Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane. INTRODUCTIONTwo major functions of epithelia in multicellular organisms are protection from the external environment and preservation of the unique biochemical composition within different body compartments (Schock and Perrimon, 2002). These functions require restriction of the passage of solutes and macromolecules (Madara, 1998) that depends on three types of specialized plasma membrane structures termed tight junction (TJ), adherens junction (AJ), and desmosomes (Tsukita et al., 2001;Matter and Balda, 2003). TJs and AJs are closely positioned at the apical-most aspect of the lateral plasma membrane and are functionally coupled in the regulation of paracellular permeability. Thus, the TJ and AJ are collectively referred to as the apical junctional complex (AJC). Both TJs and AJs represent multiprotein complexes consisting of integral membrane proteins and peripheral proteins associated with the cytosolic side of the plasma membrane (Yap et al., 1997;Tsukita et al., 2001;Pokutta and Weis, 2002;Matter and Balda, 2003). It is generally thought that integral membrane proteins of TJs and AJs interact with partners on the opposing cell plasma membrane in a trans manner providing mechanical forces for cell-cell adhesion and creating a physical barrier that limits diffusion of solutes and macromolecules. The transmembrane proteins of TJs include occludin, members of the claudin protein family and two immunoglobulin (Ig)-like proteins, junctional adhesion molecule (JAM)-A and coxsackie adenovirus receptor (Tsukita et al., 2001;Gonzalez-Mariscal et al., 2003;Matter and Balda, 2003). The major integral membrane component...

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The secondary structures of the eubacterial RNase P RNAs are being elucidated by a phylogenetic comparative approach. Sequences of genes encoding RNase P RNA from each of the recognized subgroups (a, I, y, and 8) of the proteobacteria have now been determined. These sequences allow the refinement, to nearly the base pair level, of the phylogenetic model for RNase P RNA secondary structure. Evolutionary change among the RNase P RNAs was found to occur primarily in four discrete structural domains that are peripheral to a highly conserved core structure. The new sequences were used to examine critically the proposed similarity (C. Guerrier-Takada, N. Lumelsky, and S. Altman, Science 246:1578-1584) between a portion of RNase P RNA and the "exit site" of the 23S rRNA of Escherichia cofi. Phylogenetic comparisons indicate that these sequences are not homologous and that any similarity in the structures is, at best, tenuous.RNase P cleaves leader sequences from precursor tRNAs to generate the mature 5' end of tRNA (see references 1 and 19 for reviews). In the eubacteria Bacillus subtilis and Escherichia coli, RNase P is composed of a small protein (119 amino acids) and a large RNA (-400 nucleotides). Although RNase P functions as a ribonucleoprotein in vivo, the RNA alone is an efficient and accurate catalyst at elevated concentrations of salt in vitro. RNase P contains the only RNA known to act catalytically in vivo, in the sense that each molecule of RNase P RNA acts on many substrate molecules.An accurate model for the structure of eubacterial RNase P RNA is a prerequisite for understanding the mechanism of substrate recognition, catalytic activity, and evolution of this ribozyme. A model for the secondary structure of RNase P RNA in eubacteria was originally derived by phylogenetic comparative analysis of the sequences of genes encoding RNase P RNA from several members of the y subdivision of the proteobacteria and four species of gram-positive eubacteria of the genus Bacillus (12). On the basis of the criteria used for analysis of ribosomal RNA structure (8), covariation of two base pairings within a potential continuous helix was considered proof of the occurrence of that helix. In order to increase the resolution of this model, we have cloned and sequenced the genes for RNase P RNA from a phylogenetically diverse sampling of the following additional proteobacteria: the a proteobacteria Agrobacterium tumefaciens and Rhodospirillum rubrum, the P proteobacterium Alcaligenes eutrophus, the y proteobacterium Chromatium vinosum, and the 8 proteobacterium Desulfovibrio desulfuricans (Fig. 1). Together with the previously obtained gene sequences for RNase P RNA, these analyses complete a sampling of each of the major phylogenetic branches of proteobacteria.* Corresponding author. Nucleic acid extraction. Extracts for the isolation of genomic DNAs were prepared by resuspending the cells in STE (10 mM Tris-Cl [pH 91, 100 mM NaCl, 1 mM EDTA) (15 ml/g of cells [wet weight]), treating them with lysozyme (1 mg/ml) for 5 min ...

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