Proteins achieve their biological functions in cells by cooperation in protein complexes. In this study, we employed fluorescence lifetime imaging microscopy (FLIM)‐based Förster resonance energy transfer (FRET) measurements to investigate protein complexes comprising S100A11 and different members of the annexin (ANX) family, such as ANXA1, ANXA2, ANXA4, ANXA5, and AnxA6, in living cells. Using an S100A11 mutant without the capacity for Ca2+ binding, we found that Ca2+ binding of S100A11 is important for distinct S100A11/ANXA2 complex formation; however, ANXA1‐containing complexes were unaffected by this mutant. An increase in the intracellular calcium concentration induced calcium ionophores, which strengthened the ANXA2/S100A11 interaction. Furthermore, we were able to show that S100A11 also interacts with ANXA4 in living cells. The FLIM‐FRET approach used here can serve as a tool to analyze interactions between S100A11 and distinct annexins under physiological conditions in living cells.

In this study, a series of benzopyrylium monomethine dyes bearing a benzothiazole moiety and mainly differing in their substitution pattern were characterized regarding their ability for nucleic acid binding and their use as fluorescent probes for sensing and imaging of nucleic acids. Dyes were characterized with special focus on structure and substituent effects on dye binding towards nucleic acids. Absorption maxima of the free, unbound dyes ranged from 474 to 498 nm. Fluorescence intensities of the unbound dyes were extremely low with emission maxima in the range of 512 to 584 nm depending on the polarity of the used solvent. Quantum yields of the free dyes were below 1 %. Upon binding to nucleic acids, however, dyes became highly fluorescent. Dyes with ste-rically demanding 2 -di-or-triamino substituents exhibited a significant enhancement of the fluorescence quantum yield in presence of nucleic acids. The formation of stable dye-nucleic acid complexes was confirmed by time-and spectrally-resolved fluorescence measurements using a streak camera-based setup. Dye complexation was associated with prolonged lifetimes. All dyes were cell permeable and able to stain nucleic acids present in nuclear structures in living as well as in fixed cells. Colocalization analysis with nuclear specific reporters revealed that all dyes bind selectively to nucleic acids, which confirms their applicability as highly specific fluorescent probes for sensing nucleic acids and cell nuclei imaging. In comparison to commercially available nucleic acid stains, the investigated dyes displayed superior staining properties with high cell viability and less cytoplasmic enrichment. In conclusion, the results obtained by different complementary methods demonstrate that studied dyes are useful fluorescent probes for sensing and imaging of nucleic acids in-vitro, in fixed as well as in living cells. Bulkier dye substituents like piperazinyl and the presence of quaternary ammonium salts in the 2-amino side chain enhance nucleic acid binding significantly, thereby making the parent dye structure attractive for sensing applications and ongoing studies of structure-function relationship of nucleic acid stains.

Hereditary spastic paraplegia is a spastic gait disorder that arises from degeneration of corticospinal axons. The subtype SPG48 is associated with mutations in the zeta subunit of the adaptor protein complex five (AP5). AP5 function and the pathophysiology of SPG48 are only poorly understood. Here, we report an AP5 zeta knockout mouse, which shows an age-dependent degeneration of corticospinal axons. Our analysis of knockout fibroblasts supports a trafficking defect from late endosomes to the transGolgi network and reveals a structural defect of the Golgi. We further show that both autophagic flux and the recycling of lysosomes from autolysosomes were impaired in knockout cells. In vivo, we observe an increase of autophagosomes and autolysosomes and, at later stages, the accumulation of intracellular waste in neurons. Taken together, we propose that loss of AP5 function blocks autophagy and thus leads to the aberrant accumulation of autophagic cargo, which finally results in axon degeneration.

