Influence of chain length and branching on poly(ADP-ribose)-protein interactions.

Hundreds of proteins interact with poly(ADP-ribose) (PAR) via multiple PAR interaction motifs, thereby regulating their physico-chemical properties, sub-cellular localizations, enzymatic activities, or protein stability. Here, we present a targeted approach based on fluorescence correlation spectroscopy (FCS) to characterize potential structure-specific interactions of PAR molecules of defined chain length and branching with three prime PAR-binding proteins, the tumor suppressor protein p53, histone H1, and the histone chaperone APLF. Our study reveals complex and structure-specific PAR-protein interactions. Quantitative Kd values were determined and binding affinities for all three proteins were shown to be in the nanomolar range. We report PAR chain length dependent binding of p53 and H1, yet chain length independent binding of APLF. For all three PAR binders, we found a preference for linear over hyperbranched PAR. Importantly, protein- and PAR-structure-specific binding modes were revealed. Thus, while the H1-PAR interaction occurred largely on a bi-molecular 1:1 basis, p53-and potentially also APLF-can form complex multivalent PAR-protein structures. In conclusion, our study gives detailed and quantitative insight into PAR-protein interactions in a solution-based setting at near physiological buffer conditions. The results support the notion of protein and PAR-structure-specific binding modes that have evolved to fit the purpose of the respective biochemical functions and biological contexts.

High sensitivity stimulated Raman scattering microscopy with electronic resonance enhancement.

Raman microscopy is an important tool for labelfree microscopy. However, spontaneous Raman microscopy suffers from slow image acquisition rates and susceptibility to fluorescence background. Coherent Raman microsocopy techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, by contrast, offer fast imaging capability and robustness against sample fluorescence. Yet, their rather low sensitivity impedes their broader application. This review discusses sensitivity enhancement of SRS microscopy to µM detection levels by using electronically pre-resonant excitation. We present the foundations of this approach, discuss its technological implementation, and show first successful applications. A special emphasis is given to outlining new experimental developments allowing novel types of investigations.

Characterization of polyphosphate dynamics in the widespread freshwater diatom Achnanthidium minutissimum under varying phosphorus supplies.

Polyphosphates (polyP) are ubiquitous biomolecules that play a multitude of physiological roles in many cells. We have studied the presence and role of polyP in a unicellular alga, the freshwater diatom Achnanthidium minutissimum. This diatom stores up to 2.0 pg·cell-1 of polyP, with chain lengths ranging from 130 to 500 inorganic phosphate units (Pi). We applied energy dispersive X-ray spectroscopy, Raman/fluorescence microscopy, and biochemical assays to localize and characterize the intracellular polyP granules that were present in large apical vacuoles. We investigated the fate of polyP in axenic A. minutissimum cells grown under phosphorus (P), replete (P(+)), or P deplete (P(-)) cultivation conditions and observed that in the absence of exogenous P, A. minutissimum rapidly utilizes their internal polyP reserves, maintaining their intrinsic growth rates for up to 8 days. PolyP-depleted A. minutissimum cells rapidly took up exogenous P a few hours after Pi resupply and generated polyP three times faster than cells that were not initially subjected to P limitation. Accordingly, we propose that A. minutissimum deploys a succession of acclimation strategies regarding polyP dynamics where the production or consumption of polyP plays a central role in the homeostasis of the diatom.

Observation of liquid glass in molecular dynamics simulations.

Molecular anisotropy plays an important role in the glass transition of a liquid. Recently, a novel bulk glass state has been discovered by optical microscopy experiments on suspensions of ellipsoidal colloids. "Liquid glass" is a disordered analog of a nematic liquid crystal, in which rotation motion is hindered but particles diffuse freely. Global nematic order is suppressed as clusters of aligned particles intertwine. We perform Brownian dynamics simulations to test the structure and dynamics of a dense system of soft ellipsoidal particles. As seen in the experiments and in accordance with predictions from the mode coupling theory, on the time scale of our simulations, rotation motion is frozen but translation motion persists in liquid glass. Analyses of the dynamic structure functions for translation and rotation corroborates the presence of two separate glass transitions for rotation and translation, respectively. Even though the equilibrium state should be nematic, aligned structures remain small and orientational order rapidly decays with increasing size. Long-wavelength fluctuations are remnants of the isotropic-nematic transition.

Observation of liquid glass in suspensions of ellipsoidal colloids.

