract Cis and trans-interactions in cadherins are the foundations of cellular adhesions in multicellular organisms. While the trans-interactions mediate the intercellular attachment, the cis-interaction is presumed as reinforcement to trans. Thus, trans precedes cis has been the well-accepted model in cadherin adhesion. The stronger affinity of trans-binding over cis has been the decisive influence in the trans first model. Here we show that cadherin-23, a non-classical cadherin with an extended extracellular region, can undergo cis-clustering in solution independent of trans and phase separate as liquid droplets. Using single-molecule measurements, we decipher that weaker cis-interactions favor the cis-clustering. In-cellulo, the cis-clustering is manifested as puncta, a common feature in non-classical cadherin junctions, and accelerates the cell adhesion. The cis-clustering thus kinetically controls cell-adhesion before trans-binding. Notably, M2-macrophages predominantly express cadherin-23 and rapidly attach to circulatory tumor cells during metastatic migration. However, the relation of cis-clustering with rapid cell-cell adhesion in physiology is not yet established

Identification of Allosteric Fingerprints of Alpha-Synuclein Aggregates in Matrix Metalloprotease-1 and Substrate-Specific Virtual Screening with Single Molecule Insights

2021 ◽  
Author(s):  
Sumaer Kamboj ◽  
Chase Harms ◽  
Derek Wright ◽  
Anthony Nash ◽  
Lokender Kumar ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Single Molecule ◽  
Conformational Changes ◽  
Single Point ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Alpha Synuclein ◽  
Matrix Metalloproteases ◽  
Single Point Mutation ◽  
Mmp Inhibitor

Abstract Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previously, we showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. Here we report quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is not available, we performed molecular docking of MMP1 with aSyn using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to identify an appropriate pose. We used experimentally validated simulations to define conformational changes at the catalytic site and identify allosteric residues in the hemopexin domain having strong correlations with the catalytic motif residues. We defined Shannon entropy to quantify MMP1 dynamics. We performed virtual screening against a site on selected aSyn-MMP1 binding poses and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

scholarly journals Desmoplakin Is Required Early in Development for Assembly of Desmosomes and Cytoskeletal Linkage

1998 ◽  
Vol 143 (7) ◽  
pp. 2009-2022 ◽  
Author(s):  
G. Ian Gallicano ◽  
Panos Kouklis ◽  
Christoph Bauer ◽  
Mei Yin ◽  
Valeri Vasioukhin ◽  
...  
Keyword(s):  
Critical Role ◽  
Early Embryo ◽  
Cell Junctions ◽  
Epithelial Polarity ◽  
Tissue Architecture ◽  
Desmosomal Cadherins ◽  
Tissue Integrity ◽  
Classical Cadherins ◽  
Mediate Cell ◽  
Cell Cell

Desmosomes first assemble in the E3.5 mouse trophectoderm, concomitant with establishment of epithelial polarity and appearance of a blastocoel cavity. Throughout development, they increase in size and number and are especially abundant in epidermis and heart muscle. Desmosomes mediate cell–cell adhesion through desmosomal cadherins, which differ from classical cadherins in their attachments to intermediate filaments (IFs), rather than actin filaments. Of the proteins implicated in making this IF connection, only desmoplakin (DP) is both exclusive to and ubiquitous among desmosomes. To explore its function and importance to tissue integrity, we ablated the desmoplakin gene. Homozygous −/− mutant embryos proceeded through implantation, but did not survive beyond E6.5. Mutant embryos proceeded through implantation, but did not survive beyond E6.5. Surprisingly, analysis of these embryos revealed a critical role for desmoplakin not only in anchoring IFs to desmosomes, but also in desmosome assembly and/or stabilization. This finding not only unveiled a new function for desmoplakin, but also provided the first opportunity to explore desmosome function during embryogenesis. While a blastocoel cavity formed and epithelial cell polarity was at least partially established in the DP (−/−) embryos, the paucity of desmosomal cell–cell junctions severely affected the modeling of tissue architecture and shaping of the early embryo.

