Publications related to Laboratory of Biophysical Chemistry of Macromolecules

Effective removal of the rotifer Brachionus calyciflorus from a Chlorella vulgaris microalgal culture by homogeneous solar photo-Fenton at neutral pH

Jiahua Chen, Jérémie Decker, Stefanos Giannakis, Christian Ludwig, Horst Pick, Adrian Pulgarin, Dominik Refardt

In this study, a citrate-modified photo-Fenton process was successfully applied to decontaminate a Chlorella vulgaris microalgae culture spiked with the rotifer Brachionus calyciflorus (5 individuals mL−1). The applied treatment (1 mg L−1 Fe2+, 20 mg L−1 H2O2, 17.5 mg L−1 citric acid) had only moderate effects on viability and regrowth of the microalgae since, after a short post-treatment delay of a few days, they reached final cell densities similar to that obtained for microalgae cultures that were not spiked. The decontamination was effective as no regrowth of rotifers was observed in the microalgae cultures after treatment. The efficacy of the citrate-modified photo-Fenton treatment was also studied with a higher starting concentration of 20 rotifers mL−1 and was compared with a solar light/H2O2 treatment. Results show that both treatments had similar efficacies on the rotifer elimination, but that the citrate-modified photo-Fenton treatment had a lower negative impact on the regrowth of microalgae than the solar light/H2O2 treatment. However, when microalgae cultures were spiked with 20 rotifers mL−1, rotifers were only partially inactivated and post-treatment regrowth occurred, which highlights the importance to apply the photo-Fenton process at an early stage of a contamination to achieve full rotifer elimination. In any case, a contamination with 5 rotifers mL−1 is already a significant threat as numbers above 1000 rotifers mL−1 were reached after 14 days and caused the microalgae culture to fail. Overall, our treatment suggests that the citrate-modified solar photo-Fenton process is an environmentally friendly solution to support the maintenance of contaminant-free microalgal cultures.

2022

Fluorescent D-Amino Acids for Super-resolution Microscopy of the Bacterial Cell Wall

Juliette Griffie, Suliana Manley, Jiangtao Qiao, Luc Reymond, Ophélie Rutschmann, Faustine Ambroisine Charline Ryckebusch, Willi Leopold Stepp, Chen Zhang

Fluorescent D-amino acids (FDAAs) have previ-ously been developed to enable in situ highlighting of locations of bacterial cell wall growth. Most bacterial cells lie at the edge of the diffraction limit of visible light; thus, resolving the precise details of peptidoglycan (PG) biosynthesis requires super-resolution micros-copy after probe incorporation. Single molecule localization microscopy (SMLM) has stringent requirements on the fluorophore photophysical properties and therefore has remained challenging in this context. Here, we report the synthesis and characterization of new FDAAs compatible with one-step labeling and SMLM imaging. We demonstrate the incorporation of our probes and their utility for visualizing PG at the nanoscale in Gram-negative, Gram-positive, and mycobacteria species. This improved FDAA toolkit will endow researchers with a nanoscale perspective on the spatial distribution of PG biosynthesis for a broad range of bacterial species.

AMER CHEMICAL SOC2022

Multivalent Peptide Ligands To Probe the Chromocenter Microenvironment in Living Cells

Adam Miklos Eördögh, Beat Fierz, Nora Guidotti, Maxime Nicolas Mivelaz, Pablo Rivera Fuentes

Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated “reader” proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid–liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here we present tools to interrogate the environment of chromatin subcompartments in the form of modular, cell-permeable, multivalent, and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. Here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. Image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.

2022

Development of a single-molecule approach to observe ubiquitination dynamics in defined chromatin states

Beat Fierz, Alexandra Teslenko

CELL PRESS2022

Dynamic Chromatin Invasion and Remodeling by the Transcription Factor Rap1

Maxime Nicolas Mivelaz

Pioneer transcription factors (PTF) are a subset of transcription factors with nucleosome-binding properties allowing them to bind specific sequences in condensed chromatin. Although the biological functions of PTFs including target genes and epigenetic changes have been well characterized in vivo, their mechanism of action remains unknown. Here, we investigate dynamic chromatin binding of Rap1 (an essential Yeast PTF) in highly defined in-vitro single-molecule experiments. Rap1 is an essential regulatory protein whose DNA-binding sites are found in three types of chromosomal elements: promoters, silencers and telomeres. Moreover, its DNA targets are well characterized on a genome wide scale. Further, Rap1 binding sites have been found to co-localize with fragile, i.e. unstable and partially accessible nucleosomes. Due to its role as a PTF, Rap1 might be able to directly interact with compact chromatin and open chromatin structure in the process. Alternatively, it might capture transiently open states, stabilizing fragile nucleosomes. To discriminate between these possibilities, we will investigate dynamic chromatin binding of Rap1 in highly defined in vitro single-molecule experiments. Our experiments include single-molecule FRET and single-molecule TIRF to probe the search and binding kinetics of Rap1 in different environments (i.e free DNA, mono-nucleosomes and chromatin) but also study the effect of Rap1 binding on chromatin. For this, we have designed a library of nucleosome positioning sequences containing Rap1 binding sites based on a well characterized promoter region found in vivo (Ribosomal Protein L30 promoter). Subsequently, these modular DNA sequences can be fluorescently labelled and inserted into large arrays of DNA allowing too later form defined chromatin fibres. We have so far shown that Rap1 is indeed capable of binding nucleosomal DNA and that its binding is specific and modulated by the local chromatin environment. Furthermore, the sequence composition and position of its recognition site within the nucleosome and chromatin structure alters binding kinetics. Importantly, we also find that Rap1 opens local chromatin structure upon binding, thereby potentially providing access to the gene expression machinery.