Despite the omnipresence of colloidal suspensions, little is known about the influence of colloid shape on phase transformations, especially in nonequilibrium. To date, real-space imaging results at high concentrations have been limited to systems composed of spherical colloids. In most natural and technical systems, however, particles are nonspherical, and their structural dynamics are determined by translational and rotational degrees of freedom. Using confocal microscopy of fluorescently labeled core-shell particles, we reveal that suspensions of ellipsoidal colloids form an unexpected state of matter, a liquid glass in which rotations are frozen while translations remain fluid. Image analysis unveils hitherto unknown nematic precursors as characteristic structural elements of this state. The mutual obstruction of these ramified clusters prevents liquid crystalline order. Our results give insight into the interplay between local structures and phase transformations. This helps to guide applications such as self-assembly of colloidal superstructures and also gives evidence of the importance of shape on the glass transition in general.

Next-Generation Metabolic Glycosylation Reporters Enable Detection of Protein O-GlcNAcylation in Living Cells without S-Glyco Modification.

Protein O-GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O-GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non-specific protein labeling termed S-glyco modification. Here, we report norbornene-modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O-GlcNAc reporters. They do not lead to detectable S-glyco modification, and they efficiently react in the inverse-electron-demand Diels-Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S-acetyl-2-thioethyl-protected phosphate, we demonstrate the protein-specific detection of O-GlcNAcylation of several proteins and the protein-specific imaging of O-GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).

Live Cell Imaging of Enzymatic Turnover of an Adenosine 5'-Tetraphosphate Analog.

The hydrolysis of nucleotides is of paramount importance as an energy source for cellular processes. In addition, the transfer of phosphates from nucleotides onto proteins is important as a post-translational protein modification. Monitoring the enzymatic turnover of nucleotides therefore offers great potential as a tool to follow enzymatic activity. While a number of fluorescence sensors are known, so far, there are no methods available for the real-time monitoring of ATP hydrolysis inside live cells. We present the synthesis and application of a novel fluorogenic adenosine 5'-tetraphosphate (Ap4) analog suited for this task. Upon enzymatic hydrolysis, the molecule displays an increase in fluorescence intensity, which provides a readout of its turnover. We demonstrate how this can be used for monitoring cellular processes involving Ap4 hydrolysis. To this end, we visualized the enzymatic activity in live cells using confocal fluorescence microscopy of the Ap4 analog. Our results demonstrate that the Ap4 analog is hydrolyzed in lysosomes. We show that this approach is suited to visualize the lysosome distribution profiles within the live cell and discuss how it can be employed to gather information regarding autophagic flux.

Fluorescently Labelled ATP Analogues for Direct Monitoring of Ubiquitin Activation.

Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real-time are scarce. To address this shortcoming, we developed fluorescently labelled adenosine tri-, tetra- and pentaphosphate analogues of ATP. The novel ATP analogues bear - in contrast to earlier reports - only a single acridone-based dye at the terminal phosphate group. The dye's fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP-cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin-activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate-harbouring ATP analogue supersedes the efficiency of recently reported dual-dye labelled analogues and thus, is a promising candidate for broad applications.

Preparation and Tracking of Oblate Core-Shell Polymethyl-Methacrylate Ellipsoids.

Although single-particle level studies on prolate ellipsoidal colloids are relatively abundant, similar studies on oblate ellipsoids are rare because suitable model systems are scarcely available. Here, we present the preparation of monodisperse hard core-shell oblate ellipsoids that can be imaged and tracked in 3D with confocal laser scanning microscopy. Using a thermomechanical squeezing method, we transform spherical core-shell polymethyl-methacrylate (PMMA) particles into oblate ellipsoids. We show how the shape polydispersity as well as the aspect ratio of the obtained oblate ellipsoids can be controlled. In addition, we discuss how the core-shell geometry limits the range of aspect ratios because of the different viscoelastic properties of the cross-linked PMMA core and linear PMMA shell. We further demonstrate imaging of the core-shell oblate dispersions on a single-particle level in real space and time and the tracking of position and orientation using our recently developed tracking algorithm for anisotropic core-shell colloids. Our results thus provide the tools for the future investigation of the behavior of oblate ellipsoids, especially in dense suspensions.

Formation of nematic order in 3D systems of hard colloidal ellipsoids.

Suspensions of hard ellipsoidal particles exhibit complex phase behavior as shown by theoretical predictions and simulations of phase diagrams. Here, we report quantitative confocal microscopy experiments of hard prolate colloidal ellipsoids with different aspect ratio a/b. We studied different volume fractions φ of ellipsoids in density and refractive index matched suspensions. Large 3D sample volumes were investigated and the positions as well as the orientations of all ellipsoids were extracted by image analysis routines. By evaluating the translational and orientational order in the system we determined the presence of isotropic and nematic phases. For ellipsoids with a/b = 2.0 we found that isotropic phases form at all φ, while ellipsoids with a/b = 7.0 formed nematic phases at high φ, as expected from theory and simulations. For a/b = 3.5 and a/b = 4.1, however, we observed the absence of long-range orientational order even at φ where nematic phases are expected. We show that local orientational order formed with the emergence of nematic precursors for a/b = 3.5 and short-ranged nematic domains for a/b = 4.1. Our results provide novel insight into the phase behavior and orientational order of ellipsoids with different aspect ratios.