The p120 catenin family: complex roles in adhesion, signaling and cancer

2000 ◽  
Vol 113 (8) ◽  
pp. 1319-1334 ◽  
Author(s):  
P.Z. Anastasiadis ◽  
A.B. Reynolds
Keyword(s):  
Cell Adhesion ◽  
Biological Function ◽  
Family Members ◽  
Cell Junctions ◽  
Beta Catenin ◽  
Juxtamembrane Domain ◽  
P120 Catenin ◽  
Structural Relationships ◽  
Classical Cadherins ◽  
Cell Cell

p120 catenin (p120) is the prototypic member of a growing subfamily of Armadillo-domain proteins found at cell-cell junctions and in nuclei. In contrast to the functions of the classical catenins (alpha-catenin, beta-catenin, and gamma-catenin/plakoglobin), which have been studied extensively, the first clues to p120's biological function have only recently emerged, and its role remains controversial. Nonetheless, it is now clear that p120 affects cell-cell adhesion through its interaction with the highly conserved juxtamembrane domain of classical cadherins, and is likely to have additional roles in the nucleus. Here, we summarize the data on the potential involvement of p120 both in promotion of and in prevension of adhesion, and propose models that attempt to reconcile some of the disparities in the literature. We also discuss the structural relationships and functions of several known p120 family members, as well as the potential roles of p120 in signaling and cancer.

scholarly journals Force-dependent switch in protein unfolding pathways and transition-state movements

2016 ◽  
Vol 113 (6) ◽  
pp. E715-E724 ◽  
Author(s):  
Pavel I. Zhuravlev ◽  
Michael Hinczewski ◽  
Shaon Chakrabarti ◽  
Susan Marqusee ◽  
D. Thirumalai
Keyword(s):  
Transition State ◽  
Single Molecule ◽  
Single Point ◽  
Optical Tweezer ◽  
Sh3 Domain ◽  
Single Domain ◽  
Structural Basis ◽  
Single Point Mutation ◽  
Single Chain ◽  
Upward Curvature

Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, ku(f), as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a log⁡ku(f) plot. Similar observations were reported for other proteins for the unfolding rate ku([C]). These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [C] is increased from a low to a high value. We provide a unified theory demonstrating that if log⁡ku as a function of a perturbation (f or [C]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in log⁡ku(f) to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants.

A chimeric N-cadherin/beta 1-integrin receptor which localizes to both cell-cell and cell-matrix adhesions

1992 ◽  
Vol 103 (4) ◽  
pp. 943-951 ◽  
Author(s):  
B. Geiger ◽  
D. Salomon ◽  
M. Takeichi ◽  
R.O. Hynes
Keyword(s):  
Molecular Mechanisms ◽  
Cytoplasmic Domain ◽  
Cell Junctions ◽  
Integrin Receptor ◽  
Cell Contacts ◽  
Cell Matrix ◽  
Focal Contacts ◽  
Extracellular Region ◽  
Beta 1 Integrin ◽  
Cell Cell

To study the molecular mechanisms involved in formation of cell contacts, we have transfected cultured cells with a chimeric cDNA encoding the cytoplasmic and transmembrane domains of beta 1 integrin and the extracellular region of N-cadherin and determined the subcellular distribution of the chimeric molecule. We show that the chimeric receptor associates preferentially with cell-matrix focal contacts, suggesting that its distribution is directed by its beta 1 integrin segment, presumably via interactions of the cytoplasmic domain with cytoskeletal elements characteristic of focal contacts. Transfected cells which expressed relatively high levels of the cadherin/integrin chimera underwent an apparent epithelialization and contained the molecule both in cell-matrix and cell-cell contacts. Location in cell-cell contacts indicates competence of the cadherin extracellular domain to participate in formation of cell-cell junctions using a foreign cytoplasmic domain. Labeling of these cultures for talin, which is normally associated only with matrix adhesions, revealed specific labeling along the newly formed intercellular junctions. This suggests that the local association of talin with these sites is induced by the cytoplasmic tail of beta 1 integrin receptor presented by the chimeric protein. These results suggest that the formation of adherens-type junctions is driven by the cooperative interactions of the relevant adhesion molecules (cadherins and integrins) both with the respective extracellular ligands and with the cytoskeleton.

scholarly journals Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

2016 ◽  
Vol 113 (34) ◽  
pp. E4956-E4965 ◽  
Author(s):  
Namita Bisaria ◽  
Max Greenfeld ◽  
Charles Limouse ◽  
Dmitri S. Pavlichin ◽  
Hideo Mabuchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Structural Information ◽  
Equilibrium Constants ◽  
Single Point ◽  
Single Point Mutation ◽  
Single Molecule Fret ◽  
Predictive Understanding ◽  
And Function ◽  
And Behavior

The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.

scholarly journals Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II–dependent manner

2010 ◽  
Vol 189 (7) ◽  
pp. 1107-1115 ◽  
Author(s):  
Quint le Duc ◽  
Quanming Shi ◽  
Iris Blonk ◽  
Arnoud Sonnenberg ◽  
Ning Wang ◽  
...  
Keyword(s):  
Soft Tissues ◽  
Cell Junctions ◽  
Migration And Invasion ◽  
Dependent Manner ◽  
Surface Receptors ◽  
Classical Cadherins ◽  
Wide Range ◽  
Actomyosin Cytoskeleton ◽  
E Cadherin ◽  
Cell Cell

