Search

ERK3 is a ubiquitously expressed member of the atypical mitogen activated protein kinases (MAPKs) and the physiological significance of its short half-life remains unclear. By employing gastrointestinal 3D organoids, we detect that ERK3 protein levels steadily decrease during epithelial differentiation. ERK3 is not required for 3D growth of human gastric epithelium. However, ERK3 is stabilized and activated in tumorigenic cells, but deteriorates over time in primary cells in response to lipopolysaccharide (LPS). ERK3 is necessary for production of several cellular factors including interleukin-8 (IL-8), in both, normal and tumorigenic cells. Particularly, ERK3 is critical for AP-1 signaling through its interaction and regulation of c-Jun protein. The secretome of ERK3-deficient cells is defective in chemotaxis of neutrophils and monocytes both in vitro and in vivo. Further, knockdown of ERK3 reduces metastatic potential of invasive breast cancer cells. We unveil an ERK3-mediated regulation of IL-8 and epithelial secretome for chemotaxis.

Upconverting nanoparticles (UCNPs) are a highly attractive tool owing to their unique property of showing visible luminescence when excited in the near-infrared (NIR) region. Plain UCNPs have no biorecognition capabilities, but functionalization of their surface with azido groups renders them conjugatable to ethynyl-modified oligonucleotides in a bioorthogonal fashion. Single-stranded DNA was covalently attached to the surface of UCNPs by click chemistry and purified by size exclusion chromatography (SEC) at elevated temperature. Covalent attachment was evidenced by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. DNA conjugation makes the particle soluble in water and enables it to recognize its counter strand. Such UCNPs are capable of nonspecifically crossing cell membranes. Confocal microscopy reveals the high potential of the bright UCNPs for live cell imaging in the NIR, where the UCNP–DNA conjugates can be considered to act as a kind of nano-sized lamp. Furthermore, cross-linking of those DNA nanolamps yields highly emissive aggregates.

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.

Item does not contain fulltext Signaling cascades are initiated in the plasma membrane via activation of one molecule by another. The interaction depends on the mutual availability of the molecules to each other and this is determined by their localization and lateral diffusion in the cell membrane. The cytoskeleton plays a very important role in this process by enhancing or restricting the possibility of the signaling partners to meet in the plasma membrane. In this study we explored the mode of diffusion of the cAMP receptor, cAR1, in the plasma membrane of Dictyostelium discoideum cells and how this is regulated by the cytoskeleton. Single-particle tracking of fluorescently labeled cAR1 using Total Internal Reflection Microscopy showed that 70% of the cAR1 molecules were mobile. These receptors showed directed motion and we demonstrate that this is not because of tracking along the actin cytoskeleton. Instead, destabilization of the microtubules abolished cAR1 mobility in the plasma membrane and this was confirmed by Fluorescence Recovery after Photobleaching. As a result of microtubule stabilization, one of the first downstream signaling events, the jump of the PH domain of CRAC, was decreased. These results suggest a role for microtubules in cAR1 dynamics and in the ability of cAR1 molecules to interact with their signaling partners. 01 juni 2011

Aufkonvertierende lumineszierende Materialien sind in der Lage, nah-infrarotes Licht zu absorbieren und in kurzerwellige Lumineszenz umzuwandeln. Die Effizienz dieses bemerkenswerten Effekts ist stark temperaturabhangig, was die Temperaturmessung mit einer Ortsauflosung im Nanometerbereich ermoglicht. Alle bisher bekannten Aufkonvertierungs-Materialien zeigen mehrere (meistens zwei) schmale Emissionsbanden, jede mit einer eigenen Temperaturabhangigkeit. Das Verhaltnis der Intensitaten zweier dieser Banden ist ein selbstreferenziertes Signal zur optischen Temperaturmessung, beispielsweise in Zellen.

Upconverting materials are capable of absorbing near-infrared light and converting it into short-wavelength luminescence. The efficiency of this remarkable effect is highly temperature dependent and thus can be used for temperature determination (thermometry) on a nanometer scale. All the upconverting materials discovered so far display several (mainly two) narrow emission bands, each of which has its own temperature dependence. The ratio of the intensity of two of these bands provides a referenced signal for optical sensing of temperature, for example inside cells.

Single-molecule detection and tracking is important for observing biomolecule interactions in the microenvironment. Here we report selective plane illumination microscopy (SPIM) with single-molecule detection in living organisms, which enables fast imaging and single-molecule tracking and optical penetration beyond 300 microm. We detected single nanocrystals in Drosophila larvae and zebrafish embryo. We also report our first tracking of single quantum dots during zebrafish development, which displays a transition from flow to confined motion prior to the blastula stage. The new SPIM setup represents a new technique, which enables fast single-molecule imaging and tracking in living systems.

