Search

Förster resonance energy transfer (FRET) is a powerful tool for the visualization of molecular signaling events such as protein activities and interactions in cells. In its different implementations, FRET microscopy has been mainly used for monitoring single events. Recently, there has been a trend of extending FRET imaging towards the simultaneous detection of multiple events and interactions. The concomitant increase in experimental complexity requires a deeper understanding of the biophysical background of FRET. The presence of multiple acceptors for one donor affects the well-known formalism for FRET between two molecules, increasing distance sensitivity through mechanisms that have become known as the 'antenna' and 'surplus' effect. We will discuss the nature of these effects and present the imaging methods that have been used to unravel the combined transfer rates in the multi-protein interactions of multiplexed FRET experiments. Multiplexing strategies are becoming invaluable analytical tools for the elucidation of biological complexes and for the visualization of decision points in cellular signaling networks in physiological and pathological conditions.

The steady improvement in the imaging of cellular processes in living tissue over the last 10-15 years through the use of various fluorophores including organic dyes, fluorescent proteins and quantum dots, has made observing biological events common practice. Advances in imaging and recording technology have made it possible to exploit a fluorophore's fluorescence lifetime. The fluorescence lifetime is an intrinsic parameter that is unique for each fluorophore, and that is highly sensitive to its immediate environment and/or the photophysical coupling to other fluorophores by the phenomenon Förster resonance energy transfer (FRET). The fluorescence lifetime has become an important tool in the construction of optical bioassays for various cellular activities and reactions. The measurement of the fluorescence lifetime is possible in two formats; time domain or frequency domain, each with their own advantages. Fluorescence lifetime imaging applications have now progressed to a state where, besides their utility in cell biological research, they can be employed as clinical diagnostic tools. This review highlights the multitude of fluorophores, techniques and clinical applications that make use of fluorescence lifetime imaging microscopy (FLIM).

Previous studies indicate an important role for the cellular prion protein (PrP(C)) in the development of Alzheimer's disease (AD) pathology. In the present study, we analyzed the involvement of PrP(C) in different pathological mechanisms underlying AD: the processing of the amyloid-β protein precursor (AβPP) and its interaction with AβPP, tau, and different phosphorylated forms of the tau protein (p-tau). The effect of PrP(C) on tau expression was investigated in various cellular compartments using a HEK293 cell model expressing a tau mutant (3PO-tau) or wild type (WT)-tau. We could show that PrP(C) reduces AβPP cleavage, leading to decreased levels of Aβ40 and sAβPP without changing the protein expression of AβPP, β-secretase, or γ-secretase. Tau and its phosphorylated forms were identified as interactions partners for PrP(C), raising the question as to whether PrP(C) might also be involved in tau pathology. Overexpression of PrP(C) in PRNP and 3PO-tau transfected cells resulted in a reduction of 3PO-tau and p-tau as well as a decrease of 3PO-tau-related toxicity. In addition, we used the transgenic PrP(C) knockout (Prnp0/0) mouse line to study the dynamics of tau phosphorylation, an important pathological hallmark in the pathogenesis of AD in vivo. There, an effect of PrP(C) on tau expression could be observed under oxidative stress conditions but not during aging. In summary, we provide further evidence for interactions of PrP(C) with proteins that are known to be the key players in AD pathogenesis. We identified tau and its phosphorylated forms as potential PrP-interactors and report a novel protective function of PrP(C) in AD-like tau pathology.

