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Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.

Mandai K, Nakanishi H, Satoh A, Obaishi H, Wada M, Nishioka H, Itoh M, Mizoguchi A, Aoki T, Fujimoto T, Matsuda Y, Tsukita S, Takai Y - J. Cell Biol. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan.

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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Different localization of l-afadin, ZO-1, and  desmoplakin. (a1–a3) Localization of l-afadin and ZO-1  in mouse small intestine: (a1)  l-afadin; (a2) ZO-1; (a3) l-afadin and ZO-1. arrows, the  sites where green (ZO-1),  yellow (the mixture of l-afadin and ZO-1), and red (l-afadin) signals occurred in  this order from the apical  side to the basal side. (b1–b3)  Localization of l-afadin and  desmoplakin in the isolated  bile canaliculi: (b1) l-afadin;  (b2) desmoplakin; (b3) l-afadin and desmoplakin. Asterisk, the inner space of the  bile canaliculi. Bars: (a) 15  μm; (b) 5 μm.
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Figure 8: Different localization of l-afadin, ZO-1, and desmoplakin. (a1–a3) Localization of l-afadin and ZO-1 in mouse small intestine: (a1) l-afadin; (a2) ZO-1; (a3) l-afadin and ZO-1. arrows, the sites where green (ZO-1), yellow (the mixture of l-afadin and ZO-1), and red (l-afadin) signals occurred in this order from the apical side to the basal side. (b1–b3) Localization of l-afadin and desmoplakin in the isolated bile canaliculi: (b1) l-afadin; (b2) desmoplakin; (b3) l-afadin and desmoplakin. Asterisk, the inner space of the bile canaliculi. Bars: (a) 15 μm; (b) 5 μm.

Mentions: To examine the precise localization of l-afadin at the junctional complex region, the frozen sections of small intestine were doubly stained with the monoclonal anti–ZO-1 antibody, and the bile canaliculi isolated from liver were doubly stained with the monoclonal antidesmoplakin antibody. ZO-1 and desmoplakin are known to be markers for tight junction (Stevenson et al., 1986; Itoh et al., 1993) and desmosome (Skerrow and Matoltsy, 1974; Franke et al., 1983; Mueller and Franke, 1983; Miller et al., 1987), respectively. In the absorptive epithelia, l-afadin was localized slightly more at the basal side than ZO-1 (Fig. 8 a1–a3). In the canaliculi, the localization of l-afadin did not coincide with that of desmoplakin (Fig. 8 b1–b3). These results indicate that l-afadin is localized at cell-to-cell AJ rather than at tight junction or desmosome. The localization of l-afadin at cell-to-cell AJ was finally confirmed by immunoelectron microscopy of intestine absorptive epithelia using the silver enhancement and ultrathin cryosection techniques. l-Afadin was associated with the undercoat of cell-to-cell AJ (Fig. 9, a and b).


Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.

Mandai K, Nakanishi H, Satoh A, Obaishi H, Wada M, Nishioka H, Itoh M, Mizoguchi A, Aoki T, Fujimoto T, Matsuda Y, Tsukita S, Takai Y - J. Cell Biol. (1997)

Different localization of l-afadin, ZO-1, and  desmoplakin. (a1–a3) Localization of l-afadin and ZO-1  in mouse small intestine: (a1)  l-afadin; (a2) ZO-1; (a3) l-afadin and ZO-1. arrows, the  sites where green (ZO-1),  yellow (the mixture of l-afadin and ZO-1), and red (l-afadin) signals occurred in  this order from the apical  side to the basal side. (b1–b3)  Localization of l-afadin and  desmoplakin in the isolated  bile canaliculi: (b1) l-afadin;  (b2) desmoplakin; (b3) l-afadin and desmoplakin. Asterisk, the inner space of the  bile canaliculi. Bars: (a) 15  μm; (b) 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139800&req=5

Figure 8: Different localization of l-afadin, ZO-1, and desmoplakin. (a1–a3) Localization of l-afadin and ZO-1 in mouse small intestine: (a1) l-afadin; (a2) ZO-1; (a3) l-afadin and ZO-1. arrows, the sites where green (ZO-1), yellow (the mixture of l-afadin and ZO-1), and red (l-afadin) signals occurred in this order from the apical side to the basal side. (b1–b3) Localization of l-afadin and desmoplakin in the isolated bile canaliculi: (b1) l-afadin; (b2) desmoplakin; (b3) l-afadin and desmoplakin. Asterisk, the inner space of the bile canaliculi. Bars: (a) 15 μm; (b) 5 μm.
Mentions: To examine the precise localization of l-afadin at the junctional complex region, the frozen sections of small intestine were doubly stained with the monoclonal anti–ZO-1 antibody, and the bile canaliculi isolated from liver were doubly stained with the monoclonal antidesmoplakin antibody. ZO-1 and desmoplakin are known to be markers for tight junction (Stevenson et al., 1986; Itoh et al., 1993) and desmosome (Skerrow and Matoltsy, 1974; Franke et al., 1983; Mueller and Franke, 1983; Miller et al., 1987), respectively. In the absorptive epithelia, l-afadin was localized slightly more at the basal side than ZO-1 (Fig. 8 a1–a3). In the canaliculi, the localization of l-afadin did not coincide with that of desmoplakin (Fig. 8 b1–b3). These results indicate that l-afadin is localized at cell-to-cell AJ rather than at tight junction or desmosome. The localization of l-afadin at cell-to-cell AJ was finally confirmed by immunoelectron microscopy of intestine absorptive epithelia using the silver enhancement and ultrathin cryosection techniques. l-Afadin was associated with the undercoat of cell-to-cell AJ (Fig. 9, a and b).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan.

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.

Show MeSH
Related in: MedlinePlus