Inhibitor of apoptosis proteins (IAP) are evolutionarily conserved anti-apoptotic regulators. C-RAF protein kinase is a direct RAS effector protein, which initiates the classical mitogen-activated protein kinase (MAPK) cascade. This signalling cascade mediates diverse biological functions, such as cell growth, proliferation, migration, differentiation and survival. Here we demonstrate that XIAP and c-IAPs bind directly to C-RAF kinase and that siRNA-mediated silencing of XIAP and c-IAPs leads to stabilization of C-RAF in human cells. XIAP binds strongly to C-RAF and promotes the ubiquitylation of C-RAF in vivo through the Hsp90-mediated quality control system, independently of its E3 ligase activity. In addition, XIAP or c-IAP-1/2 knockdown cells showed enhanced cell migration in a C-RAF-dependent manner. XIAP promotes binding of CHIP (carboxy terminal Hsc70-interacting protein), a chaperone-associated ubiquitin ligase, to the C-RAF-Hsp90 complex in vivo. Interfering with CHIP expression resulted in stabilization of C-RAF and enhanced cell migration, as observed in XIAP knockdown cells. Our data show an unexpected role of XIAP and c-IAPs in the turnover of C-RAF protein, thereby modulating the MAPK signalling pathway and cell migration.

While the extravasation cascade of lymphocytes is well characterized, data on their intraepithelial positioning and morphology are scant. However, the latter process is presumably crucial for many immune functions. Integrin alpha(E)(CD103)beta(7) has previously been implicated in epithelial retention of some T cells through binding to E-cadherin. Our current data suggest that alpha(E)(CD103)beta(7) also determines shape and motility of some lymphocytes. Time-lapse microscopy showed that wild-type alpha(E)(CD103)beta(7) conferred the ability to form cell protrusions/filopodia and to move in an amoeboid fashion on E-cadherin, an activity that was abrogated by alpha(E)(CD103)beta(7)-directed antibodies or cytochalasin D. The alpha(E)-dependent motility was further increased (P.001) when point-mutated alpha(E)(CD103) locked in a constitutively active conformation was expressed. Moreover, different yellow fluorescent protein-coupled alpha(E)(CD103) species demonstrated that the number and length of filopodia extended toward purified E-cadherin, cocultured keratinocytes, cryostat-cut skin sections, or epidermal sheets depended on functional alpha(E)(CD103). The in vivo relevance of these findings was demonstrated by wild-type dendritic epidermal T cells (DETCs), which showed significantly more dendrites and spanned larger epidermal areas as compared with DETCs of alpha(E)(CD103)-deficient mice (P.001). Thus, integrin alpha(E)(CD103)beta(7) is not only involved in epithelial retention, but also in shaping and proper intraepithelial morphogenesis of some leukocytes.

The p53 family is known as a family of transcription factors with functions in tumor suppression and development. Whereas the central DNA-binding domain is highly conserved among the three family members p53, p63 and p73, the C-terminal domains (CTDs) are diverse and subject to alternative splicing and post-translational modification. Here we demonstrate that the CTDs strongly influence DNA binding and transcriptional activity: while p53 and the p73 isoform p73gamma have basic CTDs and form weak sequence-specific protein-DNA complexes, the major p73 isoforms have neutral CTDs and bind DNA strongly. A basic CTD has been previously shown to enable sliding along the DNA backbone and to facilitate the search for binding sites in the complex genome. Our experiments, however, reveal that a basic CTD also reduces protein-DNA complex stability, intranuclear mobility, promoter occupancy in vivo, target gene activation and induction of cell cycle arrest or apoptosis. A basic CTD therefore provides both positive and negative regulatory functions presumably to enable rapid switching of protein activity in response to stress. The different DNA-binding characteristics of the p53 family members could therefore reflect their predominant role in the cellular stress response (p53) or developmental processes (p73).

The calcium signaling protein calmodulin (CaM) interacts with many target proteins inside the cell to regulate a wide range of biological signals. CaM's availability to propagate signals depends on its mobility, which may be regulated by interactions with multiple target proteins. We detected single molecules of CaM labeled with a fluorescent dye and injected into living HEK 293 cells, and we used high-speed, wide-field, single-molecule imaging to track single CaM molecules. Single-molecule trajectories were analyzed to characterize the motions of individual CaM molecules. Single-molecule localization resolved CaM positions with a position accuracy of100nm, permitting sub-diffraction imaging of features with localized CaM that form in response to increased free Ca(2+). Single-molecule tracking demonstrated the presence of a wide range of mobilities of individual calmodulin molecules in a cell, with diffusion coefficients ranging from0.01μm(2)s(-1) to ~5μm(2) s(-1), whereas analysis by spatio-temporal image correlation spectroscopy revealed faster-moving components with diffusion coefficients of10μm(2)s(-1). For molecules confined to small regions of the cell, super-resolved images of presumed signaling complexes were recovered. Individual trajectories were classified as normal diffusion, confined diffusion, or directed motion, and could suggest how the individual CaM molecules were bound in the cell. The results show that interactions of CaM with target proteins result in decreased translational mobilities of a significant fraction of CaM molecules inside cells. The work presented here illustrates methods that can characterize location, mobilities, and the availability of signaling molecules in live cells.