A series of benzo[b]furan-2(3H)-ones (coumaran-2-ones) bearing a urea substructure, namely derivatives of 3-(aminocarbonylamino)benzo[b]furan-2(3H)-one, was prepared for the first time. The accessibility of these compounds through an electrophilic alpha-amidoalkylation approach of phenols (Tscherniac-Einhorn reaction) in the key step as well as the chemiluminescence (CL) properties of the desired compounds are strongly dependent on the substitution patterns at the urea moiety. Competing reaction pathways are discussed and an improved one pot synthetical approach of also general interest is presented. In conclusion, especially N,N-dialkylaminocarbonylamino-derivatives of benzo[b]furan-2(3H)-ones exhibit a strong flash like blue CL upon treatment with bases such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in the presence of oxygen or hydrogen peroxide. Comparative physico-chemical investigations revealed that novel compounds outperform their urethane-analogues in terms of CL-intensity and the speed of the decay making them potentially useful as new tools for CL-based applications on the short time scale.

Cytochrome C oxidase and FoF1-ATP synthase constitute complex IV and V, respectively, of the five membrane-bound enzymes in mitochondria comprising the respiratory chain. These enzymes are located in the inner mitochondrial membrane (IMM), which exhibits large invaginations called cristae. According to recent electron cryotomography, FoF1-ATP synthases are located predominantly at the rim of the cristae, while cytochrome C oxidases are likely distributed in planar membrane areas of the cristae. Previous FLIM measurements (K. Busch and coworkers) of complex II and III unravelled differences in the local environment of the membrane enzymes in the cristae. Here, we tagged complex IV and V with mNeonGreen and investigated their mitochondrial nano-environment by FLIM and superresolution microscopy in living human cells. Different lifetimes and anisotropy values were found and will be discussed.

Interleukin-17A (IL-17A) is considered an important pro-inflammatory cytokine but its importance in joint diseases such as rheumatoid arthritis (RA) is unclear. It has also been reported that IL-17A may induce pain but it is unclear whether pro-inflammatory and pro-nociceptive effects are linked. Here we studied in wild type (WT) and IL-17A knockout (IL-17AKO) mice inflammation and hyperalgesia in antigen-induced arthritis (AIA). We found that the severity and time course of AIA were indistinguishable in WT and IL-17AKO mice. Furthermore, the reduction of inflammation by sympathectomy, usually observed in WT mice, was preserved in IL-17AKO mice. Both findings suggest that IL-17A is redundant in AIA pathology. However, in the course of AIA IL-17AKO mice showed less mechanical hyperalgesia than WT mice indicating that IL-17A contributes to pain even if it is not crucial for arthritis pathology. In support for a role of IL-17A and other members of the IL-17 family in the generation of pain we found that sensory neurones in the dorsal root ganglia (DRG) express all IL-17 receptor subtypes. Furthermore, in isolated DRG neurones most IL-17 isoforms increased tetrodotoxin- (TTX-) resistant sodium currents which indicate a role of IL-17 members in inflammation-evoked sensitization of sensory nociceptive neurones.

Many cellular processes are orchestrated by protein–protein interactions that allow the formation of protein networks involved in subcellular compartmentalization, communication, and signalling. Postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase protein (MAGuK) family, is a central scaffold protein of the postsynaptic density (PSD) of excitatory synapses in the mammalian central nervous system. PSD-95 serves as a matrix for targeting and accumulation of yet other PSD proteins including ion channels and receptors via its three N-terminal PDZ (PSD-95, discs large, zonula occludens-1) domains. However, the stoichiometry of PSD-95 and its binding partners in such complexes and the dynamic regulation of their interactions remain elusive. Here, we have investigated the protein–protein interaction between PSD-95 and the inward rectifier potassium channel Kir2.1, which we consider as a model for other PDZ domain based interactions at synapses. By using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), we show that PSD-95 and Kir2.1 directly interact within clusters which are formed at the plasma membrane of culture cells. Our in vivo FRET data indicate that Kir2.1 binds to more than one PDZ domain of PSD-95, suggesting a structural model of synergistic target binding by the first two PDZ domains of the scaffold protein. We show that the cluster formation is induced by the channel whereas PSD-95 alone does not form clusters. The interaction of PSD-95 and Kir2.1 is dynamically regulated by protein kinase A (PKA) mediated phosphorylation, which is directly visualized and quantified in living cells and in real time.

Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

PURPOSETime and spectrally resolved measurements of autofluorescence have the potential to monitor metabolism at the cellular level. Fluorophores that emit with the same fluorescence intensity can be discriminated from each other by decay time of fluorescence intensity after pulsed excitation. We performed time-resolved autofluorescence measurements on fundus samples from a donor with significant extramacular drusen.METHODSTissue sections from two human donors were prepared and imaged with a laser scanning microscope. The sample was excited with a titanium-sapphire laser, which was tuned to 860 nm, and frequency doubled by a BBO crystal to 430 nm. The repetition rate was 76 MHz and the pulse width was 170 femtoseconds (fs). The time-resolved autofluorescence was recorded simultaneously in 16 spectral channels (445-605 nm) and bi-exponentially fitted.RESULTSRPE can be discriminated clearly from Bruch's membrane, drusen, and choroidal connective tissue by fluorescence lifetime. In RPE, bright fluorescence of lipofuscin could be detected with a maximum at 510 nm and extending beyond 600 nm. The lifetime was 385 ps. Different types of drusen were found. Most of them did not contain lipofuscin and exhibited a weak fluorescence, with a maximum at 470 nm. The lifetime was 1785 picoseconds (ps). Also, brightly emitting lesions, presumably representing basal laminar deposits, with fluorescence lifetimes longer than those recorded in RPE could be detected.CONCLUSIONSThe demonstrated differentiation of fluorescent structures by their fluorescence decay time is important for interpretation of in vivo measurements by the new fluorescence lifetime imaging (FLIM) ophthalmoscopy on healthy subjects as well as on patients.

Fluorescent ion indicators are widely used to measure ion concentrations in living cells. However, despite considerable efforts in synthesizing new compounds, no ratiometric sodium indicator is available that can be excited at visible wavelengths. Ratiometric indicators have an advantage in that measured fluorescence intensities can be corrected for fluctuations of the indicator concentration and the illumination intensity, which is not possible when non-ratiometric indicators are used. One way to circumvent this problem is to measure fluorescence lifetimes, which are independent of these factors. Another way to overcome the disadvantages of a non-ratiometric indicator dye is to embed it, together with a reference dye, into nanoparticles. By relating the indicator fluorescence to the fluorescence of the reference dye, inhomogeneities in the nanosensor concentration or the illumination intensity can be cancelled out reliably. In this study we compare the benefits and drawbacks of these approaches.

Global analysis algorithms perform better than unconstrained data fitting and can improve the accuracy and precision of the experimental data. Here, we report on different strategies that can be pursued to improve the results derived from fluorescence measurements. We point out the benefits of acquiring fluorescence data in a temporally and spectrally resolved manner and show how these data sets can be used to evaluate FRET measurements. Fluorescence measurements can be also combined with other methods such as the patch-clamp technique. This combination allows to record simultaneously fluorescence signals and electrical currents of ion channels in membrane patches. Global analysis of the data can yield valuable information about the processes underlying channel activation. We used this approach to study the activation of homotetrameric CNGA2 channels in inside-out membrane patches. By using fluorescent analogues of cyclic nucleotides as ligands we were able to simultaneously determine ligand binding and channel activation.

Since its discovery, green fluorescent protein (GFP) and its variants have proven to be a good and convenient fluorescent label for proteins: GFP and other visible fluorescent proteins (VFPs) can be fused selectively to the protein of interest by simple cloning techniques and develop fluorescence without additional cofactors. Among the steadily growing collection of VFPs, several pairs can be chosen that can serve as donor and acceptor fluorophores in Forster resonance energy transfer (FRET) experiments. Among them, the cyan fluorescent proteins (ECFP/Cerulean) and the enhanced yellow fluorescent protein (EYFP) are most commonly used. We show that ECFP and Cerulean have some disadvantages despite their common use: Upon irradiation with light intensities that are commonly used for intensity- and lifetime-based FRET measurements, both the fluorescence intensity and the fluorescence lifetime of ECFP and Cerulean decrease. This can hamper both intensity- and lifetime-based FRET measurements and emphasizes the need for control measurements to exclude these artifacts.