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

Adeno-associated virus serotype 6 (AAV6) viral vectors encoding mutant and normal tau were used to produce focal tau pathology. Two mutant forms of tau were used; the P301S tau mutation is associated with neurofibrillary tangle formation in humans, and the 3PO mutation leads to rapid tau aggregation and is associated with pathogenic phosphorylation and cytotoxicity in vitro. We show that adeno-associated viral injection into entorhinal cortex of normal and tau knockout animals leads to local overexpression of tau, and the presence of human tau in axons projecting to and emanating from the entorhinal cortex. Starting at 2 months and increasing by 6 months post-injection neurons expressing mutant tau developed hyperphosphorylated tau pathology, in addition to dystrophic neurites. There was neuronal loss in tau-expressing regions, which was similar in normal and in TASTPM mice injected with mutant tau. There was neuroinflammation around plaques, and in regions expressing mutant tau. We saw no evidence that mutant tau had affected amyloid-beta pathology or vice versa. Morris water maze behavioral tests demonstrated mild memory impairment attributable to amyloid-beta pathology at 2 and 4 months, with severe impairment at 6 months in animals receiving adeno-associated viral-3PO. Therefore, TASTPM mice injected with mutant tau displayed many of the main features characteristic of human Alzheimer's disease patients and might be used as a model to test new drugs to ameliorate clinical features of Alzheimer's disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Proper illumination is essential for light microscopy. Whereas in early years incandescent light was the only illumination, today, more and more specialized light sources, such as lasers or arc lamps are used. Because of the high efficiency and brightness that light-emitting diodes (LED) have reached today, they have become a serious alternative for almost all kinds of illumination in light microscopy. LED have a high durability, do not need expensive electronics, and they can be switched in nanoseconds. Besides this, they are available throughout the UV/Vis/NIR-spectrum with a narrow bandwidth. This makes them ideal light sources for fluorescence microscopy. The white LED, with a color temperature ranging from 2,600 up to 5,000 K is an excellent choice for bright-field illumination with the additional advantage of simple brightness adjustments without changing the spectrum. This review discusses the different LED types, their use in the fluorescence microscope, and discusses LED as specialized illumination sources for Förster resonance energy transfer and fluorescent lifetime imaging microscopy., (Copyright © 2012 International Society for Advancement of Cytometry.)

This review focuses on technical advances in fluorescence microscopy techniques including laser scanning techniques, fluorescence-resonance energy transfer (FRET) microscopy, fluorescence lifetime imaging (FLIM), stimulated emission depletion (STED)-based super-resolution microscopy, scanning confocal endomicroscopes, thin-sheet laser imaging microscopy (TSLIM), and tomographic techniques such as early photon tomography (EPT) as well as on clinical laser-based endoscopic and microscopic techniques. We will also discuss the new developments in the field of fluorescent dyes and fluorescent genetic reporters that enable new possibilities in high-resolution and molecular imaging both in in vitro and in vivo. Small animal and tissue imaging benefit from the development of new fluorescent proteins, dyes, and sensing constructs that operate in the far red and near-infrared spectrum.

Malfunction of the ubiquitin-proteasome system has been implicated as a causal factor in the pathogenesis of aggregation-related disorders, e.g. Parkinson's disease. We show here that Transforming growth factor-beta 1 (TGF-beta), a multifunctional cytokine and trophic factor for dopaminergic (DAergic) neurons modulates proteasome function in primary midbrain neurons. TGF-beta differentially inhibited proteasomal subactivities with a most pronounced time-dependent inhibition of the peptidyl-glutamyl peptide hydrolyzing-like and chymotrypsin-like subactivity. Regulation of proteasomal activity could be specifically quantified in the DAergic subpopulation. Protein blot analysis revealed an accumulation of ubiquitinated proteins after TGF-beta treatment. The identity of these enriched proteins was further analyzed by 2D-gel electrophoresis and mass spectrometry. We found epidermal fatty acid binding protein (EFABP) to be strongly increased and ubiquitinated after TGF-beta treatment and confirmed this finding by co-immunoprecipitation. While application of TGF-beta increased neurite regeneration in a scratch lesion model, downregulation of EFABP by siRNA significantly decreased this effect. We thus postulate that a differential regulation of proteasomal function, as demonstrated for TGF-beta, can result in an enrichment of proteins, such as EFABP, that mediate physiological functions, such as neurite regeneration.