Smad proteins are the major signal transducers for the Transforming Growth Factor superfamily of cytokines and their serine/threonine kinase receptors. Smads mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 stimulates phosphorylation of Smad1, which interacts with Smad4. This complex translocates into the nucleus and regulates transcription of target genes. Here, we report our development of cellular fluorescence biosensors for direct visualization of Smad signaling in live mammalian cells. Fluorescence resonance energy transfer between cyan and yellow fluorescent proteins fused to the Smad1 and Smad4 proteins was used to unravel the temporal aspects of BMP/Smad signaling. A rate-limiting delay of 2–5 min occurred between BMP activation and Smad1 activity. A similar delay was observed in the Smad1/Smad4 complexation. Further experimentation indicated that the delay is dependent on the MH1 domain and linker of Smad1. These results give new insights into the dynamics of the BMP receptor –Smad1/4 signaling process and provide a new tool for studying Smads.

Planar illumination imaging allows for illumination of the focal plane orthogonal to the imaging axis in various light forms and is advantageous for high optical sectioning, high imaging speed, low light exposure, and inherently deeper imaging penetration into small organisms and tissue sections. The drawback of the technique is the low inherent resolution, which can be overcome by the incorporation of a dual-sheet stimulated emission depletion (STED) beam to the planar illumination excitation. Our initiative is the implementation of STED into the planar illumination microscope for enhanced resolution. We demonstrate some of our implementations. The depletion of STED in the microscope follows an inverse square root saturation for up to 2.5-fold axial resolution improvements with both high and low numerical aperture imaging objectives.

Arabidopsis plasma membrane nanodomains have been visualized in vivo using Remorin proteins of the taxonomic group 1b. The physiological role of such domains is to facilitate protein-protein interactions near the plasma membrane and to act as signal transducing platforms, routing information from the extra- to the intracellular side of the cell. Beneath sterols and sphingolipids as lipid components, group 1b Remorin proteins showed a strong tendency to accumulate in nanodomains. In contrast to sterol- and sphingolipid enriched membrane domaincounterparts in animals, individual Arabidopsis nanodomains did not exhibit any kind of lateral movement in the plane of the membrane. Since group 1b Remorins are reported to form microfilaments in vitro, assemblies of Remorin microfilaments with stable structures could have an impact on domains´ lateral stability in the plane of the membrane. To test a possible influence of cell wall components on the mobility of individual Remorin proteins resident in plasma membrane nanodomains FRAP-experiments were performed in Arabidopsis mesophyll tissue and protoplasts, using fluorescently labeled group 1b Remorins. It was revealed that the cell wall has a stabilizing effect on the mobility of individual Remorins resident in nanodomains.

The RAF family of kinases mediates RAS signaling, and RAF inhibitors can be effective for treating tumors with BRAF V600E mutant protein. However, RAF inhibitors paradoxically accelerate metastasis in RAS -mutant tumors and become ineffective in BRAF V600E tumors because of reactivation of downstream mitogen-activated protein kinase (MAPK) signaling. We found that the RAF isoform ARAF has an obligatory role in promoting MAPK activity and cell migration in a cell type–dependent manner. Knocking down ARAF prevented the activation of MAPK kinase 1 (MEK1) and extracellular signal–regulated kinase 1 and 2 (ERK1/2) and decreased the number of protrusions from tumor cell spheroids in three-dimensional culture that were induced by BRAF V600E -specific or BRAF/CRAF inhibitors (GDC-0879 and sorafenib, respectively). RAF inhibitors induced the homodimerization of ARAF and the heterodimerization of BRAF with CRAF and the scaffolding protein KSR1. In a purified protein solution, recombinant proteins of the three RAF isoforms competed for binding to MEK1. In cells in culture, overexpressing mutants of ARAF that could not homodimerize impaired the interaction between ARAF and endogenous MEK1 and thus prevented the subsequent activation of MEK1 and ERK1/2. Our findings reveal a new role for ARAF in directly activating the MAPK cascade and promoting tumor cell invasion and suggest a new therapeutic target for RAS- and RAF-mediated cancers.

VE-cadherin is the predominant adhesion molecule in vascular endothelial cells being responsible for maintenance of the endothelial barrier function by forming adhesive contacts (adherens junctions) to neighbouring cells. We found by use of single molecule fluorescence microscopy that VE-cadherin is localised in preformed clusters when not inside adherens junctions. These clusters depend on the integrity of the actin cytoskeleton and are localised in cholesterol rich microdomains of mature endothelial cells as found by membrane fractionation. The ability to form and maintain VE-cadherin based junctions was probed using the laser tweezer technique, and we found that cholesterol depletion has dramatical effects on VE-cadherin mediated adhesion. While a 30% reduction of the cholesterol-level results in an increase of adhesion, excessive cholesterol depletion by about 60% leads to an almost complete loss of VE-cadherin function. Nevertheless, the cadherin concentration in the membrane and the single molecule kinetic parameters of the cadherin are not changed. Our results suggest that the actin cytoskeleton, junction-associated proteins and protein–lipid assemblies in cholesterol-rich microdomains mutually stabilise each other to form functional adhesion contacts.