Double modulation SRS and SREF microscopy: signal contributions under pre-resonance conditions.

Pre-electronic resonance enhancement can increase the sensitivity of non-linear Raman microscopy to the single molecule detection limit. A major problem, however, is the generation of background signal due to unwanted linear and non-linear photophysical processes. In this work, we report the setup of a novel detection scheme for stimulated Raman scattering microspectroscopy based on the simultaneous modulation of pump and Stokes beam. Apart from allowing the parallel detection of stimulated Raman loss and gain (SRL and SRG), the setup gives access to the quantitative analysis of different sources of background signal. We report spectrally and temporally resolved measurements on three exemplary rhodamine dyes and derive the contributions of two-photon absorption and stimulated emission to their SRL, SRG, and stimulated Raman excited fluorescence signals. These results give guidelines for the further improvement of the sensitivity of non-linear Raman micospectroscopy under electronic pre-resonance conditions.

Direct Imaging of Protein-Specific Methylation in Mammalian Cells.

Abundant post-translational modification through methylation alters the function, stability, and/or localization of a protein. Malfunctions in post-translational modification are associated with severe diseases. To unravel protein methylation sites and their biological functions, chemical methylation reporters have been developed. However, until now, their usage was limited to cell lysates. Herein, we present the first generally applicable approach for imaging methylation of individual proteins in human cells, which is based on a combination of chemical reporter strategies, bioorthogonal ligation reactions, and FRET detected by means of fluorescence lifetime imaging microscopy. Through this approach, methylation of histone 4 and the non-histone proteins tumor suppressor p53, kinase Akt1, and transcription factor Foxo1 in two human cell lines has been successfully imaged. To further demonstrate its potential, the localization-dependent methylation state of Foxo1 in the cellular context has been visualized.

Yb fiber based laser source for tunable, narrow bandwidth picosecond pulses in the visible.

We present a simple Yb fiber pumped source for narrow bandwidth picosecond pulses which are tunable in the visible spectral region. This emission is obtained by frequency doubling of a soliton generated in a photonic crystal fiber. The system is attractive for different types of nonlinear optical microscopy and can easily be adapted to meet different experimental prerequisites. As an example, we demonstrate coherent anti-Stokes Raman scattering microscopy using the laser source described.

Fluorescence-Lifetime-Sensitive Probes for Monitoring ATP Cleavage.

Adenosine triphosphate (ATP) probes modified with fluorescence dyes that change their fluorescence properties upon cleavage are an interesting tool for monitoring enzymatic ATP turnover. As a readout parameter, fluorescence lifetime is attractive because it is nearly independent of concentration. In our study, we synthesised and investigated fifteen different ATP analogues, in which the fluorophores were attached to the γ-phosphate of ATP. All analogues showed distinctly different fluorescence lifetimes compared to the corresponding values of the free fluorophores. Both increases and decreases in fluorescence lifetime were observed upon attachment to ATP. To shed light on the photophysical processes governing the lifetime changes, we performed photoelectron spectroscopy in air (PESA) to determine HOMO energy levels and time-resolved fluorescence spectroscopy to obtain rate constants. We present evidence that fluorescence quenching in the compounds tested is dynamic and attributed to photoinduced electron transfer (PET), whereas fluorescence lifetime increases are caused by stacking interactions between chromophore and the nucleobase reducing non-radiative relaxation. Finally, we demonstrate that enzymatic cleavage of the ATP analogues presented can be followed by continuous monitoring of fluorescence lifetime changes.

Real-space imaging of translational and rotational dynamics of hard spheres from the fluid to the crystal.

Using real-space imaging of single particles, we investigate the interplay between translational and rotational motion of tracer particles in suspensions of colloidal particles over a wide range of volume fractions from dilute fluid to densely packed crystal. To this end, we introduce a new type of spherical colloidal tracer particles containing two differently labelled fluorescent cores. The tracer particles can be combined with host particles enclosing a single fluorescent core and chemical and physical properties identical to the tracers. This leads to a system of spherical colloidal particles, in which spatio-temporal trajectories of rotation and translation of individual particles can be recorded simultaneously with full 360° resolution of rotational dynamics. Our analysis shows that translation and rotation of colloidal particles are uncorrelated and decoupled for all volume fractions irrespective of the phase of the particle system.