In eukaryotes, most intracellular membrane fusion reactions are mediated by the interaction of SNARE proteins that are present in both fusing membranes. However, the minimal number of SNARE complexes needed for membrane fusion is not known. Here we show unambiguously that one SNARE complex is sufficient for membrane fusion. We performed controlled in vitro Förster resonance energy transfer (FRET) experiments and found that liposomes bearing only a single SNARE molecule are still capable of fusion with other liposomes or with purified synaptic vesicles. Furthermore, we demonstrated that multiple SNARE complexes do not act cooperatively, showing that synergy between several SNARE complexes is not needed for membrane fusion. Our findings shed new light on the mechanism of SNARE-mediated membrane fusion and call for a revision of current views of fusion events such as the fast release of neurotransmitters.

Kinesin-3 motor UNC-104/KIF1A is essential for transporting synaptic precursors to synapses. Although the mechanism of cargo binding is well understood, little is known how motor activity is regulated. We mapped functional interaction domains between SYD-2 and UNC-104 by using yeast 2-hybrid and pull-down assays and by using FRET/fluorescence lifetime imaging microscopy to image the binding of SYD-2 to UNC-104 in living Caenorhabditis elegans. We found that UNC-104 forms SYD-2-dependent axonal clusters (appearing during the transition from L2 to L3 larval stages), which behave in FRAP experiments as dynamic aggregates. High-resolution microscopy reveals that these clusters contain UNC-104 and synaptic precursors (synaptobrevin-1). Analysis of motor motility indicates bi-directional movement of UNC-104, whereas in syd-2 mutants, loss of SYD-2 binding reduces net anterograde movement and velocity (similar after deleting UNC-104's liprin-binding domain), switching to retrograde transport characteristics when no role of SYD-2 on dynein and conventional kinesin UNC-116 motility was found. These data present a kinesin scaffolding protein that controls both motor clustering along axons and motor motility, resulting in reduced cargo transport efficiency upon loss of interaction.

Dendritic mRNA transport coupled with local regulation of translation enables neurons to selectively alter the protein composition of individual postsynaptic sites. We have analyzed dendritic localization of shank1 mRNAs; shank proteins (shank1-3) are scaffolding molecules of the postsynaptic density (PSD) of excitatory synapses, which are crucial for PSD assembly and the formation of dendritic spines. Live cell imaging demonstrates saltatory movements of shank1 mRNA containing granules along microtubules in both anterograde and retrograde directions. A population of brain messenger ribonucleoprotein particles (mRNPs) containing shank1 mRNAs associates with the cargo-binding domain of the motor protein KIF5C. Through expression of dominant negative proteins, we show that dendritic targeting of shank1 mRNA granules involves KIF5C and the KIF5-associated RNA-binding protein staufen1. While transport of shank1 mRNAs follows principles previously outlined for other dendritic transcripts, shank1 mRNAs are distinguished by their translational regulation. Translation is strongly inhibited by a GC-rich 5(')untranslated region; in addition, internal ribosomal entry sites previously detected in other dendritic transcripts are absent in the shank1 mRNA. A concept emerges from our data in which dendritic transport of different mRNAs occurs collectively via a staufen1- and KIF5-dependent pathway, whereas their local translation is controlled individually by unique cis-acting elements.

The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPPalpha and the neurotoxic beta-amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of Förster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPPalpha, as sAPPalpha binding disrupts APP dimers, and this disruption of APP dimers by sAPPalpha is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPPalpha. These findings suggest a potentially beneficial effect of increasing sAPPalpha production or disrupting APP dimers for neuronal survival.

Fluorescence microscopy is a non-invasive technique that allows high resolution imaging of cytoskeletal structures. Advances in the field of fluorescent labelling (e.g., fluorescent proteins, quantum dots, tetracystein domains) and optics (e.g., super-resolution techniques and quantitative methods) not only provide better images of the cytoskeleton, but also offer an opportunity to quantify the complex of molecular events that populate this highly organised, yet dynamic, structure.For instance, fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging allow mapping of protein-protein interactions; furthermore, techniques based on the measurement of photobleaching kinetics (e.g., fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and fluorescence localisation after photobleaching) permit the characterisation of axonal transport and, more generally, diffusion of relevant biomolecules.Quantitative fluorescence microscopy techniques offer powerful tools for understanding the physiological and pathological roles of molecular machineries in the living cell.

