Abstract. A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY-labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N- [5-(5,7-dimethyl (Cs-DMB-Cer), which exhibited a shift in its emission maximum from green (,x,515 nm) to red (•620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller mounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that Cs-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration-dependent spectral properties of C~-DMB-Cer were used to monitor the transport of Cs-DMB-lipids to the cell surface at 37°C. . This molecule is a vital stain for the Golgi apparatus (24), and, in combination with fluorescence video imaging, has been used to study the dynamics of this organelle within living cells (8). C6-NBD-Cer also stains the Golgi apparatus of fixed cells, most likely through interaction(s) with endogenous lipids and cholesterol, and serves as a trans-Golgi marker for both light and electron microscopy in these preparations (29,33). Similar to its endoge- nous counterpart, C6-NBD-Cer is metabolized in living cells to sphingomyelin (SM) and a glycolipid, glucosylceramide (GlcCer) (17,22,23,32,44,45). These fluorescent metabolites are subsequently transported to the plasma membrane of cells from the Golgi complex by a vesicle-mediated process (17,23). In polarized cells the fluorescent Cer is metabolized to fluorescent analogues of SM and GlcCer, and the latter is preferentially delivered to the apical cell surface (44,45). This polarized delivery is consistent with the known enrichment of glycosphingolipids in apical membranes, and indicates that C6-NBD-Cer and its metabolites are recognized by the cellular sorting and transport machinery in a manner similar to their natural counterparts. BODIPY)-l-pentanoyl]-D-erythro-sphingosineAlthough much useful information has already been obtained using C6-NBD-Cer, there are several disadvantages of this probe. First, the NBD-fluorophore is rapidly bleached during observation under...

An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal 89Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with 89Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing 89Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to high selective tumor uptake (67.5 ± 5.0 %ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic.

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Developmental Dynamics 232:359 -368, 2005.