We report on the values of the two-photon excitation action cross-sections of commercially available enhanced cyan, green, yellow, and red fluorescent proteins. The two-photon absorption spectra are very similar in shape to those measured for one-photon absorption. However, they exhibit a significant blueshift, which is attributed to the participation of a vibrational mode in the two-photon absorption process. The two-photon spectra are compared to that of flavine mononucleotide, which constitutes the main source of autofluorescence in mammalian cells. The definition of a relative detection yield between the autofluorescent proteins and flavine allows us to quantify the applicability of autofluorescent proteins in two-photon single-molecule studies in living cells.

The spectral and photophysical characteristics of the autofluorescent proteins were analyzed and compared to flavinoids to test their applicability for single-molecule microscopy in live cells. We compare 1) the number of photons emitted by individual autofluorescent proteins in artificial and in vivo situations, 2) the saturation intensities of the various autofluorescent proteins, and 3) the maximal emitted photons from individual fluorophores in order to specify their use for repetitive imaging and dynamical analysis. It is found that under relevant conditions and for millisecond integration periods, the autofluorescent proteins have photon emission rates of approximately 3000 photons/ms (with the exception of DsRed), saturation intensities from 6 to 50 kW/cm2, and photobleaching yields from 10(-4) to 10(-5). Definition of a detection ratio led to the conclusion that the yellow-fluorescent protein mutant eYFP is superior compared to all the fluorescent proteins for single-molecule studies in vivo. This finding was subsequently used for demonstration of the applicability of eYFP in biophysical research. From tracking the lateral and rotational diffusion of eYFP in artificial material, and when bound to membranes of live cells, eYFP is found to dynamically track the entity to which it is anchored.

The present study offers reliable protocols for the preparation of new thiol-reactive Cy5 derivatives which are urgently needed for single molecule fluorescence microscopy. In a systematic approach, two alternate strategies were found for the extension of commercial amine-reactive Cy5 with thiol-reactive end groups. In the two-step method, Cy5 succinimidyl ester was first reacted with ethylenediamine under conditions which gave approximately 99% asymmetric "Cy5-amine" and only approximately 1% symmetric product with two Cy5 residues. Subsequently, "Cy5-amine" was derivatized with commercial heterobifunctional cross-linkers to introduce thiol-reactive end groups (maleimide or pyridyldithio). Alternatively, commercial Cy5 succinimidyl ester was reacted with a primary amine (MTSEA, methanethiosulfonylethylamine, or PDEA, pyridyldithioethylamine) or a secondary amine (PEM, piperazinylethylmaleimide) to give the corresponding thiol-reactive derivatives in a single step. Results were good for MTSEA, moderate for PEM, and poor for PDEA. An additional drawback of the one-step method was the need for rigorous removal of unreacted Cy5 succinimidyl ester, which would label lysine residues on probe molecules. It is concluded that, except for the Cy5-MTSEA conjugate, the two-step method is much more general, reliable, and easier to follow by the typical biophysicist, biologist, etc., for whose benefit, these procedures are being published. All thiol-reactive Cy5 derivatives showed similar absorption and fluorescence properties as Cy5 succinimidyl ester, and fluorescence was fully retained after binding to thiols on proteins. The kinetics of protein labeling was also examined in order to get an idea of proper labeling conditions.

A time-resolved study of polarizing energy transfer in oriented blends of a conjugated polymer [a dialkoxy-substituted poly(p-phenyleneethynylene) derivative] and an organic laser dye (7-diethylamino-4-methylcoumarin) in ultra-high molecular weight polyethylene is presented. The transfer is described in terms of a Forster mechanism, based on long-range dipole–dipole interactions. Forster radii were determined in oriented blend films and in chloroform solutions. It was found that the transfer process is critically influenced by the phase behavior of the system under investigation. A depolarizing homotransfer between donor molecules was found to be a key step in the polarizing nature of the transfer which, ultimately, allows excitation light polarized perpendicularly to the film orientation direction to be emitted with the polarization direction parallel to its orientation.

An approach to probing single-molecule ion channel kinetics and conformational dynamics, patch-clamp confocal fluorescence microscopy (PCCFM), uses simultaneous ultrafast fluorescence spectroscopy and single-channel electric current recording. PCCFM is applied to determine single-channel conformational dynamics by probing single-pair fluorescence resonant energy transfer, fluorescence self-quenching, and anisotropy of the dye-labeled gramicidin ion channel incorporated in an artificial lipid bilayer. Hidden conformational changes were observed, which strongly suggests that multiple intermediate conformation states are involved in gramicidin ion channel dynamics.