Streptococcus pneumoniae is the most frequent cause of bacterial meningitis, leading to permanent neurological damage in 30% and lethal outcome in 25% of patients. The cholesterol-dependent cytolysin pneumolysin is a major virulence factor of S. pneumoniae. It produces rapid cell lysis at higher concentrations or apoptosis at lower concentrations. Here, we show that sublytic amounts of pneumolysin produce rapid bundling and increased acetylation of microtubules (signs of excessive microtubule stabilization) in various types of cells--neuroblastoma cells, fibroblasts and primary astrocytes. The bundling started perinuclearly and extended peripherally towards the membrane. The effect was not connected to pneumolysin's capacity to mediate calcium influx, macropore formation, apoptosis, or RhoA and Rac1 activation. Cellular cholesterol depletion and neutralization of the toxin by pre-incubation with cholesterol completely inhibited the microtubule phenotype. Pharmacological inhibition of Src-family kinases diminished microtubule bundling, suggesting their involvement in the process. The relevance of microtubule stabilization to meningitis was confirmed in an experimental pneumococcal meningitis animal model, where increased acetylation was observed. Live imaging experiments demonstrated a decrease in organelle motility after toxin challenge in a manner comparable to the microtubule-stabilizing agent taxol, thus proposing a possible pathogenic mechanism that might contribute to the CNS damage in pneumococcal meningitis.

Intracellular pH is an important indicator for cellular metabolism and pathogenesis. pH sensing in living cells has been achieved using a number of synthetic organic dyes and genetically expressible sensor proteins, even allowing the specific targeting of intracellular organelles. Ideally, a class of genetically encodeable sensors need to cover relevant cellular pH ranges. We present a FRET-based pH sensor platform, based on the pH modulation of YFP acceptor fluorophores in a fusion construct with ECFP. The concurrent loss of the overlap integral upon acidification results in a proportionally reduced FRET coupling. The readout of FRET over the sensitized YFP fluorescence lifetime yields a highly sensitive and robust pH measurement that is self-calibrated. The principle is demonstrated in the existing high-efficiency FRET fusion Cy11.5, and tunability of the platform design is demonstrated by genetic alteration of the pH sensitivity of the acceptor moiety.

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.

Cell-penetrating peptides (CPPs), such as the one derived from the human immunodeficiency virus Tat protein, facilitate the delivery of cargoes across cellular membranes. However, questions about the therapeutic potential of CPP-mediated delivery remain. For instance, the impact of the purification procedure on the functionality of Tat-fusion proteins has not been systematically examined. Here, we isolated fusion proteins of the chaperone heat shock protein 70 (Hsp70) and the Tat CPP under denaturing or native conditions. To investigate the therapeutic potential of different recombinant protein preparations, we examined Tat-Hsp70 transduction efficiency and quantified Tat-Hsp70-mediated folding of a chaperone-dependent yellow fluorescent protein in vitro. Transduction efficiency and chaperone activity of Tat-Hsp70-treated cells was significantly higher compared to cells treated with Hsp70. The application of native isolated Tat-Hsp70 had the strongest effect. This chaperone activity correlates with increased viability of cells treated with the recombinant protein after cell death induction with 6-hydroxydopamine. This suggests that the method of recombinant Tat-fusion protein purification influences its functionality. For Tat-Hsp70, the method of choice seems to be isolation under native conditions, for which we present a purification protocol. Our results may contribute to improve Tat-fusion protein application in basic research and may facilitate its use as therapeutic tool, for instance in Parkinson's disease.

