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).

DNA electrotransfer is a successful technique for gene delivery into cells and represents an attractive alternative to virus-based methods for clinical applications including gene therapy and DNA vaccination. However, little is currently known about the mechanisms governing DNA internalization and its fate inside cells. The objectives of this work were to investigate the role of endocytosis and to quantify the contribution of different routes of cellular trafficking during DNA electrotransfer. To pursue these objectives, we performed flow cytometry and single-particle fluorescence microscopy experiments using inhibitors of endocytosis and endosomal markers. Our results show that ~50% of DNA is internalized by caveolin/raft-mediated endocytosis, 25% by clathrin-mediated endocytosis, and 25% by macropinocytosis. During active transport, DNA is routed through multiple endosomal compartments with, in the hour following electrotransfer, 70% found in Rab5 structures, 50% in Rab11-containing organelles and 30% in Rab9 compartments. Later, 60% of DNA colocalizes with Lamp1 vesicles. Because these molecular markers can overlap while following organelles through several steps of trafficking, the percentages do not sum up to 100%. We conclude that electrotransferred DNA uses the classical endosomal trafficking pathways. Our results are important for a generalized understanding of gene electrotransfer, which is crucial for its safe use in clinics.

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.

Poly(ADP ribos)ylation (PARylation) is an impor tant 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. Addi tionally, 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 under standing of its significance in a cellular context.

Locked nucleic acid based antisense oligonucleotides (LNA-ASOs) can reach their intracellular RNA targets without delivery modules. Functional cellular uptake involves vesicular accumulation followed by translocation to the cytosol and nucleus. However, it is yet unknown how many LNA-ASO molecules need to be delivered to achieve target knock down. Here we show by quantitative fluorescence imaging combined with LNA-ASO microinjection into the cytosol or unassisted uptake that ∼105 molecules produce >50% knock down of their targets, indicating that a substantial amount of LNA-ASO escapes from endosomes. Microinjected LNA-ASOs redistributed within minutes from the cytosol to the nucleus and remained bound to nuclear components. Together with the fact that RNA levels for a given target are several orders of magnitude lower than the amounts of LNA-ASO, our data indicate that only a minor fraction is available for RNase H1 mediated reduction of target RNA. When non-specific binding sites were blocked by co-administration of non-related LNA-ASOs, the amount of target LNA-ASO required was reduced by an order of magnitude. Therefore, dynamic processes within the nucleus appear to influence the distribution and activity of LNA-ASOs and may represent important parameters for improving their efficacy and potency.

Die Glycosylierung von Proteinen ist eine weit verbreitete posttranslationale Modifikation, die an der Regulation vieler Proteinfunktionen beteiligt ist. Um die biologischen Funktionen dieser Modifikation zu verstehen, wäre die Visualisierung des Glycosylierungszustandes spezifischer Proteine in lebenden Zellen von entscheidender Bedeutung. Bisher wurde dies noch nicht erreicht. Hier zeigen wir die Detektion proteinspezifischer Glycosylierung der intrazellulären Proteine OGT, Foxo1, p53 und Akt1 in lebenden Zellen. Unser breit anwendbarer Ansatz beruht auf der Fluoreszenzmarkierung intrazellulärer, metabolisch eingebauter Kohlenhydrate durch Diels‐Alder‐Chemie. Die gewählten Proteine sind mit grün fluoreszierendem Protein (EGFP) markiert. Fluoreszenzlebenszeitmikroskopie (FLIM) gestattet die Detektion von Förster‐Resonanzenergietransfer (FRET) zwischen EGFP und dem am Glycan gebundenen Fluorophor mit hohem Kontrast selbst in Gegenwart des großen Überschusses an Akzeptorfluorophor.

Nucleotides containing adenosine play pivotal roles in every living cell. Adenosine triphosphate (ATP), for example, is the universal energy currency, and ATP-consuming processes also contribute to posttranslational protein modifications. Nevertheless, detecting the turnover of adenosine nucleotides in the complex setting of a cell remains challenging. Here, we demonstrate the use of fluorogenic analogs of ATP and adenosine tetraphosphate to study nucleotide hydrolysis in lysates of human cell lines and in intact human cells. We found that the adenosine triphosphate analog is completely stable in lysates of human cell lines, whereas the adenosine tetraphosphate analog is rapidly turned over. The observed activity in human cell lysates can be assigned to a single enzyme, namely, the human diadenosine tetraphosphate hydrolase NudT2. Since NudT2 has been shown to be a prognostic factor for breast cancer, the adenosine tetraphosphate analog might contribute to a better understanding of its involvement in cancerogenesis and allow the straightforward screening for inhibitors. Studying hydrolysis of the analogs in intact cells, we found that electroporation is a suitable method to deliver nucleotide analogs into the cytoplasm and show that high FRET efficiencies can be detected directly after internalization. Time-dependent experiments reveal that adenosine triphosphate and tetraphosphate analogs are both processed in the cellular environment. This study demonstrates that these nucleotide analogs indeed bear the potential to be powerful tools for the exploration of nucleotide turnover in the context of whole cells.

Poly(ADP-Ribos)ylierung (PARylierung) ist eine wichtige posttranslationale Proteinmodifikation,d ie in grundlegende zelluläre Prozesse wie Genregulation und DNA-Reparatur involviert ist. Ihre Fehlregulierung wurde mit verschiedenen Krankheiten wie Krebs in Verbindung gebracht. Trotz grçßter Wichtigkeit gibt es nur wenige Methoden, um PARylierung und ihre Dynamik in Zellen zu beobachten. Mittels einer chemisch-biologischen Herangehensweise entwickelten wir ein fluoreszierendes NAD + -Analogon, das sich als kompetitiver Baustein für die PARylierung von Proteinen in vitro und in Zellen erwies.D ies ermçglichte uns,d en Umsatz von PARd irekt und in lebenden Zellen nachD NA-Schädigung durchN IR-Mikrobestrahlung zu verfolgen. Zusätzlich wurden mithilfe von FLIM-FRET-Mikroskopie kovalente und nichtkovalente Interaktionen von PARm it ausgewählten Proteinen sichtbar gemacht. Unsere Ergebnisse erçffnen neue Chancen für die schnelle zelluläre Untersuchung der Protein-PARylierung in Echtzeit und werden somit zu einem besseren Verständnis und hçherer Aussagekraft im zellulären Kontext beitragen. Abbildung 1. Struktur von A) Poly(ADP-ribose) und B) NAD + und NAD + -Analoga 1 und 2,die in dieser Studie verwendet wurden.[ + + ]D iese Autoren haben zu gleichen Teilen zu der Arbeit beigetragen.Hintergrundinformationen zu diesem Beitrag sind unter: http://dx.