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Image-based profiling has emerged as a powerful technology for various steps in basic biological and pharmaceutical discovery, but the community has lacked a large, public reference set of data from chemical and genetic perturbations. Here we present data generated by the Joint Undertaking for Morphological Profiling (JUMP)-Cell Painting Consortium, a collaboration between 10 pharmaceutical companies, six supporting technology companies, and two non-profit partners. When completed, the dataset will contain images and profiles from the Cell Painting assay for over 116,750 unique compounds, over-expression of 12,602 genes, and knockout of 7,975 genes using CRISPR-Cas9, all in human osteosarcoma cells (U2OS). The dataset is estimated to be 115 TB in size and capturing 1.6 billion cells and their single-cell profiles. File quality control and upload is underway and will be completed over the coming months at the Cell Painting Gallery:https://registry.opendata.aws/cellpainting-gallery. A portal to visualize a subset of the data is available athttps://phenaid.ardigen.com/jumpcpexplorer/.

Protein misfolding and aggregation are hallmarks of many severe neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's disease. As a supramolecular ligand that binds to lysine and arginine residues, the molecular tweezer CLR01 was found to modify the aggregation pathway of disease-relevant proteins in vitro and in vivo with beneficial effects on toxicity. However, the molecular mechanisms of how tweezers exert these effects remain mainly unknown, hampering further drug development. Here, we investigate the modulation mechanism of unfolding and aggregation pathways of SOD1, which are involved in amyotrophic lateral sclerosis (ALS), by CLR01. Using a truncated version of the wildtype SOD1 protein, SOD1bar , we show that CLR01 acts on the first step of the aggregation pathway, the unfolding of the SOD1 monomer. CLR01 increases, by ∼10 °C, the melting temperatures of the A4V and G41D SOD1 mutants, which are commonly observed mutations in familial ALS. Molecular dynamics simulations and binding free energy calculations as well as native mass spectrometry and mutational studies allowed us to identify K61 and K92 as binding sites for the tweezers to mediate the stability increase. The data suggest that the modulation of SOD1 conformational stability is a promising target for future developments of supramolecular ligands against neurodegenerative diseases.

International audience; The thermal stability of the superoxide dismutase 1 protein in a crowded solution is investigated by performing enhanced sampling molecular simulations. By complementing thermal unfolding experiments done close to physiological conditions (200 mg/mL), we provide evidence that the presence of the protein crowder bovine serum albumin in different packing states has only a minor, and essentially destabilizing, effect. The finding that quinary interactions counteract the pure stabilization contribution stemming from excluded volume is rationalized here by exploring the SOD1 unfolding mechanism in microscopic detail. In agreement with recent experiments, we unveil the importance of intermediate unfolded states as well as the correlation between protein conformations and local packing with the crowders. This link helps us to elucidate why certain SOD1 mutations involved in the ALS disease reverse the stability effect of the intracellular environment.

Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htte1). Above a threshold of 37 glutamine residues, htte1 starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htte1 (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htte1. 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 htte1 monomer and inhibits htte1 aggregation, underpinning the key role of N17 in modulating htte1 toxicity.

The human transient receptor potential vanilloid 4 (hTRPV4) ion channel plays a critical role in a variety of biological processes. Whilst the activation of hTRPV4 gating properties has been reported for a broad spectrum of stimuli, including synthetic 4α-phorbols, the molecular basis of the activation is poorly understood. Here we report the novel cryo-EM structure of the hTRPV4 determined in the presence of the archetypical phorbol acid agonist, 4α-PDD. Complementary mutagenesis experiments support the EM-identified binding site as well as allowing rationalization of disruptive mutants located outside of the 4α-PDD binding site. This work represents the first structural information of hTRPV4 in a ligand-induced open conformation. Together, our data reveal the underlying molecular mechanisms resulting in the opening of the central pore and ion-channel activation and provide a structural template for designing inhibitors targeting the open-state conformation of hTRPV4.

In cells, proteins are embedded in a crowded environment that controls their properties via manifold avenues including weak protein-macromolecule interactions. A molecular level understanding of these quinary interactions and their contribution to protein stability, function, and localization in the cell is central to modern structural biology. Using a mutational analysis to quantify the energetic contributions of single amino acids to the stability of the ALS related protein superoxide dismutase I (SOD1) in mammalian cells, we show that quinary interactions destabilize SOD1 by a similar energetic offset for most of the mutants, but there are notable exceptions: Mutants that alter its surface properties can even lead to a stabilization of the protein in the cell as compared to the test tube. In conclusion, quinary interactions can amplify and even reverse the mutational response of proteins, being a key aspect in pathogenic protein misfolding and aggregation.

Changes of the extracellular milieu could affect cellular crowding. To prevent detrimental effects, cells use adaptation mechanisms to react to such conditions. Using fluorescent crowding sensors, we show that the initial response to osmotic stress is fast but imperfect, while the slow response renders cells more tolerant to stress, particularly in the presence of osmolytes.

