Abstract
The pleckstrin-homology-domain-containing protein PLEKHA7 was recently identified as a protein linking the E-cadherin-p120 ctn complex to the microtubule cytoskeleton. Here we characterize the expression, tissue distribution and subcellular localization of PLEKHA7 by immunoblotting, immunofluorescence microscopy, immunoelectron microscopy, and northern blotting in mammalian tissues. Anti-PLEKHA7 antibodies label the junctional regions of cultured kidney epithelial cells by immunofluorescence microscopy, and major polypeptides of Mr ∼135 kDa and ∼145 kDa by immunoblotting of lysates of cells and tissues. Two PLEKHA7 transcripts (∼5.5 kb and ∼6.5 kb) are detected in epithelial tissues. PLEKHA7 is detected at epithelial junctions in sections of kidney, liver, pancreas, intestine, retina, and cornea, and its tissue distribution and subcellular localization are distinct from ZO-1. For example, PLEKHA7 is not detected within kidney glomeruli. Similarly to E-cadherin, p120 ctn, β-catenin and α-catenin, PLEKHA7 is concentrated in the apical junctional belt, but unlike these adherens junction markers, and similarly to afadin, PLEKHA7 is not localized along the lateral region of polarized epithelial cells. Immunoelectron microscopy definitively establishes that PLEKHA7 is localized at the adherens junctions in colonic epithelial cells, at a mean distance of 28 nm from the plasma membrane. In summary, we show that PLEKHA7 is a cytoplasmic component of the epithelial adherens junction belt, with a subcellular localization and tissue distribution that is distinct from that of ZO-1 and most AJ proteins, and we provide the first description of its distribution and localization in several tissues.
Highlights
Epithelial cells are characterized by an apical junctional complex, comprising tight junctions (TJ), adherens junctions (AJ) and desmosomes [1]
PLEKHA7 is restricted to the AJ apical belt, and is absent from the lateral membrane To further examine the junctional localization of PLEKHA7, we examined by double immunofluorescence microscopy tissue sections stained with antibodies against PLEKHA7 and several markers of AJ [6,25], including the transmembrane protein Ecadherin, and several cytoplasmic plaque AJ proteins: 1) a-catenin, which interacts with the cytoplasmic domain of E-cadherin through b-catenin; 2) b-catenin, which interacts with E-cadherin and a-catenin; 3) p120 ctn, which interacts with the juxtamembrane region of E-cadherin, and with PLEKHA7 [19]; 4) afadin, which interacts with nectin and a-catenin [12]
The AJ localization of PLEKHA7 was inferred in cultured intestinal epithelial cells from the profile of distribution of PLEKHA7 immunofluorescent labeling along the lateral membrane, which was partially co-localized with E-cadherin [19]
Summary
Epithelial cells are characterized by an apical junctional complex, comprising tight junctions (TJ), adherens junctions (AJ) and desmosomes [1]. TJ control the barrier function of epithelia and maintain cell polarity, and AJ regulate cell-cell adhesion and morphogenesis [2,3]. Actomyosin filaments modulate the TJ barrier and orchestrate the signaling and adhesive functions of AJ, through the interaction with several actin-binding junctional proteins, including Zonula-Occludens-1 (ZO-1), acatenin, and afadin [9,10,11,12]. It was shown that Ecadherin, the major transmembrane protein of AJ, associates with microtubules through a protein complex comprising p120 ctn, and the newly identified proteins PLEKHA7 and nezha [19]. It was shown that PLEKHA7 partially co-localizes with E-cadherin in cultured intestinal cells [19], nothing is known about its pattern of expression and its subcellular localization in epithelial tissues. The localization of PLEKHA7 at the ultrastructural level has not been determined in any tissue
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