A method for spectral analysis of Förster resonance energy transfer (FRET) signals is presented, taking into consideration both the contributions of unpaired donor and acceptor fluorophores and the influence of incomplete labeling of the interacting partners. It is shown that spectral analysis of intermolecular FRET cannot yield accurate values of the Förster energy transfer efficiency E, unless one of the interactors is in large excess and perfectly labeled. Instead, analysis of donor quenching yields a product of the form Ef(d)p(a), where f(d) is the fraction of donor-type molecules participating in donor-acceptor complexes and p(a) is the labeling probability of the acceptor. Similarly, analysis of sensitized emission yields a product involving Ef(a). The analysis of intramolecular FRET (e.g., of tandem constructs) yields the product Ep(a). We use our method to determine these values for a tandem construct of cyan fluorescent protein and yellow fluorescent protein and compare them with those obtained by standard acceptor photobleaching and fluorescence lifetime measurements. We call the method lux-FRET, since it relies on linear unmixing of spectral components.

The common neurotransmitter serotonin controls different aspects of early neuronal differentiation, although the underlying mechanisms are poorly understood. Here we report that activation of the serotonin 5-HT 7 receptor promotes synaptogenesis and enhances synaptic activity in hippocampal neurons at early postnatal stages. An analysis of G␣ 12 -deficient mice reveals a critical role of G 12 -protein for 5-HT 7 receptor-mediated effects in neurons. In organotypic preparations from the hippocampus of juvenile mice, stimulation of 5-HT 7 R/G 12 signaling potentiates formation of dendritic spines, increases neuronal excitability, and modulates synaptic plasticity. In contrast, in older neuronal preparations, morphogenetic and synaptogenic effects of 5-HT 7 /G 12 signaling are abolished. Moreover, inhibition of 5-HT 7 receptor had no effect on synaptic plasticity in hippocampus of adult animals. Expression analysis reveals that the production of 5-HT 7 and G␣ 12 -proteins in the hippocampus undergoes strong regulation with a pronounced transient increase during early postnatal stages. Thus, regulated expression of 5-HT 7 receptor and G␣ 12 -protein may represent a molecular mechanism by which serotonin specifically modulates formation of initial neuronal networks during early postnatal development.

In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and studied oligomerization dynamics in living cells. We also investigated the role of receptor palmitoylation in this process. Biochemical analysis performed in neuroblastoma N1E-115 cells demonstrated that both palmitoylated and non-palmitoylated 5-HT1A receptors form homo-oligomers and that the prevalent receptor species at the plasma membrane are dimers. A combination of an acceptor-photobleaching FRET approach with fluorescence lifetime measurements verified the interaction of CFP- and YFP-labeled wild-type as well as acylation-deficient 5-HT1A receptors at the plasma membrane of living cells. Using a novel FRET technique based on the spectral analysis we also confirmed the specific nature of receptor oligomerization. The analysis of oligomerization dynamics revealed that apparent FRET efficiency measured for wild-type oligomers significantly decreased in response to agonist stimulation, and our combined results suggest that this decrease was mediated by accumulation of FRET-negative complexes rather than by dissociation of oligomers to monomers. In contrast, the agonist-mediated decrease of FRET signal was completely abolished in oligomers composed by non-palmitoylated receptor mutants, demonstrating the importance of palmitoylation in modulation of the structure of oligomers.

Early stages of the adhesion of human endothelial cells onto a set of smooth polymer films were analyzed to reveal the modulation of cell-matrix interactions by the physicochemical constraints of predeposited fibronectin (FN). Hydrophobic and hydrophilic polymer substrates, consisting of poly(octadecene-alt-maleic anhydride) and poly(propene-alt-maleic anhydride) films, were coated with similar amounts of FN at conditions of either covalent or noncovalent immobilization. The well-defined substrates permit variation of the anchorage of FN at invariant topography, pliability, and molecular composition. Although all of the compared FN coatings were effective in stimulating attachment of endothelial cells, the initial formation of cell-matrix adhesions was found to be controlled by the type of interaction between predeposited FN and the underlying substrate. Covalent linkage and hydrophobic interactions of the predeposited FN with the polymer films interfered with the rapid generation of focal and fibrillar adhesions. It was demonstrated that this was caused by the fact that only weakly bound FN could become readily reorganized by the adherent cells. Upon prolonged culture periods at standard cell culture conditions, secretion and deposition of organized extracellular matrix by the attached cells was found to balance out the differences of the substrates.

During development of the nervous system, short- and long-range signals cooperate to promote axonal growth, guidance, and target innervation. Particularly, a short-range signal transducer, the neural cell adhesion molecule (NCAM), stimulates neurite outgrowth via mechanisms that require posttranslational modification of NCAM and signaling via receptors to a long-range messenger, the fibroblast growth factor (FGF). In the present study we further characterized a mechanism which regulates the functional interplay between NCAM and FGF receptor(s). We show that activation of FGF receptor(s) by FGF2 leads to palmitoylation of the two major transmembrane NCAM isoforms, NCAM140 and NCAM180, translocation of NCAM to GM1 ganglioside-containing lipid rafts, and stimulation of neurite outgrowth of hippocampal neurons. Ablation of NCAM, mutation of NCAM140 or NCAM180 palmitoylation sites, or pharmacological suppression of NCAM signaling inhibited FGF2-stimulated neurite outgrowth. Of the 23 members of the aspartate-histidine-histidine-cysteine (DHHC) domain containing proteins, DHHC-7 most strongly stimulated palmitoylation of NCAM, and enzyme activity was enhanced by FGF2. Thus, our study uncovers a molecular mechanism by which a growth factor regulates neuronal morphogenesis via activation of palmitoylation, which in turn modifies subcellular location and thus signaling via an adhesion molecule.

No abstract

Förster resonance energy transfer (FRET)-based biosensors for the quantitative analysis of intracellular signaling, including sensors for monitoring cyclic adenosine monophosphate (cAMP), are of increasing interest. The measurement of the donor/acceptor emission ratio in tandem biosensors excited at the donor excitation wavelength is a commonly used technique. A general problem, however, is that this ratio varies not only with the changes in cAMP concentration but also with the changes of the ionic environment or other factors affecting the folding probability of the fluorophores. Here, we use a spectral FRET analysis on the basis of two excitation wavelengths to obtain a reliable measure of the absolute cAMP concentrations with high temporal and spatial resolution by using an "exchange protein directly activated by cAMP". In this approach, FRET analysis is simplified and does not require additional calibration routines. The change in FRET efficiency (E) of the biosensor caused by [cAMP] changes was determined as DeltaE = 15%, whereas E varies between 35% at low and 20% at high [cAMP], allowing quantitative measurement of cAMP concentration in the range from 150 nM to 15 microM. The method described is also suitable for other FRET-based biosensors with a 1:1 donor/acceptor stoichiometry. As a proof of principle, we measured the specially resolved cAMP concentration within living cells and determined the dynamic changes of cAMP levels after stimulation of the Gs-coupled serotonin receptor subtype 7 (5-HT7).

Background: Characterization of hematopoietic stem cells (HSCs) by laser scanning cytometry (LSC) was compared with conventional flow cytometry (FCM). The method was evaluated for application in the development of advanced cell culture substrates that were supposed to support the ex vivo expansion of HSC. For this purpose, adherent HSCs were grown in culture on thin polymer films coated with reconstituted collagen I fibrils and subsequently analyzed by LSC. Methods: CD34ϩ HSCs were isolated from cord blood by immunomagnetic separation and cultivated on polymer films coated with reconstituted collagen I fibrils. Cell surface antigens (CD34, CD29) were stained with antibodies, and nuclei were labeled with a DNA stain (TO-PRO-3 iodide) that does not interfere with the fluorochromes of the antibodies. Fluorescence intensity of the adherent cells was measured by means of LSC. Before and after in vitro expansion for time periods of up to 7 days, suspension cells were analyzed with LSC and FCM. Results: LSC-based analysis enabled reliable quantification of CD34 ϩ cells with bright antigen expression before cell