In pemphigus vulgaris, a life-threatening autoimmune skin disease, epidermal blisters are caused by autoantibodies primarily targeting desmosomal cadherins desmoglein 3 (DSG3) and DSG1, leading to loss of keratinocyte cohesion. Due to limited insights into disease pathogenesis, current therapy relies primarily on nonspecific long-term immunosuppression. Both direct inhibition of DSG transinteraction and altered intracellular signaling by p38 MAPK likely contribute to the loss of cell adhesion. Here, we applied a tandem peptide (TP) consisting of 2 connected peptide sequences targeting the DSG adhesive interface that was capable of blocking autoantibody-mediated direct interference of DSG3 transinteraction, as revealed by atomic force microscopy and optical trapping. Importantly, TP abrogated autoantibody-mediated skin blistering in mice and was effective when applied topically. Mechanistically, TP inhibited both autoantibody-induced p38 MAPK activation and its association with DSG3, abrogated p38 MAPK-induced keratin filament retraction, and promoted desmosomal DSG3 oligomerization. These data indicate that p38 MAPK links autoantibody-mediated inhibition of DSG3 binding to skin blistering. By limiting loss of DSG3 transinteraction, p38 MAPK activation, and keratin filament retraction, which are hallmarks of pemphigus pathogenesis, TP may serve as a promising treatment option.

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.

Understanding the spatiotemporal changes of cellular and molecular events within an organism is crucial to elucidate the complex immune processes involved in infections, autoimmune disorders, transplantation, and neoplastic transformation and metastasis. Here we introduce a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule–1 (MAdCAM-1) and T cell responses in Peyer’s patches following stimulation of the immune system. In addition, we employed LSFM to map individual T cell subsets after hematopoietic cell transplantation and detected rare cellular events. Thus, we present a versatile imaging technology that should be highly beneficial in biomedical research.

Bone (or body) morphogenetic proteins (BMPs) belong to the TGFβ superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.

Evidence exists that cAMP stabilizes the endothelial barrier, in part via activation of the small GTPase Rac1. However, despite the high medical relevance of this signaling pathway, the mechanistic effects on intercellular contacts on the ultrastructural level are largely unknown. In microvascular endothelial cell monolayers, in which increased cAMP strengthened barrier properties, similar to intact microvessels in vivo, both forskolin and rolipram (F/R) to increase cAMP and 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphorothioate (O-Me-cAMP) to stimulate exchange protein directly activated by cAMP/Ras proximate-1 (EPac/Rap 1) signaling enhanced transendothelial electrical resistance and induced activation of Rac1. Concurrently, augmented immunofluorescence intensity and linearization of signals at cell borders were observed for intercellular junction proteins VE-cadherin and claudin 5. Ultrastructural analysis of the intercellular contact zone architecture documented that exposure to F/R or O-Me-cAMP led to a significant increase in the proportion of contact sites displaying complex interdigitations of cell borders, in which membranes of neighboring cells were closely apposed over comparatively long distances; in addition, they were stabilized by numerous intercellular junctions. Interference with Rac1 activation by NSC-23766 completely abolished both barrier stabilization and contact zone reorganization in response to O-Me-cAMP, whereas F/R-mediated Rac1 activation and barrier enhancement were not affected by NSC-23766. In parallel experiments using macrovascular endothelium, increased cAMP failed to induce Rac1 activation, barrier enhancement, and contact zone reorganization. These results indicate that, in microvascular endothelium, Rac1-mediated alterations in contact zone architecture contribute to cAMP-induced barrier stabilization.

The fluorescein arsenical hairpin binder (FlAsH) shows much promise to determine the relative orientations of protein regions and structures even in living cells and in the plasma membrane. In this study, we characterized FlAsH's photophysical properties by steady-state anisotropy and time-resolved single photon counting for further applications with G-protein coupled receptors. We find that FlAsH has a relatively high initial anisotropy of 0.31 ± 0.01 and a three-component fluorescence lifetime with an average of 4.1 ± 0.1 ns. We characterized the FlAsH fluorophore orientation in the α2A adrenergic receptor revealing rigid orientations of FlAsH in the membrane plane for rotational correlation times of ∼50 ns in living cells. To elucidate the fluorophore-membrane orientation and rotational correlation time, an anisotropy treatment similar to that of another researcher (Axelrod, D. 1979. Biophys. J. 26:557–573) was developed. The rotational correlation times were observed to increase by up to 16 ns after agonist addition. The rotational correlation time also allowed for a comparison to the theoretical relationship between translational and rotational diffusion (originally proposed by Saffman, P. G., and M. Delbrück. 1975. Proc. Natl. Acad. Sci. USA. 72:3111–3113) and revealed a discrepancy of a factor between 10 and 100.

Single molecule detection and tracking provides at times the only possible method to observe the interactions of low numbers of biomolecules, inlcuding DNA, receptors and signal mediating proteins in living systems. However, most existing imaging methods do not enable both high sensitivity and non-invasive imaging of large specimens. In this study we report a new setup for selective plane illumination microscopy (SPIM), which enables fast imaging and single molecule tracking with the resolution of confocal microscopy and the optical penetration beyond 300 μm. We detect and report our instrumental figures of merit, control values of fluorescence properties of single nano crystals in comparison to both standard widefield configurations, and also values of nanocrystals in multicellular "fruiting bodies" of Dictyostelium, an excellent control as a model developmental system. In the Dictyostelium , we also report some of our first tracking of single nanocrystals with SPIM. The new SPIM setup represents a new technique, which enables fast single molecule imaging and tracking in living systems.

Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive. There is no evidence of both the functional axonal impairments in vivo and their connection with prion disease. We studied the functional axonal impairments in motor neurons at the onset of clinical prion disease using the combination of tracing as a functional assay for axonal transport with immunohistochemistry experiments. Well-established and novel confocal and ultramicroscopy techniques were used to image and quantify labeled neurons. Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally—after inoculation into the right sciatic nerve. Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods. Our data connect axonal transport impairments with disease symptoms for different prion strains and inoculation routes and establish further insight on the development of prion pathology in vivo. The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway. These findings suggest novel targets for therapeutic and diagnostic approaches in the early stages of prion disease., Author Summary For almost a century, prion disease symptoms, such as dementia and motor system defects, have been accompanied with neuropathological hallmarks in the central nervous system. In past decades, discrepancies between neuropathological changes and clinical symptoms showed that the processes triggering the disease remain elusive. We concentrated on the hypothesis that nervous system integrity and axonal properties may play an important role in the disease development. Since motor system defects are typical for prion disease, we investigated the centers of the motor system, red nucleus and hindlimb area of motor cortex. Although intracerebral prion infection led to a 30% to 45% bilateral reduction of labeled neurons in the red nucleus, infection into the right sciatic nerve—the major hindlimb nerve—led to unilateral reduction of labeled neurons in the red nucleus. Up to 94% reduction was found in the neurons of motor cortex hindlimb area. This reduction is probably caused by functional axonal impairments in motor neurons. Prion-induced alterations in protein distribution implicate a mechanism of transport disruption at cargo attachment to the retrograde axonal transport complex. Our work reveals a connection between axonal transport impairments and disease symptoms in vivo, providing further insight in the development of prion pathology and suggesting novel targets for therapeutic and diagnostic approaches.

We present a method and an apparatus of polarized fluorescence resonance energy transfer (FRET) and anisotropy imaging microscopy done in parallel for improved interpretation of the photophysical interactions. We demonstrate this apparatus to better determine the protein-protein interactions in the pleckstrin homology domain and the conformational changes in the Parathyroid Hormone Receptor, a G-protein coupled receptor, both fused to the cyan and yellow fluorescent proteins for either inter- or intramolecular FRET. In both cases, the expression levels of proteins and also background autofluorescence played a significant role in the depolarization values measured in association with FRET. The system has the sensitivity and low-noise capability of single-fluorophore detection. Using counting procedures from single-molecule methods, control experiments were performed to determine number densities of green fluorescence protein variants CFP and YFP where homo resonance energy transfer can occur. Depolarization values were also determined for flavins, a common molecule of cellular background autofluorescence. From the anisotropy measurements of donor and acceptor, the latter when directly excited or when excited by energy transfer, we find that our instrumentation and method also characterizes crucial effects from homotransfer, polarization specific photobleaching and background molecules.

Multiphoton microscopy has become a standard method for noninvasive imaging of thick specimens with subcellular resolution. Higher harmonic generation microscopy (HHGM), based on nonlinear multiphoton excitation, is a contrast mechanism for the structural and molecular imaging of native samples in cell culture and in fixed and live tissues, for both, three-dimensional and four-dimensional reconstructions. HHGM comprises second and third harmonic generation (SHG, THG) of ordered molecules, can be obtained without exogenous labels, and provides detailed real-time optical reconstruction of fibrillar collagen, myosin, microtubules, and membrane potential, as well as cell depolarization. This unit presents the principles of SHG and THG and the basic setup of a HHGM system, and summarizes current applications in cell biology. Multimodal multiphoton microscopy using HHGM together with two-photon excited fluorescence will develop into a key approach to real-time imaging of cell dynamics in the context of live tissues.

Integrin-bound Src tyrosine kinase mediates alpha(IIb)beta(3) out-side-in signaling to the cytoskeleton required for platelet adhesion and thrombus formation. Src activation (signal initiation) by phosphorylation of Tyr-418 occurs at lamellipodia leading edges. However, little is known about Src inactivation mediated by C-terminal Src kinase (Csk) Tyr-529 phosphorylation. In an established platelet model cell line (A5-Chinese hamster ovary), we studied the inactivation of Src during alpha(IIb)beta(3)-mediated adhesion to fibrinogen with live cell fluorescence resonance energy transfer (FRET) microscopy. Imaging revealed highly dynamic Src-Csk interactions at the leading edges of active lamellipodia. The Src-Csk interaction followed a highly dynamic pattern. Every 2-3 min, Src-Csk complexes moved inward in the cell, reorganized, and formed stable focal adhesions. These accumulations were primarily seen during retraction of lamellipodia, whereas no interaction was observed during protrusions. Western blot analysis during the run time of FRET signaling revealed an increase in Csk-mediated SrcTyr-529 phosphorylation with a parallel decline of tyrosine 418 phosphorylation. Mutation analysis provided additional insights into the role of Src. Although inactivation of Csk (CskK222R) had no effect on cell adhesion and spreading efficiency, cells with constitutively active expressed Src (SrcY529F) exhibited hardly any adhesion and no spreading. The few adherent cells showed weak focal adhesions that were disorganized and oversized. The data clearly demonstrate the important role of tight Src control by Csk for functional cell adhesion and spreading. The novel experimental FRET approach reported here for the inactivation of Src can be readily applied to other integrin and signaling pathways, including closely related Src family kinase members.

