Excitation spectra and brightness optimization of two-photon excited probes.

Two-photon probe excitation data are commonly presented as absorption cross section or molecular brightness (the detected fluorescence rate per molecule). We report two-photon molecular brightness spectra for a diverse set of organic and genetically encoded probes with an automated spectroscopic system based on fluorescence correlation spectroscopy. The two-photon action cross section can be extracted from molecular brightness measurements at low excitation intensities, while peak molecular brightness (the maximum molecular brightness with increasing excitation intensity) is measured at higher intensities at which probe photophysical effects become significant. The spectral shape of these two parameters was similar across all dye families tested. Peak molecular brightness spectra, which can be obtained rapidly and with reduced experimental complexity, can thus serve as a first-order approximation to cross-section spectra in determining optimal wavelengths for two-photon excitation, while providing additional information pertaining to probe photostability. The data shown should assist in probe choice and experimental design for multiphoton microscopy studies. Further, we show that, by the addition of a passive pulse splitter, nonlinear bleaching can be reduced--resulting in an enhancement of the fluorescence signal in fluorescence correlation spectroscopy by a factor of two. This increase in fluorescence signal, together with the observed resemblance of action cross section and peak brightness spectra, suggests higher-order photobleaching pathways for two-photon excitation.

Fluorescence cross-correlation spectroscopy reveals mechanistic insights into the effect of 2'-O-methyl modified siRNAs in living cells.

RNA interference (RNAi) offers a powerful tool to specifically direct the degradation of complementary RNAs, and thus has great therapeutic potential for targeting diseases. Despite the reported preferences of RNAi, there is still a need for new techniques that will allow for a detailed mechanistic characterization of RNA-induced silencing complex (RISC) assembly and activity to further improve the biocompatibility of modified siRNAs. In contrast to previous reports, we investigated the effects of 2'-O-methyl (2'OMe) modifications introduced at specific positions within the siRNA at the early and late stages of RISC assembly, as well as their influence on target recognition and cleavage directly in living cells. We found that six to 10 2'OMe nucleotides on the 3'-end inhibit passenger-strand release as well as target-RNA cleavage without changing the affinity, strand asymmetry, or target recognition. 2'OMe modifications introduced at the 5'-end reduced activated RISC stability, whereas incorporations at the cleavage site showed only minor effects on passenger-strand release when present on the passenger strand. Our new fluorescence cross-correlation spectroscopy assays resolve different steps and stages of RISC assembly and target recognition with heretofore unresolved detail in living cells, which is needed to develop therapeutic siRNAs with optimized in vivo properties.

Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.

Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2-associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

Independence of maximum single molecule fluorescence count rate on the temporal and spectral laser pulse width in two-photon FCS.

We investigate the fluorescence emission characteristics of standard dye tetramethylrhodamine (TMR) in two-photon fluorescence correlation spectroscopy for different temporal and spectral properties of the femtosecond excitation pulses. After determining the second-order dispersion of our setup, including the microscope objective, a pulse stretcher was employed to control the temporal width at the location of the specimen. As expected, the fluorescence per molecule and therefore the signal-to-noise ratio of an FCS-measurement can be improved at constant average excitation power by altering either the temporal or spectral width of the excitation pulses. We found however, that the maximum achievable molecular brightness is largely independent of the temporal and spectral width in the regime analyzed. This observation confirms the current working hypothesis for two-photon fluorescence correlation spectroscopy that bleaching and saturation, and thus, the inherent properties of the dye system, are the dominant effects limiting the quality of measurements. As a practical consequence, elaborate optimization of temporal and spectral laser pulse width, e.g. by introducing pulse stretchers in the beam path, is less critical than previously expected.

Nuclear translocation of the transcription factor STAT5 in the rat brain after systemic leptin administration.

Leptin binding to its functional receptor stimulates JAK-STAT-signaling pathway, which finally results in activation and nuclear translocation of transcription factors of the signal transducer and activator of transcription (STAT) family, namely of STAT3. Here we report for the first time that systemic treatment with leptin (5 mg/kg; intraperitoneal injection) also increased the number of nuclear STAT5 signals in the hypothalamus. In particular, the entire arcuate nucleus (ARC), the ventral premammilary nucleus (PMV), and the supraoptic nucleus (SO) showed an enhanced nuclear STAT5 translocation in response to leptin when compared to saline, 120 min after the respective injection. Co-localization studies revealed that a high percentage of those STAT5-responsive cells proved to be neurons. In addition, some astrocytes within the ARC showed nuclear STAT5 signals. The functional relevance of leptin-induced nuclear STAT5 activation in hypothalamic cells still has to be determined.

Pyrexia, anorexia, adipsia, and depressed motor activity in rats during systemic inflammation induced by the Toll-like receptors-2 and -6 agonists MALP-2 and FSL-1.

Macrophage-activating lipopeptide-2 (MALP-2) from Mycoplasma fermentans has been identified as a pathogen-associated molecular pattern of Mycoplasmas that causes activation of the innate immune system through the activation of the heterodimeric Toll-like receptors (TLRs)-2 and -6. The aim of this study was to characterize the ability of MALP-2 and a synthetic analog fibroblast-stimulating lipopeptide-1 (FSL-1; represents the NH2-terminal sequence of a lipoprotein from M. salivarium) to act as exogenous pyrogens, to induce formation of cytokines (endogenous pyrogens), and to cause sickness behavior, such as depressed motor activity, anorexia, and adipsia. For this purpose, body temperature, activity, food intake, and water intake were recorded for 3 days by use of telemetry devices in several groups of rats treated with MALP-2/FSL-1 or the respective control solutions. Intraperitoneal injections of FSL-1 caused fever at doses of 10 or 100 microg/kg, which was preceded by a pronounced phase of hypothermia in response to a dose of 1,000 microg/kg. The maximal fever (a peak of 1.5 degrees C above baseline) was caused by the 100 microg/kg dose with almost identical responses to both MALP-2 and FSL-1. Fever was accompanied by pronounced rises of the proinflammatory cytokines TNF and IL-6 in plasma. Treatment with the TLR-2 and -6 agonists further induced a dose-dependent manifestation of anorexia and adipsia, as well as a reduction of motor activity. We could thus demonstrate that activation of TLR-2 and -6 can induce systemic inflammation in rats accompanied by the classical signs of brain-controlled illness responses.

Immunohistochemical evidence of functional leptin receptor expression in neuronal and endothelial cells of the rat brain.

Leptin binding to its functional receptor leads to activation of the JAK-STAT-signaling pathway and especially to the activation of the signal transducer and activator of transcription factor 3 (STAT3). The immunohistochemical detection of nuclear STAT3 translocation is used as a neuroanatomical mapping tool to determine leptin-responsive cells in the rat brain. This study neuroanatomically identifies those brain cell phenotypes showing STAT3 activation after intraperitoneal leptin treatment (5 mg/kg) using immunohistochemical colocalization with neuronal and endothelial cell marker proteins. Leptin treatment induced nuclear STAT3 signals with the strongest response observed 90min after the treatment. The caudobasal hypothalamus showed a particularly pronounced STAT3 response. Leptin-induced nuclear STAT3 signals were additionally determined in the solitary tract nucleus, the choroid plexus and in the brain endothelium. The vast majority of STAT3-responsive cells proved to be neurons located in the caudobasal hypothalamus, however, a marked number of brain endothelial cells distributed throughout the entire brain got activated as well. In conclusion, neurons and non-neuronal brain cells, e.g., endothelial or choroid plexus cells, seem to express functional leptin receptors and might thereby mediate leptin-dependent functions in the rat brain.