Membrane proteins of cytotoxic T cells specifically reorganize to form an immunological synapse (IS) on interaction with their specific target. In this paper, we investigated the redistribution of Kv1.3 channels, which are the dominant voltage-gated potassium channels, in the plasma membrane of allogen-activated human cytotoxic T lymphocytes (CTLs) on interacting with their specific target cells. Kv1.3 channels bearing a FLAG epitope were expressed in the CTLs and the cell-surface distribution of fluorescently labeled ion channels was determined from confocal laser-scanning microscopy images. FLAG epitope-tagged Kv1.3 channels showed a patchy distribution in CTLs not engaged with target cells, whereas the channels were accumulated in the IS formed between CTLs and specific target lymphocytes. Localization of Kv1.3 channels in the IS might open an unrevealed possibility in the regulation of ion channel activity by signaling molecules accumulated in the IS.

P-glycoprotein (Pgp), coded for by the mdr1 gene, is one of the ABC transporters held responsible for the phenomenon of multidrug resistance (mdr), which is reflected by a rapidly escalating failure of chemotherapy with different classes of cytotoxic agents: anthracyclins, vinca alkaloids, taxanes, epipodophylotoxins. Although overcoming resistance conveyed by Pgp alone may not be sufficient for reaching effective treatment, the abundance of observations available for this paradigmatic multidrug transporter at both in vitro and in vivo setting is a tempting ground for an updated assessment of the main currents of mdr research. In this review we attempt to help keep track of the features of Pgp-mediated drug transport that serve as the major starting points for ongoing efforts of mdr reversal. We will analyze the slowly narrowing gaps that prevail between our ever increasing understanding at the protein, cell and organism level, focusing on the molecular interactions involving Pgp.

Major histocompatibility complex (MHC) class II molecules displayed clustered patterns at the surfaces of T (HUT-102B2) and B (JY) lymphoma cells characterized by interreceptor distances in the micrometer range as detected by scanning force microscopy of immunogold-labeled antigens. Electron microscopy revealed that a fraction of the MHC class II molecules was also heteroclustered with MHC class I antigens at the same hierarchical level as described by the scanning force microscopy data, after specifically and sequentially labeling the antigens with 30- and 15-nm immunogold beads. On JY cells the estimated fraction of co-clustered HLA II was 0.61, whereas that of the HLA I was 0.24. Clusterization of the antigens was detected by the deviation of their spatial distribution from the Poissonian distribution representing the random case. Fluorescence resonance energy transfer measurements also confirmed partial co-clustering of the HLA class I and II molecules at another hierarchical level characterized by the 2- to 10-nm Förster distance range and providing fine details of the molecular organization of receptors. The larger-scale topological organization of the MHC class I and II antigens may reflect underlying membrane lipid domains and may fulfill significant functions in cell-to-cell contacts and signal transduction.

Background: A novel flow cytometric assay has been described in an accompanying report (Gombos et al.,Methods: The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft‐preserving Triton X‐100 or Nonidet P‐40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft‐mediated and non–raft‐related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents.Results: The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2‐labeled cell‐surface Pgps to Triton X‐100 versus Nonidet P‐40. Approximately 34% of the UIC2 Fab‐labeled Pgp molecules were associated with the cytoskeleton through detergent‐resistant, cholesterol‐sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and G_M1‐gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping.Conclusions: By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS‐174‐T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels.