Apical cell adhesion molecule, trophinin, localizes to the nuclear envelope

Abstract

Trophinin mediates homophilic and apical cell adhesion between trophoblastic cells and endometrial epithelial cells, which is potentially the initial attachment step in human embryo implantation. Since trophinin is an atypical membrane protein without the signal sequence, it is possible that trophinin localizes to the cytoplasm. By treating trophinin-expressing trophoblastic cells with a series of detergents, we found significant levels of endogenous trophinin in the cytoplasm, particularly at the nuclear envelope (NE). Fluorescence photobleaching of GFP-trophinin expressed in COS-1 cells showed the stable association of trophinin with the NE, suggesting an additional role of trophinin besides apical cell adhesion.

Nuclear localization of magphinins, alternative splicing products of the human trophinin gene

“…Previously we showed that endogenous magphinin‐α in human trophoblastic teratocarcinoma HT‐H cells is nuclear [Aoyama et al, 2005]. However, here magphinin‐α expressed in mouse NIH3T3 cells was cytoplasmic (Fig.…”

“…To determine whether magphinin‐γ associates with the nuclear matrix, we undertook sequential extraction [He et al, 1990; Aoyama et al, 2005; Nakayama et al, 2006] (Fig. 5).…”

“…COS‐1 or NIH3T3 cells were cultured in Iscove's modified Dulbecco's medium (IMDM) containing 5% fetal bovine serum (FBS) or 10% calf serum (CS) at 37°C in a humidified incubator under 5% CO 2 . Cells were transfected with a plasmid vector encoding each magphinin isoform or mutant forms using LipofectAMINE™2000 Reagent (Life Technologies) or TransIT Transfection Reagent (Mirus), according to the manufacturer's instructions, as reported [Yamaguchi et al, 2001; Kasahara et al, 2004; Aoyama et al, 2005; Nakayama and Yamaguchi, 2005].…”

“…Extracted proteins were resolved by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated with anti‐HA (Y‐11) or anti‐GFP antibody, followed by horseradish peroxidase‐conjugated F(ab') 2 fragments of goat anti‐rabbit IgG antibody [Yamaguchi et al, 2001; Aoyama et al, 2005; Kasahara et al, 2007b]. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham Biosciences) using an image analyzer LAS‐1000‐plus (Fujifilm).…”

“…Nuclear matrix fractionation was performed essentially as described [He et al, 1990; Aoyama et al, 2005; Nakayama et al, 2006]. Cells were washed and extracted for 3 min at 4°C with cytoskeleton (CSK) buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl 2 , and 1 mM EGTA) supplemented with 0.5% Triton X‐100 and protease inhibitors.…”