A new algorithm is presented for the detection of single, fluorescence-labeled proteins in the analysis of images from living cells. It is especially suited for images with just a few (

Three promising variants of autofluorescent proteins have been analyzed photophysically for their proposed use in single-molecule microscopy studies in living cells to compare their superiority to other fluorescent proteins previously reported regarding the number of photons emitted. The first variant under investigation the F46L mutant of eYFP has a 10% greater photon emission rate and > 50% slower photobleaching rate on average than the standard eYFP fluorophore. The monomeric red fluorescent protein (mRFP) has a fivefold lower photon emission rate, likely due to the monomeric content, and also a tenfold faster photobleaching rate than the DsRed fluorescent protein. In contrast, the previously reported eqfp611 has a 50% lower emission rate yet photobleaches more than a factor 2 slowly. We conclude that the F46L YFP and the eqfp611 are superior new options for single molecule imaging and tracking studies in living cells. Studies were also performed on the effects of forced quenching of multiple fluorescent proteins in sub-micrometer regions that would show the effects of dimerization at low concentration levels of fluorescent proteins and also indicate corrections to stoichiometry patterns with fluorescent proteins previously in print. We also introduce properties at the single molecule level of new FRET pairs with combinations of fluorescent proteins and artificial fluorophores.

[structure: see text] A urea-containing tetraphenoxy-substituted perylene bisimide has been synthesized, and its gelation ability has been studied. This functional dye forms fluorescent organogels in toluene and tetrachloromethane through self-assembly by hydrogen-bonding and pi-pi-stacking interactions. AFM and confocal laser scanning microscopy reveal the formation of fiberlike aggregates.

Complex conformational changes influence and regulate the dynamics of ion channels. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. Here, we report our studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy, which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrate patch-clamp fluorescence microscopy by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of single-pair fluorescence resonance energy transfer and self-quenching is widely distributed, which reflects a broad distribution of conformations. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer.

We report the observation of single-molecule colocalization and quantitative fluorescence resonant energy transfer by simultaneously imaging the emission and polarization characteristics of two colocalized fluorophores using a simple optical design. The methodology was tested using the ligand-receptor system streptavidin, fluorescence labeled with the dye Cy5, and biotin labeled with tetramethylrhodamine. Discrimination of the two dyes permitted the observation of single-pair fluorescence resonant energy transfer with an efficiency of 89%. The multidimensional character of our fluorescence microscopy combined with the robustness of our design provides a simple method suitable to study biomolecular interactions on the single molecule level.

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0

Arf assembles a complex containing Miz1, heterochromatin, and histone H3K3 to block expression of genes involved in cell adhesion and signal transduction. The resulting blockade of cell–cell and cell–matrix interactions facilitates elimination of cells carrying oncogenic mutations., Oncogenic stress induces expression of the alternate reading frame (Arf) tumor suppressor protein. Arf then stabilizes p53, which leads to cell cycle arrest or apoptosis. The mechanisms that distinguish both outcomes are incompletely understood. In this study, we show that Arf interacts with the Myc-associated zinc finger protein Miz1. Binding of Arf disrupts the interaction of Miz1 with its coactivator, nucleophosmin, induces the sumoylation of Miz1, and facilitates the assembly of a heterochromatic complex that contains Myc and trimethylated H3K9 in addition to Miz1. Arf-dependent assembly of this complex leads to the repression of multiple genes involved in cell adhesion and signal transduction and induces apoptosis. Our data point to a tumor-suppressive pathway that weakens cell–cell and cell–matrix interactions in response to expression of Arf and that may thereby facilitate the elimination of cells harboring an oncogenic mutation.