Fluorescence lifetime imaging is a valuable and versatile tool for the investigation of the molecular environment of fluorophores in living cells. It is ideally suited-and is therefore increasingly used-for the quantification of the occurrence of Förster Resonance Energy Transfer, a powerful microscopy method for the detection of subnanometer conformational changes, protein-protein interactions, and protein biochemical status. However, careful quantitative analysis is required for the correct and meaningful interpretation of fluorescence lifetime data. This can be a daunting task to the nonexpert user, and is the source for many avoidable errors and unsound interpretations. Digman and colleagues (Digman et al., 2007, Biophys. J. 94, L14-6) present an analysis technique that avoids data fitting in favor of a simple graphical polar data representation. In this "phasor" space, the physics of lifetime imaging becomes more intuitive and accessible also to the inexperienced user. The cumulated information from image pixels, even over different cells, describes patterns and trajectories that can be visually interpreted in physically meaningful ways. Its usefulness is demonstrated in the study of the dimerization of the uPAR receptor (Caiolfa et al., 2007, J. Cell Biol. 179, 1067-1082).

The signal-to-noise ratio of a measurement is determined by the photon economy of the detection technique and the available photons that are emitted by the sample. We investigate the efficiency of various frequency-domain lifetime detection techniques also in relation to time-domain detection. Nonlinear effects are discussed that are introduced by the use of image intensifiers and by fluorophore saturation. The efficiency of fluorescence lifetime imaging microscopy setups is connected to the speed of acquisition and thus to the imaging throughput. We report on the optimal conditions for balancing signal-to-noise ratio and acquisition speed in fluorescence lifetime sensing.

The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half-ATP-binding cassette (ABC) transporters in the mammalian peroxisome membrane. Mutations in the gene encoding ALDP result in a devastating neurodegenerative disorder, X-linked adrenoleukodystrophy (X-ALD) that is associated with elevated levels of very long chain fatty acids because of impaired peroxisomal beta-oxidation. The interactions of peroxisomal ABC transporters, their role in the peroxisomal membrane, and their functions in disease pathogenesis are poorly understood. Studies on ABC transporters revealed that half-transporters have to dimerize to gain functionality. So far, conflicting observations are described for ALDP. By the use of in vitro methods (yeast two-hybrid and immunoprecipitation assays) on the one hand, it was shown that ALDP can form homodimers as well as heterodimers with PMP70 and ALDR, while on the other hand, it was demonstrated that ALDP and PMP70 exclusively homodimerize. To circumvent the problems of artificial interactions due to biochemical sample preparation in vitro, we investigated protein-protein interaction of ALDP in its physiological environment by FRET microscopy in intact living cells. The statistical relevance of FRET data was determined in two different ways using probability distribution shift analysis and Kolmogorov-Smirnov statistics. We demonstrate in vivo that ALDP and PMP70 form homodimers as well as ALDP/PMP70 heterodimers where ALDP homodimers predominate. Using C-terminal deletion constructs of ALDP, we demonstrate that the last 87 C-terminal amino acids harbor the most important protein domain mediating these interactions, and that the N-terminal transmembrane region of ALDP has an additional stabilization effect on ALDP homodimers. Loss of ALDP homo- or heterodimerization is highly relevant for understanding the disease mechanisms of X-ALD.

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of alpha-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant alpha-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.

NGF-induced differentiation of PC12 cells is mediated by actin-polymerisation-driven membrane protrusion, involving GTPase signalling pathways that activate actin nucleation promoting factors such as the neural Wiskott-Aldrich syndrome protein (N-WASP). Expression of the exocyst subunit Exo70 in PC12 cells and neurons leads to the generation of numerous membrane protrusions, an effect that is strongly potentiated upon NGF-induced differentiation. Förster resonance energy transfer (FRET) imaging by fluorescence lifetime microscopy (FLIM) reveals an NGF-induced interaction of activated TC10 with Exo70. Expression of dominant-negative mutants and siRNA-mediated knockdown implicates N-WASP in NGF-induced Exo70-TC10-mediated membrane protrusion. However, FRET imaging of N-WASP activation levels of cells expressing Exo70 and/or constitutively active TC10 reveals that this complex locally antagonises the NGF-induced activation of N-WASP in membrane protrusions. Experiments involving siRNA-mediated knockdown of Cdc42 and overexpression of constitutively active Cdc42 confirm that the Exo70-TC10 complex mainly targets the NGF-induced Cdc42-dependent activation of N-WASP. Our results show that Exo70 is responsible for the correct targeting of the Exo70-TC10 complex to sites of membrane protrusion. The functional uncoupling between both pathways represents a novel regulatory mechanism that enables switching between morphologically distinct - Cdc42- or TC10-dominated - forms of cellular membrane outgrowth.

