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1.
Proc Natl Acad Sci U S A ; 117(49): 31157-31165, 2020 12 08.
Article in English | MEDLINE | ID: mdl-33229577

ABSTRACT

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-α2ß1, 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.


Subject(s)
Cadherins/genetics , Ephrin-B1/genetics , Protein Binding/genetics , Protein Interaction Maps/genetics , Cadherins/ultrastructure , Cell Adhesion/genetics , Cytoplasm/genetics , Cytoplasm/ultrastructure , Desmocollins , Desmoglein 2/genetics , Desmoglein 2/ultrastructure , Desmoplakins/genetics , Desmoplakins/ultrastructure , Desmosomes/genetics , Desmosomes/ultrastructure , Ephrin-B1/ultrastructure , Humans , Integrins/genetics , Integrins/ultrastructure , Microscopy, Atomic Force , Protein Domains/genetics , Single Molecule Imaging
2.
Nat Commun ; 10(1): 1181, 2019 03 12.
Article in English | MEDLINE | ID: mdl-30862836

ABSTRACT

Attachment of human adenovirus (HAd) to the host cell is a critical step of infection. Initial attachment occurs via the adenoviral fibre knob protein and a cellular receptor. Here we report the cryo-electron microscopy (cryo-EM) structure of a <100 kDa non-symmetrical complex comprising the trimeric HAd type 3 fibre knob (HAd3K) and human desmoglein 2 (DSG2). The structure reveals a unique stoichiometry of 1:1 and 2:1 (DSG2: knob trimer) not previously observed for other HAd-receptor complexes. We demonstrate that mutating Asp261 in the fibre knob is sufficient to totally abolish receptor binding. These data shed new light on adenovirus infection strategies and provide insights for adenoviral vector development and structure-based design.


Subject(s)
Adenoviruses, Human/metabolism , Capsid Proteins/metabolism , Desmoglein 2/metabolism , Receptors, Virus/metabolism , Virus Attachment , Adenoviridae Infections/pathology , Adenoviridae Infections/virology , Adenoviruses, Human/pathogenicity , Asparagine/genetics , Capsid Proteins/ultrastructure , Cryoelectron Microscopy , Desmoglein 2/ultrastructure , HEK293 Cells , Humans , Models, Molecular , Protein Domains , Receptors, Virus/ultrastructure , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure
3.
Eur J Cell Biol ; 86(3): 127-42, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17275137

ABSTRACT

For cell and molecular biological studies of heart formation and function cell cultures of embryonal, neonatal or adult hearts of various vertebrates, notably rat and chicken, have been widely used. As the myocardium-specific cell-cell junctions, the intercalated disks (ID), have recently been found to be particularly sensitive to losses of - or mutations in - certain cytoskeletal proteins, resulting in cardiac damages, we have examined the ID organization in primary cultures of cardiomyocytes obtained from neonatal rats. Using immunofluorescence and immunoelectron microscopy, we have studied the major ID components for up to 2 weeks in culture, paying special attention to spontaneously beating, individual cardiomyocytes and myocardial cell colonies. While our results demonstrate the formation of some ID-like cardiomyocyte-connecting junction arrays, they also reveal a variety of structural disorders such as rather extended, junction-free ID regions, sac-like invaginations and endocytotic blebs as well as accumulations of intracytoplasmic structures suggestive of endocytosed forms of junction-derived vesicles or of junction fragments resembling fascia adhaerens elements. Moreover, we have noticed a novel type of small, obviously plaque-free cytoplasmic vesicles containing one or both of the desmosomal cadherins, desmocollin Dsc2 and desmoglein Dsg2. We conclude that cardiomyocyte cultures are useful model systems for studies of certain aspects of myocardiac differentiation and functions but, on the other hand, show progressive disintegration and deterioration. The potential value of molecular markers and reagents in studies of myocardial pathology as well as in the monitoring of myocardial differentiation of so-called stem cells is discussed.


Subject(s)
Adherens Junctions/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Vertebrates/metabolism , Adherens Junctions/ultrastructure , Animals , Animals, Newborn , Antibody Specificity/immunology , Cadherins/ultrastructure , Cells, Cultured , Desmoglein 2/ultrastructure , Desmoplakins/ultrastructure , Fluorescent Antibody Technique , Immunoblotting , Myocytes, Cardiac/ultrastructure , Rats , Rats, Wistar
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