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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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

Coherent Raman scattering microscopy is an attractive new technology for label‐free imaging. Both coherent anti‐Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy offer the possibility to record hyperspectral imaging data. While for the analysis of spontaneous Raman microscopy data multivariate methods are nowadays routinely employed, until to date most of the coherent Raman imaging data are interpreted using univariate data analysis. In this work, we report a quantitative comparison of the performance of different multivariate methods used for the analysis of hyperspectral SRS data from different model samples. Our data show for all samples that multivariate methods outperform univariate analysis. Using metrics to quantify method performance, we find that of the methods tested, multivariate curve resolution (MCR) gives the best results. We show that the combination with a selection of essential components based on first‐order autocorrelation, gives a simple workflow for the MCR‐based analysis of hyperspectral coherent Raman imaging data. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Monitoring of ATP consumption in real time: Fluorescently modified adenosine triphosphate (ATP) probes that change their fluorescence properties upon enzymatic turnover have been synthesised and applied in a simple enzymatic cleavage reaction. Both increases and decreases in fluorescence lifetime were observed upon attachment to ATP. We present evidence that fluorescence quenching in the compounds tested is dynamic and caused by photoinduced electron transfer (PET), whereas fluorescence lifetime increases are a result of stacking interactions between chromophore and the nucleobase reducing non‐radiative relaxation. [ABSTRACT FROM AUTHOR]

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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. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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). [ABSTRACT FROM AUTHOR]

The reaction of POCl3-activated, readily soluble diketopyrrolopyrrole (DPP) with 2-aminoheteroaromatics to yield 1:1 and 1:2 hydrogen chelates is described. Complexation of these hydrogen chelates with boron reagents results in thermally and photochemically stable fluorescent dyes (PP-azacyanines). The 1:2 complexes in particular absorb at long wavelengths and are brightly fluorescing. The rich photophysics of the new compounds are presented. Both the pronounced vibrational fine structure of the S0→S1 transitions and the observed fluorescence phenomena allow detailed conclusions to be made on the correlation between molecular structure and optical properties. [ABSTRACT FROM AUTHOR]

Photobleaching of organic fluorophores commonly used in fluorescence microscopy puts a limit to the number of images which can be acquired. Label-free imaging techniques therefore offer advantages both for rapid image acquisition and for long-term observations. CARS microscopy is a label-free imaging technique offering molecule specific contrast. Here we demonstrate that CARS microscopy allows video-rate tracking of intracellular transport of lipid droplets, but also continuous long-term observation of cells over several hours. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

Copyright of Nachrichten aus der Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)