scholarly journals Maintaining epithelial integrity

2003 ◽  
Vol 162 (7) ◽  
pp. 1305-1315 ◽  
Author(s):  
Katja Röper ◽  
Nicholas H. Brown
Keyword(s):  
Cell Adhesion ◽  
Adherens Junction ◽  
Binding Domain ◽  
Actin Binding ◽  
Cytoskeletal Protein ◽  
Epithelial Integrity ◽  
Actin Binding Domain ◽  
Mutant Cells ◽  
Spectrin Repeats ◽  
Novel Isoforms

The Short stop (Shot/Kakapo) spectraplakin is a giant cytoskeletal protein, which exists in multiple isoforms with characteristics of both spectrin and plakin superfamilies. Previously characterized Shot isoforms are similar to spectrin and dystrophin, with an actin-binding domain followed by spectrin repeats. We describe a new large exon within the shot locus, which encodes a series of plakin repeats similar to the COOH terminus of plakins such as plectin and BPAG1e. We find that the plakin repeats are inserted between the actin-binding domain and spectrin repeats, generating isoforms as large as 8,846 residues, which could span 400 nm. These novel isoforms localized to adherens junctions of embryonic and follicular epithelia. Loss of Shot within the follicle epithelium leads to double layering and accumulation of actin and ZO-1 in between, and a reduction of Armadillo and Discs lost within, mutant cells, indicative of a disruption of adherens junction integrity. Thus, we identify a new role for spectraplakins in mediating cell–cell adhesion.

scholarly journals Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform

2011 ◽  
Vol 2011 ◽  
pp. 1-18
Author(s):  
Richard A. Zuellig ◽  
Beat C. Bornhauser ◽  
Ralf Amstutz ◽  
Bruno Constantin ◽  
Marcus C. Schaub
Keyword(s):  
Actin Cytoskeleton ◽  
Tissue Expression ◽  
Protein Product ◽  
Binding Domain ◽  
Actin Binding ◽  
Rat Tissues ◽  
Cortical Actin ◽  
Actin Binding Domain ◽  
Spectrin Repeats

Utrophin and dystrophin present two large proteins that link the intracellular actin cytoskeleton to the extracellular matrix via the C-terminal-associated protein complex. Here we describe a novel short N-terminal isoform of utrophin and its protein product in various rat tissues (N-utro, 62 kDa, amino acids 1–539, comprising the actin-binding domain plus the first two spectrin repeats). Using different N-terminal recombinant utrophin fragments, we show that actin binding exhibits pronounced negative cooperativity (affinity constantsK1=∼5×106andK2=∼1×105 M-1) and is Ca2+-insensitive. Expression of the different fragments in COS7 cells and in myotubes indicates that the actin-binding domain alone binds exlusively to actin filaments. The recombinant N-utro analogue binds in vitro to actin and in the cells associates to the membranes. The results indicate that N-utro may be responsible for the anchoring of the cortical actin cytoskeleton to the membranes in muscle and other tissues.

scholarly journals Force-dependent allostery of the α-catenin actin-binding domain controls adherens junction dynamics and functions

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Noboru Ishiyama ◽  
Ritu Sarpal ◽  
Megan N. Wood ◽  
Samantha K. Barrick ◽  
Tadateru Nishikawa ◽  
...  
Keyword(s):  
Adherens Junction ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Binding Domain

scholarly journals The Cryogenic Electron Microscopy Structure of the Cell Adhesion Regulator Metavinculin Reveals an Isoform-Specific Kinked Helix in Its Cytoskeleton Binding Domain

2021 ◽  
Vol 22 (2) ◽  
pp. 645
Author(s):  
Erumbi S. Rangarajan ◽  
Tina Izard
Keyword(s):  
Electron Microscopy ◽  
Cell Adhesion ◽  
Binding Domain ◽  
Cytoskeletal Proteins ◽  
Cell Junctions ◽  
Actin Binding ◽  
Cell Matrix ◽  
Actin Binding Domain ◽  
Α Helix ◽  
Cell Cell

Vinculin and its heart-specific splice variant metavinculin are key regulators of cell adhesion processes. These membrane-bound cytoskeletal proteins regulate the cell shape by binding to several other proteins at cell–cell and cell–matrix junctions. Vinculin and metavinculin link integrin adhesion molecules to the filamentous actin network. Loss of both proteins prevents cell adhesion and cell spreading and reduces the formation of stress fibers, focal adhesions, or lamellipodia extensions. The binding of talin at cell–matrix junctions or of α-catenin at cell–cell junctions activates vinculin and metavinculin by releasing their autoinhibitory head–tail interaction. Once activated, vinculin and metavinculin bind F-actin via their five-helix bundle tail domains. Unlike vinculin, metavinculin has a 68-amino-acid insertion before the second α-helix of this five-helix F-actin–binding domain. Here, we present the full-length cryogenic electron microscopy structure of metavinculin that captures the dynamics of its individual domains and unveiled a hallmark structural feature, namely a kinked isoform-specific α-helix in its F-actin-binding domain. Our identified conformational landscape of metavinculin suggests a structural priming mechanism that is consistent with the cell adhesion functions of metavinculin in response to mechanical and cellular cues. Our findings expand our understanding of metavinculin function in the heart with implications for the etiologies of cardiomyopathies.

