Article

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

Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan.
The Journal of Cell Biology (Impact Factor: 9.83). 11/1997; 139(2):517-28. DOI: 10.1083/jcb.139.2.517
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ABSTRACT

A novel actin filament (F-actin)-binding protein with a molecular mass of approximately 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.

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    • "Tissue-and cell type–specific alternative splicing profoundly contributes to many aspects of animal development, and this is nowhere more evident than in the nervous system, especially in production of synaptic diversity at the later stages of neuronal development (Grabowski and Black, 2001; Schreiner et al., 2014). Northern and Western blot analyses showed that the two afadin isoforms display distinct tissue expression patterns; l-afadin is ubiquitously expressed, whereas s-afadin is brain specific (Mandai et al., 1997). It was recently shown that s-afadin is expressed in neurons but not in and subsequent local accumulation of F-actin, called actin patches, along the axon shaft, where the formation of actin-driven protrusions , such as filopodia and lamellipodia, and subsequent branch generation occur (Loudon et al., 2006). "
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    ABSTRACT: Precise wiring patterns of axons are one of the remarkable features of neuronal circuit formation, and establishment of the proper neuronal network requires control of outgrowth, branching, and guidance of axons. R-Ras is a Ras family small GTPase that has essential roles in multiple phases of axonal development. We recently identified afadin, an F-actin-binding protein, as an effector of R-Ras mediating axon branching through F-actin reorganization. Afadin comprises two isoforms, l-afadin having F-actin-binding domain and s-afadin lacking the F-actin-binding domain. Compared with l-afadin, s-afadin, the short splicing variant of l-afadin, contains RA domains but lacks the F-actin-binding domain. Neurons express both isoforms, however the function of s-afadin in brain remains unknown. Here we identify s-afadin as an endogenous inhibitor of cortical axon branching. In contrast to the abundant and constant expression of l-afadin throughout neuronal development, the expression of s-afadin is relatively low when cortical axons branch actively. Ectopic expression and knockdown of s-afadin suppresses and promotes branching, respectively. s-Afadin blocks the R-Ras-mediated membrane translocation of l-afadin and axon branching through inhibiting the binding of l-afadin to R-Ras. Thus, s-afadin acts as a dominant-negative isoform in R-Ras-afadin-regulated axon branching. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Mar 2015 · Molecular biology of the cell
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    • "Rabbit anti-l-afadin and rabbit anti-l/s-afadin were prepared as described [13]. The Abs listed below were purchased from commercial sources: rat anti-nectin-1, clone 48–12 (MBL); rat anti-nectin-3, clone 103-A1 (MBL); mouse anti-N-cadherin, clone 32 (BD Biosciences); rabbit anti-N-cadherin (Takara); rabbit anti-β-catenin (Sigma); rabbit anti-synapsin I (Millipore); guinea pig anti-VGLUT1 (Millipore); mouse anti-bassoon (Enzo Life Sciences); mouse anti-PSD-95, clone 7E3-1B8 (Enzo Life Sciences) and clone K28/43 (NeuroMab); mouse anti-actin (clone C4) (Millipore); and chicken anti-MAP2 (Abcam). "
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    ABSTRACT: The formation and remodeling of mossy fiber-CA3 pyramidal cell synapses in the stratum lucidum of the hippocampus are implicated in the cellular basis of learning and memory. Afadin and its binding cell adhesion molecules, nectin-1 and nectin-3, together with N-cadherin, are concentrated at puncta adherentia junctions (PAJs) in these synapses. Here, we investigated the roles of afadin in PAJ formation and presynaptic differentiation in mossy fiber-CA3 pyramidal cell synapses. At these synapses in the mice in which the afadin gene was conditionally inactivated before synaptogenesis by using nestin-Cre mice, the immunofluorescence signals for the PAJ components, nectin-1, nectin-3 and N-cadherin, disappeared almost completely, while those for the presynaptic components, VGLUT1 and bassoon, were markedly decreased. In addition, these signals were significantly decreased in cultured afadin-deficient hippocampal neurons. Furthermore, the interevent interval of miniature excitatory postsynaptic currents was prolonged in the cultured afadin-deficient hippocampal neurons compared with control neurons, indicating that presynaptic functions were suppressed or a number of synapse was reduced in the afadin-deficient neurons. Analyses of presynaptic vesicle recycling and paired recordings revealed that the cultured afadin-deficient neurons showed impaired presynaptic functions. These results indicate that afadin regulates both PAJ formation and presynaptic differentiation in most mossy fiber-CA3 pyramidal cell synapses, while in a considerable population of these neurons, afadin regulates only PAJ formation but not presynaptic differentiation.
    Full-text · Article · Feb 2014 · PLoS ONE
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    • "Afadin is localized at AJs in epithelial and endothelial cells and regulates the formation of AJs in cooperation with nectins and cadherins [14]. Afadin is an actin filament-binding protein, encoded by the MLLT4/AF-6 gene [15]. Afadin has some splicing isoforms and the longest one, l-afadin, hereafter referred to as afadin, is ubiquitously expressed including epithelial cells. "
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    ABSTRACT: Adherens junctions (AJs) play a role in mechanically connecting adjacent cells to maintain tissue structure, particularly in epithelial cells. The major cell-cell adhesion molecules at AJs are cadherins and nectins. Afadin binds to both nectins and α-catenin and recruits the cadherin-β-catenin complex to the nectin-based cell-cell adhesion site to form AJs. To explore the role of afadin in radial glial and ependymal cells in the brain, we generated mice carrying a nestin-Cre-mediated conditional knockout (cKO) of the afadin gene. Newborn afadin-cKO mice developed hydrocephalus and died neonatally. The afadin-cKO brain displayed enlarged lateral ventricles and cerebral aqueduct, resulting from stenosis of the caudal end of the cerebral aqueduct and obliteration of the ventral part of the third ventricle. Afadin deficiency further caused the loss of ependymal cells from the ventricular and aqueductal surfaces. During development, radial glial cells, which terminally differentiate into ependymal cells, scattered from the ventricular zone and were replaced by neurons that eventually covered the ventricular and aqueductal surfaces of the afadin-cKO midbrain. Moreover, the denuded ependymal cells were only occasionally observed in the third ventricle and the cerebral aqueduct of the afadin-cKO midbrain. Afadin was co-localized with nectin-1 and N-cadherin at AJs of radial glial and ependymal cells in the control midbrain, but these proteins were not concentrated at AJs in the afadin-cKO midbrain. Thus, the defects in the afadin-cKO midbrain most likely resulted from the destruction of AJs, because AJs in the midbrain were already established before afadin was genetically deleted. These results indicate that afadin is essential for the maintenance of AJs in radial glial and ependymal cells in the midbrain and is required for normal morphogenesis of the cerebral aqueduct and ventral third ventricle in the midbrain.
    Preview · Article · Nov 2013 · PLoS ONE
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