Involvement of actinin-4 in the recruitment of JRAB/MICAL-L2 to cell-cell junctions and the formation of functional tight junctions.
Tight junctions (TJs) are cell-cell adhesive structures that undergo continuous remodeling. We previously demonstrated that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) localized at TJs and mediated the endocytic recycling of the integral TJ protein occludin and the formation of functional TJs. Here, we investigated how JRAB/MICAL-L2 was targeted to TJs. Using a series of deletion mutants, we found the plasma membrane (PM)-targeting domain within JRAB/MICAL-L2. We then identified actinin-4, which was originally isolated as an actin-binding protein associated with cell motility and cancer invasion/metastasis, as a binding protein for the PM-targeting domain of JRAB/MICAL-L2, using a yeast two-hybrid system. Actinin-4 was colocalized with JRAB/MICAL-L2 at cell-cell junctions and linked JRAB/MICAL-L2 to F-actin. Although actinin-4 bound to JRAB/MICAL-L2 without Rab13, the actinin-4-JRAB/MICAL-L2 interaction was enhanced by Rab13 activation. Depletion of actinin-4 by using small interfering RNA inhibited the recruitment of occludin to TJs during the Ca(2+) switch. During the epithelial polarization after replating, JRAB/MICAL-L2 was recruited from the cytosol to cell-cell junctions. This JRAB/MICAL-L2 recruitment as well as the formation of functional TJs was delayed in actinin-4-depleted cells. These results indicate that actinin-4 is involved in recruiting JRAB/MICAL-L2 to cell-cell junctions and forming functional TJs.
Nakatsuji
Hiroyoshi
H
Department of Biochemistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan.
Nishimura
Noriyuki
N
Yamamura
Rie
R
Kanayama
Hiro-Omi
HO
Sasaki
Takuya
T
D000185:Actinin / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D016764:Cell Polarity; D006224:Cricetinae; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors; D007365:Intercellular Junctions / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D062793:Occludin; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D019108:Tight Junctions / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; C491752:Actn4 protein, mouse; D003598:Cytoskeletal Proteins; C512298:JRAB protein, mouse; D008565:Membrane Proteins; D008840:Microfilament Proteins; D062793:Occludin; C568986:Ocln protein, mouse; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000185:Actinin; C484177:Rab13 protein, mouse; D020691:rab GTP-Binding Proteins; D002118:Calcium