Vom Reagenzglas in die Zelle

Die molekularen Prozesse des Lebens in Zellen finden auf engstem Raum statt, unter Bedingungen, die im Reagenzglas kaum nachgestellt werden können. In-Zell-Experimente und Simulationen entschlüsseln Funktion und Struktur von Proteinen. Eine neue spektroskopische Methode auf Basis hochauflösender Rotationsspektroskopie unterscheidet Enantiomere in der Gasphase. Sie ist auf Moleküle mit mehreren stereogenen Zentren anwendbar wie auf chirale Mischungen, und zwar ohne Aufbereitung. Neuentwicklungen in der ultraschnellen Spektroskopie verbinden zeitliche mit räumlicher Information. Dies eröffnet Möglichkeiten, photoinduzierte Prozesse in Funktionsmaterialien, etwa Polymerhalbleitern, aufzuklären. Isolierte Moleküle können mit Hilfe starker elektrischer Felder gezielt nach Größe, Form, Struktur und sogar nach ihrem Quantenzustand sortiert und dann im Raum orientiert werden

Excluded-Volume Effects in Living Cells

Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in-cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET-labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded-volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in-cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in-cell crowding effect as a modulator of biomolecular reactions

Macromolecular Crowding Measurements with Genetically Encoded Probes Based on Förster Resonance Energy Transfer in Living Cells

Genetically encoded Förster resonance energy transfer (FRET)-based probes allow a sensitive readout for different or specific parameters in the living cell. We previously demonstrated how FRET-based probes could quantify macromolecular crowding with high spatio-temporal resolution and under various conditions. Here, we present a protocol developed for the use of FRET-based crowding probes in baker’s yeast, but the general considerations also apply to other species, as well as other FRET-based sensors. This method allows straightforward detection of macromolecular crowding under challenging conditions often presented by living cells

Inhibition of huntingtin exon-1 aggregation by the molecular tweezer CLR01

Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htt(e1)). Above a threshold of 37 glutamine residues, htt(e1) starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htt(e1) (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htt(e1). Here we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htt(e1) monomer and inhibits htt(e1) aggregation, underpinning the key role of N17 in modulating htt(e1) toxicity

Optimizing the Cell Painting assay for image-based profiling

AbstractIn image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations based on their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells, and predicting assay outcomes using machine learning, among many others. Here we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum, and mitochondria. The original protocol was updated in 2016 based on several years’ experience running it at two sites, after optimizing it by visual stain quality. Here we describe the work of the Joint Undertaking for Morphological Profiling (JUMP) Cell Painting Consortium, aiming to improve upon the assay via quantitative optimization, based on the measured ability of the assay to detect morphological phenotypes and group similar perturbations together. We find that the assay gives very robust outputs despite a variety of changes to the protocol and that two vendors’ dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1–2 weeks for a typically sized batch of 20 or fewer plates; feature extraction and data analysis take an additional 1–2 weeks.Key references using this protocolVirtual screening for small-molecule pathway regulators by image-profile matching(https://doi.org/10.1016/j.cels.2022.08.003) - recent work examining the ability to use collected Cell Painting profiles to screen for regulators of a number of diverse biological pathways.JUMP Cell Painting dataset: images and profiles from two billion cells perturbed by 140,000 chemical and genetic perturbations(DOI) - the description of the main JUMP master public data set, using this protocol in the production of &gt;200 TB of image data and &gt;200 TB of measured profiles.Key data used in this protocolCell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes(https://doi.org/10.1038/nprot.2016.105) - this paper provides the first step-by-step Cell Painting protocol ever released.</jats:sec

Microorganisms maintain crowding homeostasis

Macromolecular crowding affects the mobility of biomolecules, protein folding and stability, and the association of macromolecules with each other. Local differences in crowding that arise as a result of subcellular components and supramolecular assemblies contribute to the structural organization of the cytoplasm. In this Opinion article we discuss how macromolecular crowding affects the physicochemistry of the cytoplasm and how this, in turn, affects microbial physiology. We propose that cells maintain the overall concentration of macromolecules within a narrow range and discuss possible mechanisms for achieving crowding homeostasis. In addition, we propose that the term 'homeocrowding' is used to describe the process by which cells maintain relatively constant levels of macromolecules