Abstract. Molecular mechanisms linking pre-and postsynaptic membranes at the interneuronal synapses are little known. We tested the cadherin adhesion system for its localization in synapses of mouse and chick brains. We found that two classes of cadherin-associated proteins, aN-and 13-catenin, are broadly distributed in adult brains, colocalizing with a synaptic marker, synaptophysin. At the ultrastructural level, these proteins were localized in synaptic junctions of various types, forming a symmetrical adhesion structure. These structures sharply bordered the transmitter release sites associated with synaptic vesicles, although their segregation was less clear in certain types of synapses. N-cadherin was also localized at a similar site of synaptic junctions but in restricted brain nuclei. In developing synapses, the catenin-bearing contacts dominated their junctional structures. These findings demonstrate that interneuronal synaptic junctions comprise two subdomains, transmitter release zone and cateninbased adherens junction. The catenins localized in these junctions are likely associated with certain cadherin molecules including N-cadherin, and the cadherin/catenin complex may play a critical role in the formation or maintenance of synaptic junctions.T HE synapse is a site where the axon terminal of a neuron comes into functional contact with a target cell. To generate the synapses, each neuron selectively contacts and communicates with other particular neurons. Despite the importance of understanding how specific neuronal connections are established, little is known about what kinds of adhesion molecules are essential for the formation and maintenance of interneuronal synaptic junctions. This lack of information is contrasted with a great deal of knowledge about other synaptic structures and functions, including synaptic vesicle traffic, channel/ receptor function, and signal transduction in synapses (Jessell and Kandel, 1993; Stidhof, 1995).Various electron microscopic studies have revealed the ultrastructural features of interneuronal synaptic junctions. Electron-dense cytoplasmic materials accumulate under both pre-and postsynaptic plasma membranes; and the former is, in general, associated with a cluster of synaptic vesicles. These junctional structures are considered to be the sites where synaptic transmission takes place. The interneuronal synaptic junctions exhibit some variations in morphological characteristics of the cytoplasmic density. Gray (1959) and Colonnier (1968) showed that there are two types of synapses, asymmetric (Gray's type I) and symmetric (Gray's type II). The intercellular space beAddress all correspondence to Masatoshi Takeichi, Department of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-01, Japan. Tel.: (81) 75 751 2111. Fax: (81) 75 753 4197. e-maih takeichi@take.biophys.kyoto-u.ac.jp. tween the membrane thickenings, called the synaptic cleft, is occupied by filamentous materials.Molecular analysis of the synaptic junctions has thus far b...

Recent studies have shown that mutations at amino-acid 482 in the ABCG2 gene affect the substrate specificity of the protein. To delineate the effects of these mutations clearly, human embryonic kidney cells (HEK-293) were stably transfected with wild-type 482R or mutant 482G and 482T ABCG2. By flow cytometry, mitoxantrone, BODIPY-prazosin, and Hoechst 33342 were found to be substrates of all ABCG2 proteins, while rhodamine 123, daunorubicin, and LysoTracker Green were transported only by mutant ABCG2. In cytotoxicity assays, all ABCG2 proteins conferred high levels of resistance to mitoxantrone, SN-38, and topotecan, while mutant ABCG2 also exhibited a gain of function for mitoxantrone as they conferred a four-fold greater resistance compared to wild type. Cells transfected with mutant ABCG2 were 13-to 71-fold resistant to the P-glycoprotein substrates doxorubicin, daunorubicin, epirubicin, bisantrene, and rhodamine 123 compared to cells transfected with wild-type ABCG2, which were only three-to four-fold resistant to these compounds. ABCG2 did not confer appreciable resistance to etoposide, taxol or the histone deacetylase inhibitor depsipeptide. None of the transfected cell lines demonstrated resistance to flavopiridol despite our previous observation that ABCG2-overexpressing cell lines are cross-resistant to the drug. Recently reported inhibitors of ABCG2 were evaluated and 50 mM novobiocin was found to reverse wild-type ABCG2 completely, but only reverse mutant ABCG2 partially. The studies presented here serve to underscore the importance of amino-acid 482 in defining the substrate specificity of the ABCG2 protein and raise the possibility that amino-acid 482 mutations in human cancers could affect the clinical application of antagonists for ABCG2.

The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compound in a series of MXR-overexpressing drug-selected cell lines and in ten unselected cell lines which were used to determine if the four fluorescent compounds were sensitive enough to detect the low MXR levels found in drug-sensitive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was found in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and low but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both selected and unselected cell lines was found to correlate well with MXR levels as determined by Northern blotting, r(2)=0.89 and r(2)=0.70 respectively. In contrast, rhodamine and LysoTracker were not able to reliably detect MXR. Cytotoxicity assays performed on two of the four unselected cell lines confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibition of MXR-associated ATPase activity at 1 microM FTC. Short term selections of the SF295, KM12, NCI-H460 and A549 cell lines in mitoxantrone resulted in a small but measurable increase in MXR by both Northern blot and functional assay. These studies show that flow cytometric measurement of FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and unselected cell lines.