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Molecular and Cellular Biology, May 2008, p. 3324-3335, Vol. 28, No. 10
0270-7306/08/$08.00+0     doi:10.1128/MCB.00144-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Involvement of Actinin-4 in the Recruitment of JRAB/MICAL-L2 to Cell-Cell Junctions and the Formation of Functional Tight Junctions {triangledown}

Hiroyoshi Nakatsuji,1,2 Noriyuki Nishimura,1 Rie Yamamura,1 Hiro-omi Kanayama,2 and Takuya Sasaki1*

Departments of Biochemistry,1 Urology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan2

Received 28 January 2008/ Accepted 2 March 2008

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 Ca2+ 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.

* Corresponding author. Mailing address: Department of Biochemistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan. Phone: 81-88-633-9223. Fax: 81-88-633-9227. E-mail: sasaki{at}basic.med.tokushima-u.ac.jp

{triangledown} Published ahead of print on 10 March 2008.

Molecular and Cellular Biology, May 2008, p. 3324-3335, Vol. 28, No. 10
0270-7306/08/$08.00+0     doi:10.1128/MCB.00144-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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