2005;16:1071C1081. that regulate the intracellular fate of ZO-2. INTRODUCTION Zona occludens (ZO)-2 is a 160-kDa protein that localizes at the cytoplasmic plaque of tight junctions (TJs) (Gumbiner (catalog no. 230196, Artic Express RP competent cells; Stratagene, La Jolla, CA). Protein expression was induced for 24 h at 10C with 0.5 mM isopropyl -d-thiogalactoside. Fusion proteins were purified by standard methods. Generation of ZO-2 Mutant S369A The QuikChange multisite-directed mutagenesis kit (catalog no 200513; Stratagene) was used according to manufacturer’s instructions to produce a serine for alanine mutation at site 369 (S369A) of canine ZO-2. For this purpose, the following primer was used: 1486TAGTGGTGTTGAGAGACGCCAAGCAAACGCTCATCAAC1523, where the numbers indicate the corresponding nucleotides in ZO-2 canine cDNA, the nucleotide triplet that gives rise to the substitute amino acid is underlined, and nucleotides in bold highlight the nucleotides that differ from the canine ZO-2 sequence. This mutation was done in the expression plasmid pGW1 containing full-length canine ZO-2 (HA-ZO-2 S369A) and in the pGEX-3X plasmid containing the amino-ZO-2-GST construct (amino-ZO-2-GST S369A). Analysis of the Subcellular Distribution of HA-ZO-2 At different time points taken after transfecting MDCK cells with hemagglutinin (HA)-ZO-2 or HA-ZO-2 Mut. S369A, the cells were fixed and processed for immunofluorescence with a mouse monoclonal immunoglobulin (Ig) G against HA (HA-probe F-7, sc-7392; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:50) followed by fluorescein isothiocyanate (FITC)-conjugated goat anti mouse (62-6511; Zymed Laboratories, South San Francisco, CA; dilution 1:100). The observations were initiated at 6 h after transfection (time 0). In all experimental conditions, at each time point the subcellular distribution patterns of HA-ZO-2 were analyzed in 100 transfected cells observed in an Eclipse E600 microscope (Nikon, Tokyo, Japan) by using Aranidipine 60 and 100 objective lenses. The nuclear recruitment index refers to the percentage of transfected cells exhibiting nuclear stain and is integrated by cells displaying nuclear distribution in any of the following patterns: only nuclear (N), membrane and nuclear (M+N), cytoplasm and nuclear (C+N), and cytoplasm, nuclear and membrane (C+N+M) (Figure 1A). The fluorescence Aranidipine images were taken in a confocal microscope (SP2; Leica, Wetzlar, Germany), with argon and helium-neon lasers and using the Leica confocal software. Open in a separate window Figure 1. The presence of ZO-2 at the nucleus diminishes with time in a process sensitive to LMB and dependent on Aranidipine ZO-2 Ser369 phosphorylation. (A) Newly synthesized HA-ZO-2 displays several subcellular patterns of distribution in MDCK cells. Nuclei were stained with ethidium bromide (red), and HA-ZO-2 was detected with a specific antibody against HA (green). N, nuclear; M, membrane; C, cytoplasm; M+C membrane and cytoplasm; M+N membrane and nucleus; C+N, cytoplasm and nucleus; and C+N+M cytoplasm, nucleus and membrane. (B) Percentage of cells with nuclear Rabbit polyclonal to PAK1 ZO-2 as a function of time. The percentage of cells with nuclear ZO-2 was determined by immunofluorescence using an anti-HA antibody. Monolayers were fixed at the indicated times. Time 0 corresponds to the 6th h after transfection. Experiments were done with cells transfected with full-length HA-ZO-2 without (full squares) or with 50 nM LMB added for the last 2 h (triangles), and with full-length HA-ZO-2 containing a point mutation at Ser369 (HA-ZO-2 Mut. S369A, circles). In parentheses, we indicate the number of independent experiments performed. In each experiment, the distribution pattern of transfected ZO-2 was analyzed in 100 cells for each time point. *p < 0.05; **p < 0.005; and ***p < 0.0005, using a Fisher exact test comparing experimental to control values. Nuclear Microinjection Assay To analyze the departure of ZO-2 from the nucleus, we designed a novel nuclear microinjection assay schematically illustrated in Figures 2A and ?and3A3A in which the antibody against ZO-2 is injected into the nucleus of live MDCK cells together with a cDNA HA-ZO-2 construct and rhodaminated albumin. Number 6A schematically illustrates another microinjection assay carried out as explained previously (Gonzalez-Mariscal for 10 min, the immunoprecipitates were processed according to the protein A-Agarose pearls manufacturer's instructions. The pellets were then solubilized in 100 l of radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP40, 1.0% sodium deoxycholate, 0.1% SDS, 0.4 mg/ml PMSF, and the protease inhibitor cocktail Complete) and 1 electrophoresis sample buffer and boiled for 10 Aranidipine min. Samples were then centrifuged for 15 min at 4C and 9000 .