alpha-Synuclein is a primarily neuronal protein that is enriched at the pre-synapse. alpha-Synuclein and the microtubule binding protein tau have been implicated in neurodegenerative diseases. alpha-Synuclein is known to associate with phospholipid vesicles, regulates dopamine metabolism and exhibits chaperone activity, but its main role remains largely unknown. Furthermore, knowledge on its interactions and post-translational modifications is essential for a molecular understanding of alpha-synucleinopathies. We investigated alpha-synuclein mutations, causative for autosomal dominant forms of Parkinson's disease (A30P, A53T and E46K), and phosphorylation mutants at serine 129 (S129A and S129D) using fluorescently labelled alpha-synuclein, actin and tau. The investigation of colocalization, and protein-protein interactions by Förster resonance energy transfer and fluorescence lifetime imaging showed that alpha-synuclein associates with the actin cytoskeleton and interacts with tau. The A30P mutation and cytoskeletal destabilization decreased this interaction. Given the concurrent loss of membrane binding by this mutation, we propose a membrane-bound functional complex with tau that might involve the actin cytoskeleton.

The directionality of axonal transport represents an important question in neurophysiological and neuropathological research. Various approaches such as videomicroscopy, radioisotopic and fluorescence-based techniques are used. Recently, a novel FRAP-based (fluorescent recovery after photobleaching) technique using synaptophysin-EGFP expression in primary neurons was applied, allowing reliable and sensitive evaluation of gross axonal transport changes using confocal live-imaging microscopy. Here, we describe a novel FLIP-based (fluorescence loss in photobleaching) approach using a synaptophysin-EGFP probe that allows the differential evaluation of the ante- and retrograde transport parameters. Furthermore, we improved the sensitivity of the probe by substituting EGFP with an ECFP/VenusYFP fusion FRET (fluorescence resonance energy transfer) pair. The use of this FRET couple improves the precision of axonal transport measurements by combining FLIP and FLAP (fluorescence localization after photobleaching) techniques and eliminating the need for pre-bleaching images and thus pixel shifts between various exposures, and by reducing the deleterious effect of photobleaching.

The Streptococcus pneumoniae toxin pneumolysin belongs to the group of cholesterol-dependent cytolysins. It produces rapid cell lysis at higher concentrations or apoptosis at lower concentrations. In cell membranes, it forms prepores and pores. Here, we show that sublytic concentrations of pneumolysin produce rapid activation of Rho and Rac GTPases and formation of actin stress fibers, filopodia, and lamellipodia. That Rac1-specific and Rho-associated kinase (ROCK)-specific inhibitors reverted the formation of lamellipodia and stress fibers, respectively, identifies RhoA and Rac1 as key toxin effectors. Live imaging excluded macropore formation (as judged by membrane impermeability toward calcein) but indicated very early membrane depolarization [as judged by bis-(1,3-dibutylbarbituric acid)trimethine oxanol staining], indicative of formation of micropores with ion channel properties. That Rac1-dependent lamellipodia formation was reverted by the voltage-gated calcium channel inhibitor SKF96365 and by toxin exposure in calcium-free medium suggests a role for calcium influx via endogenous calcium channels in the Rac1 activation. Cellular cholesterol depletion by methyl-beta-cyclodextrin or incubation of the toxin with cholesterol before cell treatment eliminated its membrane binding and the subsequent GTPase activation. Thus, that our experiments show small GTPase activation by a cholesterol-dependent cytolysin suggests a membrane cholesterol-dependent activation mechanism.

