A number of cell functions, such as flagellar beating, swimming velocity, acrosome reaction, etc., are triggered by a Ca2+ influx across the cell membrane. For appropriate physiological functions, the motile human sperm maintains the intracellular free calcium concentration ([Ca2+]i) at a submicromolar level. The objective of this study was to determine the role of the Na+/Ca2+ exchanger (NCX) in the maintenance of [Ca2+]i in human spermatozoa. Spermatozoa maintained in extracellular medium containing>or=1 microM Ca2+ exhibited motility similar to that of the control. In addition to several calcium transport mechanisms described earlier, we provide evidence that the NCX plays a crucial role in the maintenance of [Ca2+]i. Three chemically unrelated inhibitors of the NCX (bepridil, DCB (3',4'-dichlorobenzamil hydrochloride), and KB-R7943) all blocked human sperm motility in a dose and incubation time dependent manner. The IC50 values for bepridil, DCB, and KB-R7943 were 16.2, 9.8, and 5.3 microM, respectively. The treatment with the above-mentioned blockers resulted in an elevated [Ca2+]i and a decreased [Na+]i. The store-operated calcium channel (SOCC) inhibitor SKF 96365 also blocked the sperm motility (IC50=2.44 microM). The presence of the NCX antigen in the human spermatozoa was proven by flow cytometry, confocal laser scanning microscopy, and immunoblotting techniques. Calcium homeostasis of human spermatozoa is maintained by several transport proteins among which the SOCC and the NCX may play a major role.

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The role of the expression patterns of proteins involved in oncogenesis can be understood after characterizing their multimolecular interactions. Conventional FRET methods permit the analysis of interaction between two molecular species at the most, which necessitates the introduction of new approaches for studying multicomponent signaling complexes. Flow cytometric as well as microscopic donor (dbFRET) and acceptor (abFRET) photobleaching FRET measurements were performed to determine the association states of ErbB2, b1-integrin, and CD44 receptors. Based on consecutively applied abFRET and dbFRET methods (two-sided FRET), the relationship of b1-integrin-ErbB2 heteroassociation to ErbB2 homoassociation and of b1-integrin-ErbB2 heteroassociation to ErbB2-CD44 heteroassociation was studied by correlating pixel-bypixel FRET values of the corresponding abFRET and dbFRET images in contour plots. Anticorrelation was observed between b1-integrin-ErbB2 heteroassociation and ErbB2 homoassociation on trastuzumab sensitive N87 and SK-BR-3 cells, while modest positive correlation was found between b1-integrin-ErbB2 and ErbB2-CD44 heteroassociation on trastuzumab resistant MKN-7 cells. The FRET efficiency values of b1-integrinErbB2 heteroassociation were markedly higher at the focal adhesion regions on attached cells than those measured by flow cytometry on detached cells. In conclusion, we implemented an experimental set-up termed two-sided FRET for correlating two pairwise interactions of three arbitrarily chosen molecular species. On the basis of our results, we assume that the homoassociation state of ErbB2 is dynamically modulated by its interaction with b1-integrins. ' International Society for Analytical CytologyKey terms receptor tyrosine kinase; ErbB2, b1-integrin; CD44; trastuzumab resistance; laser scanning confocal microscopy; fluorescence resonance energy transfer; tsFRET; dbFRET; abFRET UNDERSTANDING the molecular mechanisms of signal transduction processes requires the elucidation of assembling and/or disassembling of protein complexes including the conformational changes accompanying these processes. One of the best approaches for studying these molecular rearrangements is fluorescence resonance energy transfer (FRET), which is a sensitive method for measuring intra-or intermolecular distances. FRET-based methods are capable of resolving molecular associations and conformational changes in the 1-10 nm range far exceeding the diffraction limit of the conventional microscope. FRET is a nonradiative process in which energy is transferred from the first excited electronic state of a donor molecule (D) to a nearby acceptor molecule (A) in a dipole-dipole interaction under favorable conditions, resulting in the quenching of donor fluorescence accompanied by the increase (also known as sensitization) of acceptor fluorescence (1-3). For mapping the physical associations of various membrane molecules, flow cytometric and microscopic

The IL-9R consists of the α-subunit and the γc-chain shared with other cytokine receptors, including IL-2R, an important regulator of T cells. We have previously shown that IL-2R is expressed in common clusters with MHC glycoproteins in lipid rafts of human T lymphoma cells raising the question what the relationship between clusters of IL-2R/MHC and IL-9R is. Confocal microscopic co-localization and FRET experiments capable of detecting membrane protein organization at different size scales revealed non-random association of IL-9R with IL-2R/MHC clusters at the surface of human T lymphoma cells. Accommodation of IL-9Rα in membrane areas segregated from the IL-2R/MHC domains could also be detected. The bipartite nature of IL-9R distribution was mirrored by STAT activation results. Our data indicate that co-compartmentation with MHC glycoproteins is a general property of γc receptors. Distribution of receptor chains between different membrane domains may regulate their function.