EPFL2021

Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor

Louise Catherine Bryan, Beat Fierz, Kristina Makasheva

Single-molecule measurements provide detailed mechanistic insights into molecular processes, for example in genome regulation where DNA access is controlled by nucleosomes and the chromatin machinery. However, real-time single-molecule observations of nuclear factors acting on defined chromatin substrates are challenging to perform quantitatively and reproducibly. Here we present XSCAN (multiplexed single-molecule detection of chromatin association), a method to parallelize single-molecule experiments by simultaneous imaging of a nucleosome library, where each nucleosome type carries an identifiable DNA sequence within its nucleosomal DNA. Parallel experiments are subsequently spatially decoded, via the detection of specific binding of dyelabeled DNA probes. We use this method to reveal how the Cas9 nuclease overcomes the nucleosome barrier when invading chromatinized DNA as a function of PAM position.

AMER CHEMICAL SOC2021

Rapid Noninvasive Skin Monitoring by Surface Mass Recording and Data Learning

Natalia Gasilova, Hubert Girault, Milica Jovic, Andreas Stephan Lesch, Tzu-En Lin, Horst Pick, Yingdi Zhu

Skin problems are often overlooked due to a lack of robust and patient-friendly monitoring tools. Herein, we report a rapid, noninvasive, and high-throughput analytical chemical methodology, aiming at real-time monitoring of skin conditions and early detection of skin disorders. Within this methodology, adhesive sampling and laser desorption ionization mass spectrometry are coordinated to record skin surface molecular mass in minutes. Automated result interpretation is achieved by data learning, using similarity scoring and machine learning algorithms. Feasibility of the methodology has been demonstrated after testing a total of 117 healthy, benign-disordered, or malignant-disordered skins. Remarkably, skin malignancy, using melanoma as a proof of concept, was detected with 100% accuracy already at early stages when the lesions were submillimeter-sized, far beyond the detection limit of most existing noninvasive diagnosis tools. Moreover, the malignancy development over time has also been monitored successfully, showing the potential to predict skin disorder progression. Capable of detecting skin alterations at the molecular level in a nonsurgical and time-saving manner, this analytical chemistry platform is promising to build personalized skin care.

2021

Soft-probe-scanning electrochemical microscopy reveals electrochemical surface reactivity of E. coli biofilms

Sorour Darvishi, Hubert Girault, Andreas Stephan Lesch, Emadeddin Oveisi, Horst Pick

Investigating and understanding dynamic biofilm growth mechanisms is challenging, often because state-of-the-art optical characterization tools provide limited information. Micrometric electrochemical imaging of Escherichia coli biofilms using Soft-Probe-Scanning Electrochemical Microscopy (Soft-Probe-SECM) is herein presented as a complementary technique. A soft microelectrode is scanned over biofilms in a gentle contact mode, which is essential to provide a constant working distance. The on-film reduction of an electro-active compound, here the oxidized form of ferrocene methanol, is used to create in situ biofilm metabolic activity maps by applying the feedback mode of SECM. SECM approach curves of identically grown biofilms suggest that the SECM-based detection of metabolic activity is surface-confined. The analysis could therefore be carried out on entire biofilms as well as on tape-stripped biofilm surface layers. The method is further capable of distinguishing between biofilms containing E. coli cells either with or without ampicillin-resistance. Finally, the SECM detection of the degradation of an E. coli biofilm in the presence of different gentamicin concentrations is presented.

2021

Cathepsin inhibitors and uses thereof

Elena Battistello, Elisa Oricchio, Luc Reymond

The present invention relates to new agents and compositions thereof useful for anti-cancer and immunological disorder therapy and uses thereof. The invention further relates to a method of identifying agents useful in the potentiation of immunotherapy, in particular Cathepsin S inhibitors.

2021

Here, the authors use smFRET to assess the structural dynamics of metabotropic glutamate receptor mGlu2 and show that a positive allosteric modulator or the Gi protein stabilize mGlu2 in the glutamate-induced active state, leading to the full activation of the receptor. Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the G(i) protein. Our results provide important insights on the role of allosteric modulators in mGlu activation, by stabilizing the active state of a receptor that is otherwise rapidly oscillating between active and inactive states.

NATURE PORTFOLIO2021