Fluorescence lifetime imaging microscopy (FLIM) allows the investigation of the physicochemical environment of fluorochromes and protein-protein interaction mapping by Forster resonance energy transfer (FRET) in living cells. However, simpler and cheaper solutions are required before this powerful analytical technique finds a broader application in the life sciences. Wide-field frequency-domain FLIM represents a solution whose application is currently limited by the need for multichannel-plate image intensifiers. We recently showed the feasibility of using a charge-coupled device/complementory metal-oxide semiconductor (CCD/CMOS) hybrid lock-in imager, originally developed for 3-D vision, as an add-on device for lifetime measurements on existing wide-field microscopes. In the present work, the performance of the setup is validated by comparison with well-established wide-field frequency-domain FLIM measurements. Furthermore, we combine the lock-in imager with solid-state light sources. This results in a simple, inexpensive, and compact FLIM system, operating at a video rate and capable of single-shot acquisition by virtue of the unique parallel retrieval of two phase-dependent images. This novel FLIM setup is used for cellular and FRET imaging, and for high-throughput and fast imaging applications. The all-solid-state design bridges the technological gap that limits the use of FLIM in areas such as drug discovery and medical diagnostics.

Förster resonance energy transfer (FRET) microscopy is a powerful technique that enables the visualization of signaling intermediates, protein interactions, and protein conformational and biochemical status. With the availability of an ever-increasing collection of fluorescent proteins, pairs of spectrally different variants have been used for the study of FRET in living cells. However, suitable spectral overlap, necessary for efficient FRET, is limited by the requirement for proper emission separation. Currently used FRET pairs represent compromises between these opposing spectral demands that reduce the maximally attainable FRET sensitivity. We present a previously undescribed FRET acceptor, a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein). REACh allows the use of the photophysically superior FRET donor EGFP, with which it exhibits optimal spectral overlap, which obviates the need for narrow spectral filtering and allows additional fluorescent labels to be used within the same cell. The latter allows the generation of sophisticated bioassays for complex biological questions. We show that this dark acceptor is ideally suited for donor fluorescence lifetime imaging microscopy (FLIM) and confirm these measurements with an independent intensity-based donor fluorescence quenching resonance energy transfer (FqRET) assay. REACh also can be used in donor photobleaching kinetics-based FRET studies. By detecting FRET between a GFP-tagged ubiquitination substrate and REACh-labeled ubiquitin, we imaged the active ubiquitination machinery inside cells. This assay therefore can be used to study proteins whose function is regulated by ubiquitination.

Detection of specific protein-protein interactions has long been restricted to bulk biochemical methods such as immunoprecipitation and immunoblotting. Even more sensitive methods using general immunofluorescence are limited, and it is difficult to infer protein-protein interactions from the results of these tests. Fluorescence Resonance Energy Transfer (FRET) is a photophysical process that can be exploited to obtain highly sensitive information about such interactions. It can sense the presence of acceptor fluorophores in the vicinity of a donor fluorophore within a separation distance that is the size of a single protein molecule. This unit details FRET microscopy based on release of quenched donor fluorescence after acceptor photobleaching, microinjection of reagents into the nucleus or cytosol, and labeling of antibodies for these procedures.

Bax ihibitor-1 (BI-1) has been characterized as an inhibitor of Bax-induced cell death in plants and various mammalian cell systems. To explore the function of BI-1 in neurons, we overexpressed BI-1 tagged to HA or GFP in rat nigral CSM14.1 and human SH-SY5Y neuroblastoma cells. Stable BI-1 expression proved marked protection from cell death induced by thapsigargine, a stress agent blocking the Ca2+-ATPase of the endoplasmic reticulum (ER) but failed to inhibit cell death induced by staurosporine, a kinase inhibitor initiating mitochondria-dependent apoptosis. Moreover, BI-1 was neuroprotective in a paradigm mimicking ischemia, namely oxygen-glucose as well as serum deprivation. Examination of the subcellular distribution revealed that BI-1 predominantly locates to the ER and nuclear envelope but not mitochondria. Taken together, BI-1 overexpression in the ER is protective in neurons, making BI-1 an interesting target for future studies aiming at the inhibition of neuronal cell death during neurodegenerative diseases and stroke.