scholarly journals Binding to F-actin guides cadherin cluster assembly, stability, and movement

2013 ◽  
Vol 201 (1) ◽  
pp. 131-143 ◽  
Author(s):  
Soonjin Hong ◽  
Regina B. Troyanovsky ◽  
Sergey M. Troyanovsky
Keyword(s):  
Adherens Junction ◽  
Random Motion ◽  
Binding Domain ◽  
Actin Binding ◽  
Cluster Assembly ◽  
Structural Elements ◽  
Binding Interface ◽  
Extracellular Region ◽  
Actin Binding Domain ◽  
Intracellular Binding

The cadherin extracellular region produces intercellular adhesion clusters through trans- and cis-intercadherin bonds, and the intracellular region connects these clusters to the cytoskeleton. To elucidate the interdependence of these binding events, cadherin adhesion was reconstructed from the minimal number of structural elements. F-actin–uncoupled adhesive clusters displayed high instability and random motion. Their assembly required a cadherin cis-binding interface. Coupling these clusters with F-actin through an α-catenin actin-binding domain (αABD) dramatically extended cluster lifetime and conferred direction to cluster motility. In addition, αABD partially lifted the requirement for the cis-interface for cluster assembly. Even more dramatic enhancement of cadherin clustering was observed if αABD was joined with cadherin through a flexible linker or if it was replaced with an actin-binding domain of utrophin. These data present direct evidence that binding to F-actin stabilizes cadherin clusters and cooperates with the cis-interface in cadherin clustering. Such cooperation apparently synchronizes extracellular and intracellular binding events in the process of adherens junction assembly.

scholarly journals Characterisation ofSchizosaccharomyces pombe α-actinin

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1858 ◽  
Author(s):  
Barbara Addario ◽  
Linda Sandblad ◽  
Karina Persson ◽  
Lars Backman
Keyword(s):  
Salt Bridge ◽  
Structure And Function ◽  
Binding Domain ◽  
Actin Binding ◽  
Structural Domain ◽  
Structural Domains ◽  
Actin Binding Domain ◽  
Ef Hand ◽  
And Function ◽  
Spectrin Repeats

The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms,α-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain ofα-actinin from the fission yeastSchizosaccharomyces pombeconsists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventionalα-actinin, we have cloned and characterized each structural domain. Our results show that this a-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventionalα-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in otherα-actinins, which may reduce the affinity for actin.

scholarly journals Afadin: A Novel Actin Filament–binding Protein with One PDZ Domain Localized at Cadherin-based Cell-to-Cell Adherens Junction

1997 ◽  
Vol 139 (2) ◽  
pp. 517-528 ◽  
Author(s):  
Kenji Mandai ◽  
Hiroyuki Nakanishi ◽  
Ayako Satoh ◽  
Hiroshi Obaishi ◽  
Manabu Wada ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Rat Brain ◽  
Actin Filament ◽  
Pdz Domain ◽  
Adherens Junction ◽  
Binding Domain ◽  
Actin Binding ◽  
Calculated Molecular Mass ◽  
Splicing Variant ◽  
Actin Binding Domain

A novel actin filament (F-actin)–binding protein with a molecular mass of ∼205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin–binding domain at 1,631–1,829 aa residues and one PDZ domain at 1,016– 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009–1,093 aa residues but lacking the F-actin–binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin–cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.

scholarly journals A Coiled-Coil Domain of Melanophilin Is Essential for Myosin Va Recruitment and Melanosome Transport in Melanocytes

2006 ◽  
Vol 17 (11) ◽  
pp. 4720-4735 ◽  
Author(s):  
Alistair N. Hume ◽  
Abul K. Tarafder ◽  
José S. Ramalho ◽  
Elena V. Sviderskaya ◽  
Miguel C. Seabra
Keyword(s):  
Coiled Coil ◽  
Binding Domain ◽  
Actin Binding ◽  
Binding Studies ◽  
Null Cell ◽  
Coiled Coil Region ◽  
Actin Binding Domain ◽  
Transport Assay ◽  
Myosin Va

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

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