Uptake and intracellular transport of D-penicillamine coated quantum dots (DPA-QDs) of 4 nm radius by live HeLa cells have been investigated systematically by spinning disk and 4Pi confocal microscopies. Unlike larger nanoparticles, these small DPA-QDs were observed to accumulate at the plasma membrane prior to internalization, and the uptake efficiency scaled nonlinearly with the nanoparticle concentration. Both observations indicate that a critical threshold density has to be exceeded for triggering the internalization process. By using specific inhibitors, we showed that DPA-QDs were predominantly internalized by clathrin-mediated endocytosis and to a smaller extent by macropinocytosis. Clusters of DPA-QDs were found in endosomes, which were actively transported along microtubules toward the perinuclear region. Later on, a significant fraction of endocytosed DPA-QDs were found in lysosomes, while others were actively transported to the cell periphery and exocytosed with a half-life of 21 min.

We report a facile strategy to synthesize water-soluble, fluorescent gold nanoclusters (AuNCs) in one step by using a mild reductant, tetrakis(hydroxymethyl)phosphonium chloride (THPC). A zwitterionic functional ligand, D-penicillamine (DPA), as a capping agent endowed the AuNCs with excellent stability in aqueous solvent over the physiologically relevant pH range. The DPA-capped AuNCs displayed excitation and emission bands at 400 and 610 nm, respectively; the fluorescence quantum yield was 1.3%. The effect of borohydride reduction on the optical spectra and X-ray photoelectron spectroscopy (XPS) results indicated that the AuNC luminescence is closely related to the presence of Au(I) on their surfaces. In a first optical imaging application, we studied internalization of the AuNCs by live HeLa cells using confocal microscopy with two-photon excitation. A cell viability assay revealed good biocompatibility of these AuNCs. Our studies demonstrate a great potential of DPA-stabilized AuNCs as fluorescent nanoprobes in bioimaging and related applications.

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Raster image correlation spectroscopy is a powerful tool to study fast molecular dynamics such as protein diffusion or receptor-ligand interactions inside living cells and tissues. By analysing spatio-temporal correlations of fluorescence intensity fluctuations from rasterscanned microscopy images, molecular motions can be revealed in a spatially resolved manner. Because of the diffraction-limited optical resolution, however, conventional raster image correlation spectroscopy can only distinguish larger regions of interest and requires low fluorophore concentrations in the nanomolar range. Here, to overcome these limitations, we combine raster image correlation spectroscopy with stimulated emission depletion microscopy. With imaging experiments on model membranes and live cells, we show that stimulated emission depletion-raster image correlation spectroscopy offers an enhanced multiplexing capability because of the enhanced spatial resolution as well as access to 10-100 times higher fluorophore concentrations.

In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non-nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip-pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid-independent and -dependent recruitment of the GR-GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane-bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid-mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid-mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling.

Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner.

Cellular communication in multi-cellular organisms is mediated to a large extent by a multitude of cell-surface receptors that bind specific ligands. An in-depth understanding of cell signaling networks requires quantitative information on ligand-receptor interactions within living systems. In principle, fluorescence correlation spectroscopy (FCS) based methods can provide such data, but live-cell applications have proven extremely challenging. Here, we have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Absolute ligand and receptor concentrations and their diffusion coefficients within the cell membrane can be quantified without the need to perform additional calibration experiments. We also determine the concentration of ligands diffusing in the medium outside the cell within the same experiment by using a raster image correlation spectroscopy (RICS) based analysis. We have applied this robust technique to study the interactions of two Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), to their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of living HEK293T cells. We obtained significantly lower affinities than previously reported using in vitro studies, underscoring the need to measure such data on living cells or tissues.

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