Fluorescence lifetime imaging microscopy presents a powerful tool in biology and biophysics because it allows the investigation of the local environment of a fluorochrome in living cells in a quantitative manner. Furthermore, imaging Förster-type resonance energy transfer (FRET) by fluorescence lifetime imaging microscopy enables protein-protein interactions and intermolecular distances to be mapped under physiological conditions. Quantitative and precise data analysis methods are required to access the richness of information that is contained in FRET data on biological samples. Lifetime detection in the frequency-domain yields two lifetime estimations. The lifetime moments analysis (LiMA) provides a quantitative measure of the lifetime distribution broadness by exploiting the analytical relationship between the phase- and demodulation-lifetime estimations and relating them to the weighted average and variance of the lifetime distribution. The LiMA theoretical framework is validated by comparison with global analysis and by applying it to a constrained two-component FRET system using simulations and experiments. Furthermore, a novel LIMA-based error analysis and a more intuitive formalism for global analysis are presented. Finally, a new method to resolve a FRET system is proposed and experimentally applied to the investigation of protein-protein interactions.

It was recently shown that Bcl-2-associated athanogene 1 (BAG1) is a potent neuroprotectant as well as a marker of neuronal differentiation. Since there appears to exist an equilibrium within the cell between BAG1 binding to heat shock protein 70 (Hsp70) and BAG1 binding to Raf-1 kinase, we hypothesized that changing BAG1 binding characteristics might significantly alter BAG1 function. To this end, we compared rat CSM14.1 cells and human SHSY-5Y cells stably overexpressing full-length BAG1 or a deletion mutant (BAGDeltaC) no longer capable of binding to Hsp70. Using a novel yellow fluorescent protein-based foldase biosensor, we demonstrated an upregulation of chaperone in situ activity in cells overexpressing full-length BAG1 but not in cells overexpressing BAGDeltaC compared to wild-type cells. Interestingly, in contrast to the nuclear and cytosolic localizations of full-length BAG1, BAGDeltaC was expressed exclusively in the cytosol. Furthermore, cells expressing BAGDeltaC were no longer protected against cell death. However, they still showed accelerated neuronal differentiation. Together, these results suggest that BAG1-induced activation of Hsp70 is important for neuroprotectivity, while BAG1-dependent modulation of neuronal differentiation in vitro is not.

Fluorescent lifetime imaging microscopy is a powerful tool to enhance the contrast in images of biological samples and to investigate the local environment of a fluorochrome. FLIM allows the detection of protein-protein interactions and their biochemical state by the quantitative detection of Förster resonance energy transfer (FRET) between molecules in living cells or tissues. The availability of different spectral variants of the visible fluorescent proteins (VFPs) allows the investigation of molecular activities and protein-protein interactions in living cells by FRET as measured by FLIM.

The major task of modern cell biology is to identify the function and relation of the many different gene products, discovered by genomics and proteomics approaches, in the context of the living cell. To achieve this goal, an increasing toolbox of custom-designed biosensors based on fluorescent labels is available to study the molecular activities of the cellular machinery. An overview of the current status of the young field of molecular-cellular physiology is presented that includes the application of fluorescent labels in the design of biosensors and the major detection schemes used to extract their sensing information. In particular, the use of the photophysical phenomenon of Förster resonance energy transfer (FRET) as a powerful indicator of cellular biochemical events is discussed. In addition, we will point out the challenges and directions of the field and project the short-term future for the application of fluorescence-based biosensors in biology.

Detection of specific protein-protein interactions has long been restricted to bulk biochemical methods such as immunoprecipitation and immunoblotting. Even more sensitive methods using general immunofluorescence are limited, and it is difficult to infer protein-protein interactions from the results of these tests. Fluoresence Resonance Energy Transfer (FRET) is a photophysical process that can be exploited to obtain highly sensitive information about such interactions. It can sense the presence of acceptor fluorophores in the vicinity of a donor fluorophore within a separation distance that is the size of a single protein molecule. This unit details FRET microscopy based on release of quenched donor fluorescence after acceptor photobleaching, microinjection of reagents into the nucleus or cytosol, and labeling of antibodies for these procedures.