Abstract 61 In vivo models of complex immune processes like stem cell engraftment, host-pathogen interactions or anti-tumor responses face the challenge to either provide dynamic information in low resolution (e.g. in vivo bioluminescence Imaging, BLI) or provide high resolution information with a limited field of view (e.g. multi-photon laser scanning microscopy/confocal microscopy). To overcome these limitations we applied a novel selective plane illumination microscopy (SPIM) technique (also termed ultramicroscopy), which enabled us to visualize structural and cellular changes in intact organs in high resolution. Here we investigated dynamic shifts at sites of graft-versus-host disease (GVHD) initiation in mice after allogenic hematopoietic cell transplantation (allo-HCT). For in vivo and ex vivo imaging we transplanted 1,2×106 luciferase+ DsRed+ transgenic C57Bl/6 T cells (H-2b, Thy1.1+) plus 5×106 bone marrow (BM) cells (H-2b, Thy1.2+) into myeloablative conditioned allogeneic Balb/c recipients (H-2d, Thy1.2+, 8 Gy) to induce aGVHD. Utilizing BLI we followed the development of GVHD in vivo. At the transition from GVHD initiation phase to effector phase (day+3 and day+4) we prepared the small bowel and Peyer‘s Patches (PPs) of allo-HCT recipients for whole organ microscopy and compared these to organs of untreated mice. Staining for T cell populations (CD4) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) allowed us to visualize MAdCAM-1 expression in relation to T cell areas in whole PPs and surrounding intestinal mucosa (> 8mm3) in a high throughput format by creating approx. 1000 optical sections using multiple lasers and a sensitive CCD camera within 10 minutes. Adding another color channel revealed structural details through measurement of intrinsic autofluorescence. The individual color stacks were overlaid via computational image processing for three dimensional tissue reconstruction, volume measurements and quantification of protein expression. Ultramicroscopy exposed microanatomical structures like intestinal villi, crypts and PPs with its subepithelial dome regions and follicles by giving detailed information about MAdCAM-1 expressing blood vessels and T cell areas. The 3D reconstruction of small intestines in non-conditioned mice revealed that the MAdCAM-1 expression averaged at 1-2% of the entire PP and was predominantly restricted to the high endothelial venules (HEV). During the transition from the initiation to the effector phase of acute GVHD (day3-4) the overall MAdCAM-1 expression in PPs increased by 50fold. At this point MAdCAM-1 expression was also found in parafollicular and subepithelial cell populations, which still need to be characterized further concerning their lineage differentiation. MAdCAM-1 is well known as a vascular addressin molecule. Because of its importance in homing of α4β7+ T and B cell into the gastrointestinal tract we investigated the dynamic expression of MAdCAM-1 during acute GVHD within intact PPs. It is well established that MAdCAM-1 is an important regulator in the homing of α4β7+ T cell to PPs via HEVs. Additionally our data lead to the hypothesis that MAdCAM-1 might contribute to retain proliferating α4β7+ T cells during the initiation of an adaptive immune response and might support a synchronized exit of alloreactive T cells in the aGVHD effector phase. Conventional histological sections are labor intensive, time consuming and often restricted to a very limited region of interest and therefore, can result in missing rare events and under- or overestimation of biological processes. Ultramicroscopy helped us to overcome these limitations and enabled us to visualize immunological processes with subcellular resolution in whole organs. CB and MF, GSH and AB contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

When platelets adhere to fibrinogen, they undergo profound actin rearragements and spreading. These responses require signals triggered by aIIbb3, a process known as outside-in signalling. Although aIIbb3 signaling is required for normal hemostasis, its molecular basis is incompletely understood. Recently we demonstrated by coimmunoprecipitation techniques that several Src family kinases are constitutively associated with aIIbb3 in platelets. Fibrinogen binding to aIIbb3 leads to rapid activation of Src and recruitement and activation of the Syk tyrosine kinase. Theses interactions are significant because murine platelets defiecient in multiple Src kinases or Syk fail to spread on fibrinogen. One of the earliest events upon binding of fibrinogen to the receptor is the assembly of a multimolecular protein complex inculding the tyrosine kinases Src and Syk. Phosphorylation of Src at Tyrosine 418 leads to Src activation, phosphorylation of Tyrosine 529 to inactivation. The inactivation of Src is mediated by Csk. Previous studies have shown that in resting platelets Csk forms a complex with integrin α IIb β3 and Src. After fibrinogen binding Csk leaves this complex. The aim of this study is to elucidate the dynamics of Src inactivation and the interaction with Csk in this process by using Fluorescence Resonance Energy Transfer (FRET). A5 CHO cells stably expressing integrin α IIb β3 were transfected with Csk-YFP and various Src mutatnts in a GFP vector. After cell adhesion on fibrinogen, a strong FRET signal could be observed at the edges of nascent lamellipodia of the spreading cell. The FRET signal was dynamic since it was transported in waves inside the cell within seconds. After completion of the adhesion process a weak FRET signal remained visible inside the cell. A different pattern could be observed when cells were transfected with Csk and the active Src mutant Y529F. The observed FRET signal was much weaker and less dynamic after cells were allowed to spread on fibrinogen suggesting that the muation of Tyrosine 529 to Phenylalanine inhibited Csk binding and inactivation of Src. This was not due to a spreading defect since the cells appeared to spread on fibrinogen normally. The use of the inactive Src mutant K295R revealed a FRET signal comparable to WT Src. These studies suggest that Src inactivation is dynamically regulated within seconds after cell adhesion on fibrinogen and that Tyrosine 529 is required for the interaction of Csk with Src in this process.

The functional imaging of neuronal circuits of the central nervous system is crucial for phenotype screenings or investigations of defects in neurodegenerative disorders. Current techniques yield either low penetration depth, yield poor resolution, or are restricted by the age of the animals. Here, we present a novel ultramicroscopy protocol for fluorescence imaging and three-dimensional reconstruction in the central nervous system of adult mice. In combination with tracing as a functional assay for axonal transport, retrogradely labeled descending motor neurons were visualized with >4 mm penetration depth. The analysis of the motor cortex shortly before the onset of clinical prion disease revealed that >80% neurons have functional impairments in axonal transport. Our study provides evidence that prion disease is associated with severe axonal transport defects in the cortical motor neurons and suggests a novel mechanism for prion-mediated neurodegeneration.