Cell surface receptors integrate chemical and mechanical cues to regulate a wide range of biological processes. Integrin complexes are the mechanotransducers between the extracellular matrix and the actomyosin cytoskeleton. By analogy, cadherin complexes may function as mechanosensors at cell–cell junctions, but this capacity of cadherins has not been directly demonstrated. Furthermore, the molecular composition of the link between E-cadherin and actin, which is needed to sustain such a function, is unresolved. In this study, we describe nanomechanical measurements demonstrating that E-cadherin complexes are functional mechanosensors that transmit force between F-actin and E-cadherin. Imaging experiments reveal that intercellular forces coincide with vinculin accumulation at actin-anchored cadherin adhesions, and nanomechanical measurements show that vinculin potentiates the E-cadherin mechanosensory response. These investigations directly demonstrate the mechanosensory capacity of the E-cadherin complex and identify a novel function for vinculin at cell–cell junctions. These findings have implications for barrier function, morphogenesis, cell migration, and invasion and may extend to all soft tissues in which classical cadherins regulate cell–cell adhesion.

scholarly journals Cis-clustering of cadherin-23 controls the kinetics of cell-cell adhesion

2022 ◽  
Author(s):  
Cheerneni Sai Srinivas ◽  
Gayathri Sindhuri Singaraju ◽  
Sayan Das ◽  
Amin Sagar ◽  
Anuj Kumar ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Intercellular Junctions ◽  
Liquid Droplets ◽  
Extracellular Region ◽  
Cadherin 23 ◽  
Mediate Cell ◽  
Cis And Trans ◽  
Kinetics Of ◽  
Cis Interaction ◽  
Cell Cell

Cis and trans-interactions in cadherins are the foundations of multicellularity. While the trans-interaction mediate cell-cell adhesion, the cis-interaction is postulated as strengthening to trans by clustering. The well-accepted model in cadherin-adhesion is that the trans precedes cis via a diffusion-trap kinetic model. Here we report that cadherin-23, a non-classical cadherin with an extended extracellular region, undergoes clustering in solution via lateral interactions independent of trans and phase separate as liquid droplets. In cellulo using fluorescence-recovery after the photobleaching, we noticed a significantly slow-diffusion of cadherin-23 at the intercellular junctions, indicating the diffusion of a cluster. The cis-clustering accelerates the cell-cell adhesion and, thus, kinetically controls cell-adhesion via cis precedes trans model. Though the connection of cis-clustering with the rapid adhesion is yet to explore, M2-macrophages that predominantly express cadherin-23 undergo fast attachments to circulatory tumor cells during metastasis.

scholarly journals A Single Point Mutation in the V3 Region Affects Protein Kinase Cα Targeting and Accumulation at Cell-Cell Contacts

2001 ◽  
Vol 21 (10) ◽  
pp. 3351-3363 ◽  
Author(s):  
Alice Vallentin ◽  
Thi-Chang Lo ◽  
Dominique Joubert
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Single Point ◽  
Single Point Mutation ◽  
Actin Network ◽  
Wild Type ◽  
Cell Contacts ◽  
Regulatory Processes ◽  
Protein Kinase Cα ◽  
Cell Cell

ABSTRACT Given the importance of intercellular adhesion for many regulatory processes, we have investigated the control of protein kinase Cα (PKCα) targeting to the cell-cell contacts. We have previously shown that, upon treatment of the pituitary cell line GH3B6 with thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate (PMA), human PKCα (hPKCα) is selectively targeted to the cell-cell contacts (42). Here we show that the D294G mutation of hPKCα, previously identified in a subpopulation of human tumors, induces the loss of this selective targeting. The D294G mutant is instead targeted to the entire plasma membrane, including the cell-cell contacts, and the duration of the first rapid and transient translocation induced by TRH (42) is longer than that of the wild-type enzyme (93.3 versus 22.5 s), coinciding with the duration of the [Ca2+]i increase. We found that in the presence or absence of PMA, RACK1 is never localized at the cell-cell contacts nor was it coimmunoprecipitated with hPKCα wild type or the D294G mutant. In contrast, PMA treatment or long-term TRH stimulation resulted in the presence of F-actin and β-catenin at the cell-cell contacts and their exclusion from the rest of the plasma membrane. Upon disruption of the F-actin network with phalloidin or cytochalasin D, wild-type hPKCα translocates but did not accumulate at the plasma membrane and β-catenin did not accumulate at the cell-cell contacts. In contrast, the disruption of the F-actin network affected neither translocation nor accumulation of the D294G mutant. These results show that the presence of PKCα at the cell-cell contacts is a regulated process which depends upon the integrity of both PKCα and the actin microfilament network.

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