Stimulated Raman scattering microscopy by Nyquist modulation of a two-branch ultrafast fiber source.

A highly stable setup for stimulated Raman scattering (SRS) microscopy is presented. It is based on a two-branch femtosecond Er:fiber laser operating at a 40 MHz repetition rate. One of the outputs is directly modulated at the Nyquist frequency with an integrated electro-optic modulator (EOM). This compact source combines a jitter-free pulse synchronization with a broad tunability and allows for shot-noise limited SRS detection. The performance of the SRS microscope is illustrated with measurements on samples from material science and cell biology.

Bioorthogonally Functionalized NAD(+) Analogues for In-Cell Visualization of Poly(ADP-Ribose) Formation.

Poly(ADP-ribos)ylation (PARylation) is a major posttranslational modification and signaling event in most eukaryotes. Fundamental processes like DNA repair and transcription are coordinated by this transient polymer and its binding to proteins. ADP-ribosyltransferases (ARTs) build complex ADP-ribose chains from NAD(+) onto various acceptor proteins. Molecular studies of PARylation thus remain challenging. Herein, we present the development of bioorthogonally functionalized NAD(+) analogues for the imaging of PARylation in vitro and in cells. Our results show that 2-modified NAD(+) analogues perform remarkably well and can be applied to the in-cell visualization of PARylation simultaneously in two colors. This tool gives insight into the substrate scope of ARTs and will help to further elucidate the biological role of PARylation by offering fast optical, multichannel read-outs.

Real-Time Cellular Imaging of Protein Poly(ADP-ribos)ylation.

Poly(ADP-ribos)ylation (PARylation) is an important posttranslational protein modification, and is involved in major cellular processes such as gene regulation and DNA repair. Its dysregulation has been linked to several diseases, including cancer. Despite its importance, methods to observe PARylation dynamics within cells are rare. By following a chemical biology approach, we developed a fluorescent NAD(+) analogue that proved to be a competitive building block for protein PARylation in vitro and in cells. This allowed us to directly monitor the turnover of PAR in living cells at DNA damage sites after near-infrared (NIR) microirradiation. Additionally, covalent and noncovalent interactions of selected target proteins with PAR chains were visualized in cells by using FLIM-FRET microscopy. Our results open up new opportunities for the study of protein PARylation in real time and in live cells, and will thus contribute to a better understanding of its significance in a cellular context.

Visualization of Protein-Specific Glycosylation inside Living Cells.

Protein glycosylation is a ubiquitous post-translational modification that is involved in the regulation of many aspects of protein function. In order to uncover the biological roles of this modification, imaging the glycosylation state of specific proteins within living cells would be of fundamental importance. To date, however, this has not been achieved. Herein, we demonstrate protein-specific detection of the glycosylation of the intracellular proteins OGT, Foxo1, p53, and Akt1 in living cells. Our generally applicable approach relies on Diels-Alder chemistry to fluorescently label intracellular carbohydrates through metabolic engineering. The target proteins are tagged with enhanced green fluorescent protein (EGFP). Förster resonance energy transfer (FRET) between the EGFP and the glycan-anchored fluorophore is detected with high contrast even in presence of a large excess of acceptor fluorophores by fluorescence lifetime imaging microscopy (FLIM).

PMMA/PMMA core-shell particles with ellipsoidal, fluorescent cores: accessing rotational dynamics.

For several decades, nonaqueous dispersions of PMMA particles have played an important role in colloid research. They have found application as colloidal model systems, which are used to probe glassy dynamics or to explore crystal nucleation. To date, most research has focused on spherical particles, in which only translational motion can be investigated. Recently, however, there has been a surge of interest in analyzing also rotational dynamics. In this contribution, we introduce a new class of core-shell particles, which can be used as rotational probes. The colloids described herein are composed of shape anisotropic, fluorescent cores covered with nonfluorescent PMMA shells. The core-shell particles are built up in four steps. In a first step, we produce fluorescent and photo-cross-linkable PMMA colloids. In the second step, these particles are thermomechanically elongated and fixed in defined ellipsoidal shapes by photo-cross-linking. Subsequently, we cover the cross-linked, fluorescent core with a nonfluorescent PMMA shell. The shape of the resulting core-shell colloids is tunable between the initial anisotropic and perfect spherical shape. For shaping, we apply a simple solvent swelling procedure. As one option, this method yields perfect PMMA spheres with ellipsoidal, fluorescent centers. We also report morphological particle characterization using various fluorescence microscopy techniques. Finally, we demonstrate that the rotational dynamics of individual colloids can be tracked and analyzed.