DNA segregation in mammalian cells during mitosis is an essential cellular process that is mediated by a specific subchromosomal protein complex, the kinetochore. Malfunction of this complex results in aneuploidy and can cause cancer. A subkinetochore complex, the “inner kinetochore”, is present at the centromere during the entire cell cycle. Its location seems to be defined by the settlement of CENP‐A (CENH3), which replaces histone H3 in centromeric nucleosomes. This suggests that CENP‐A can recruit further inner kinetochore proteins by direct binding. Surprisingly, intense in vitro studies could not identify an interaction of CENP‐A with any other inner kinetochore protein. Instead, centromere identity seems to be maintained by a unique nucleosome, which might have a modified structure or epigenetic state that serves to distinguish the centromere from the rest of the chromosome. We investigated the association of CENP‐A and CENP‐B by fluorescence intensity and lifetime‐based FRET measurements in living human HEp‐2 cells. We observed Förster resonance energy transfer (FRET) between CENP‐A and CENP‐B at centromere locations; this indicates that these proteins are in the molecular vicinity (<10 nm) of each other. In addition, we analysed protein–protein interactions within the centromeric nucleosome. We could detect energy transfer between CENP‐A and histone H4 as well as between CENP‐A molecules themselves. On the other hand, no FRET was detected between CENP‐A and H2A.1 or H3.1. Our data support the view that two CENP‐A molecules are packed with H4, but not with H3, in a single centromeric nucleosome.

In this study, we present two different approaches that can be used for multi-wavelength fluorescence lifetime measurements in the time domain. One technique is based on a streak-camera system, the other technique is based on the time-correlated-single-photon-counting (TCSPC) approach. The setup consists of a confocal laser-scanning microscope and a Titanium:Sapphire-laser that is used for pulsed one- and two-photon excitation. Fluorescence light emitted by the sample is fed back through the scan head and guided to one of the confocal channels, where it is coupled into an optical fiber and directed to a polychromator. The polychromator disperses the emitted light according to its wavelength and focuses the resulting spectrum on the entrance slit of a streak camera or a 16 channel PMT array, which is connected to a TCSPC imaging module. With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. We applied these methods to Förster resonance energy transfer (FRET) measurements and discuss the advantages and pitfalls of fluorescence lifetime measurements.

When and where proteins associate with each other in living cells are key questions in many biological research projects. One way to address these questions is to measure the extent of Förster resonance energy transfer (FRET) between proteins that have been labeled with appropriate donor and acceptor fluorophores. When both proteins interact, donor and acceptor fluorophores are brought into close vicinity so that the donor can transmit a part of its excitation energy to the acceptor. As a result, both the intensity and the lifetime of the donor fluorescence decrease, whereas the intensity of the acceptor emission increases. This offers different approaches to determine FRET efficiency: One is to detect changes in the intensity of donor and acceptor emission, the other is to measure changes in the lifetime of the donor molecule. One important advantage of the fluorescence lifetime approach is that it allows to distinguish between free and associated donor molecules. However, like intensity measurements it lacks an intrinsic control ensuring that changes in the measured parameters are only due to FRET and not other quenching processes. Here, we show how this limitation can be overcome by spectrally resolved fluorescence lifetime measurements in the time domain. One technique is based on a streak camera system, the other technique is based on a time‐correlated‐single‐photon‐counting approach. Both approaches allow biologists to record both donor and acceptor fluorescence emitted by the sample in a single measurement. Microsc. Res. Tech., 2007. © 2007 Wiley‐Liss, Inc.