scholarly journals Involvement of Actinin-4 in the Recruitment of JRAB/MICAL-L2 to Cell-Cell Junctions and the Formation of Functional Tight Junctions

2008 ◽  
Vol 28 (10) ◽  
pp. 3324-3335 ◽  
Author(s):  
Hiroyoshi Nakatsuji ◽  
Noriyuki Nishimura ◽  
Rie Yamamura ◽  
Hiro-omi Kanayama ◽  
Takuya Sasaki
Keyword(s):  
Tight Junctions ◽  
Binding Protein ◽  
Cell Junctions ◽  
Actin Binding ◽  
Deletion Mutants ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Interfering Rna ◽  
Two Hybrid ◽  
Cell Cell

ABSTRACT 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.

scholarly journals α-Catenin-independent Recruitment of ZO-1 to Nectin-based Cell-Cell Adhesion Sites through Afadin

2001 ◽  
Vol 12 (6) ◽  
pp. 1595-1609 ◽  
Author(s):  
Shigekazu Yokoyama ◽  
Kouichi Tachibana ◽  
Hiroyuki Nakanishi ◽  
Yasunori Yamamoto ◽  
Kenji Irie ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Tight Junctions ◽  
Cell Lines ◽  
Adhesion Molecule ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Adhesion Sites ◽  
Cell Cell

ZO-1 is an actin filament (F-actin)–binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through β- and α-catenins as one of many F-actin–binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin–binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin–β-catenin complex through α-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and α-catenin–deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only α-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of α-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with α-catenin but also with the nectin-afadin system.

scholarly journals Steroidogenic Acute Regulatory Protein-binding Protein Cloned by a Yeast Two-hybrid System

2003 ◽  
Vol 278 (43) ◽  
pp. 42487-42494 ◽  
Author(s):  
Teruo Sugawara ◽  
Hiroshi Shimizu ◽  
Nobuhiko Hoshi ◽  
Ayako Nakajima ◽  
Seiichiro Fujimoto
Keyword(s):  
Protein Binding ◽  
Hybrid System ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Steroidogenic Acute Regulatory Protein ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Steroidogenic Acute Regulatory ◽  
Two Hybrid

scholarly journals The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein

1998 ◽  
Vol 141 (7) ◽  
pp. 1515-1527 ◽  
Author(s):  
Ping Lin ◽  
Helen Le-Niculescu ◽  
Robert Hofmeister ◽  
J. Michael McCaffery ◽  
Mingjie Jin ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Yeast Two Hybrid ◽  
Golgi Membranes ◽  
Yeast Two Hybrid System ◽  
Golgi Region ◽  
Ef Hand ◽  
Golgi Network ◽  
Two Hybrid

We have identified CALNUC, an EF-hand, Ca2+-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gαi3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gαi3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca2+-binding protein detected by 45Ca2+-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.

scholarly journals Rac3-induced Neuritogenesis Requires Binding to Neurabin I

2006 ◽  
Vol 17 (5) ◽  
pp. 2391-2400 ◽  
Author(s):  
Donata Orioli ◽  
Ivan N. Colaluca ◽  
Miria Stefanini ◽  
Silvano Riva ◽  
Carlos G. Dotti ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Binding Protein ◽  
Growth Cones ◽  
Actin Binding ◽  
Primary Neurons ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Gtp Binding ◽  
Rho Family ◽  
Two Hybrid

Rac3, a neuronal GTP-binding protein of the Rho family, induces neuritogenesis in primary neurons. Using yeast two-hybrid analysis, we show that Neurabin I, the neuronal F-actin binding protein, is a direct Rac3-interacting molecule. Biochemical and light microscopy studies indicate that Neurabin I copartitions and colocalizes with Rac3 at the growth cones of neurites, inducing Neurabin I association to the cytoskeleton. Moreover, Neurabin I antisense oligonucleotides abolish Rac3-induced neuritogenesis, which in turn is rescued by exogenous Neurabin I but not by Neurabin I mutant lacking the Rac3-binding domain. These results show that Neurabin I mediates Rac3-induced neuritogenesis, possibly by anchoring Rac3 to growth cone F-actin.

Identification of α-tubulin as an hsp105α-binding protein by the yeast two-hybrid system

2003 ◽  
Vol 286 (2) ◽  
pp. 233-240 ◽  
Author(s):  
Youhei Saito ◽  
Nobuyuki Yamagishi ◽  
Keiichi Ishihara ◽  
Takumi Hatayama
Keyword(s):  
Hybrid System ◽  
Binding Protein ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

scholarly journals Assembly and function of AP-3 complexes in cells expressing mutant subunits

2002 ◽  
Vol 156 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Andrew A. Peden ◽  
Rachel E. Rudge ◽  
Winnie W.Y. Lui ◽  
Margaret S. Robinson
Keyword(s):  
Binding Site ◽  
Deletion Mutant ◽  
Deletion Mutants ◽  
Yeast Two Hybrid ◽  
Point Mutant ◽  
Mouse Mutants ◽  
Yeast Two Hybrid System ◽  
Truncation Mutant ◽  
And Function ◽  
Two Hybrid

The mouse mutants mocha and pearl are deficient in the AP-3 δ and β3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the β3 and μ3 subunits coassemble into a heterodimer, whereas the σ3 subunit remains monomeric. In pearl cells, the δ and σ3 subunits coassemble into a heterodimer, whereas μ3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of β3 and μ3 can interact with each other. Pearl cell lines were generated that express β3A, β3B, a β3Aβ2 chimera, two β3A deletion mutants, and a β3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only β3A, β3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The β3Aβ2 chimera and the β3A short deletion mutant gave partial functional rescue, whereas the β3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of β3 are important for function, but the clathrin binding site is not needed.

Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

2013 ◽  
Vol 38 (9) ◽  
pp. 1583-1591
Author(s):  
Li-Yan XUE ◽  
Bing LUO ◽  
Li-Quan ZHU ◽  
Yong-Jun YANG ◽  
He-Cui ZHANG ◽  
...  
Keyword(s):  
Hybrid System ◽  
Dna Sequences ◽  
Extracellular Domain ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid
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