The influence of the cellular milieu, a complex and crowded solvent, is often neglected when biomolecular structure and function are studied in vitro. To mimic the cellular environment, crowding effects are commonly induced in vitro using artificial crowding agents like Ficoll or dextran. However, it is unclear if such effects are also observed in cellulo. Diverging results on protein stability in living cells point out the need for new quantitative methods to investigate the contributions of excluded volume and nonspecific interactions to the cellular crowding effect. We show how new crowding sensitive probes may be utilized to directly investigate crowding effects in living cells. Moreover, we discuss processes where crowding effects could play a crucial role in molecular cell biology.

Biomolekule entstehen und funktionieren in einer dicht gepackten und heterogenen zellularen Umgebung. Diese Bedingungen werden im Reagenzglas oft durch Zugabe kunstlicher makromolekularer “Crowding”-Reagentien imitiert. Dennoch ist es unklar, ob solche Kosolventien tatsachlich die physikalischen und chemischen Eigenschaften der zellularen Umgebung widerspiegeln, da der zellulare “Crowding”-Effekt bisher nicht quantifiziert wurde. Wir haben einen makromolekularen “Crowding”-Sensor, der auf einem FRET-markiertem Polymer basiert, entwickelt, um diese Effekte zu untersuchen. Die Ergebnisse zeigen, dass die Volumenausschlusseffekte zu keiner Kompression des Sensors in der Zelle fuhren. Die durchschnittliche Konformation des Sensors ist dabei ahnlich zu der im verdunnten Puffer sowie im Zell-Lysat. Ferner ist der zellulare “Crowding”-Effekt heterogen verteilt und verandert sich signifikant wahrend eines osmotischen Schocks. Mithilfe dieser Methode lasst sich der zellulare “Crowding”-Effekt als Regulator von biomolekularen Reaktionen systematisch untersuchen.

Here, we study the stabilities of different naturally fragile mutants of 3' phosphoadenosine 5' phosphosulfate (PAPS) synthases. The goal is to establish a relationship between the stability of the mutants in the cellular environment and their enzymatic activity. In vitro stability measurements have suggested that protein stability is a major factor for cellular PAPS availability [1]. We aim to specifically analyze different cellular factors for in-cell PAPSS stability. One crucial factor is the macromolecular crowding effect. The cell is filled up to a volume of 40 % with macromolecules. Often, artificial macromolecular crowding agents are used to mimic these conditions. We previously studied macromolecular crowding effects via a thermodynamic analysis of the thermal unfolding of ubiquitin [2]. We observed enthalpic stabilization and entropic destabilization forces for all tested crowders. Further, we tested how such artificial cosolutes reflect the physicochemical properties of the complex cellular environment. Therefore, we developed a FRET-based macromolecular crowding sensor to study the crowding effect in living cells [3]. We found that the in-cell crowding effect is distributed heterogeneously and can change significantly upon osmotic stress. In comparison, we now used Fast Relaxation Imaging [4] to study the stability of PAPSS within the cellular environment and compare the results to in vitro crowding studies.References:1. J. Boom, D. Heider, S.R. Martin, A. Pastore, and J.W. Mueller. PAPS synthases - naturally fragile enzymes specifically stabilized by nucleotide binding., J. Biol. Chem. 2012, 287(21):17645-55.2. M. Senske, L. Tork, B. Born, M. Havenith, C. Herrmann, S. Ebbinghaus, J. Am. Chem. Soc. 2014, 136, 9036-9041.3. D. Gnutt, M. Gao, O. Brylski, M. Heyden, S. Ebbinghaus, Angew. Chem Int. Ed. 2015, 54(8):2548-2551.4. S. Ebbinghaus, A. Dhar, J.D. McDonald and M. Gruebele. “Protein folding stability and dynamics imaged in a living cell.” Nature Methods 2010, 7:319-323.

Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin‐structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high‐molecular‐weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer.

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.

While extensive biochemical and biophysical studies have been carried out to elucidate protein-RNA binding mechanisms and dynamics in vitro, most of these studies do not take into consideration the effect of the cellular environment. Here we have experimentally tested the role of the cellular environment on protein transport and binding affinity in one of the most widely studied RNA recognition motifs, the spliceosomal protein U1A, and its binding partner, stem loop 2 (SL2) of the U1 small nuclear RNA. U1A-SL2 localization, stability and binding kinetics were monitored in live U2OS cells by fast relaxation imaging (FreI), which combines a temperature jump with fluorescence microscopy of the FRET (Fluorescence resonance energy transfer)-labeled RNA and protein. U1A protein alone was found to diffuse across the cell, whereas SL2 RNA and the U1A-SL2 complex localized in the nucleus. This demonstrates that SL2 RNA mediates transport of the U1A protein to the nucleus. The binding affinity in live cells was reduced compared to in vitro. The dissociation rate was unchanged in cells; however, the association rate was an order of magnitude lower, resulting in a two order of magnitude decrease in the dissociation constant. Introduction of a macromolecular crowder, Ficoll 70, in vitro further stabilized the complex. This suggests that differences between binding affinities as measured in vitro and in live cells cannot be explained by crowding alone. Instead, high binding affinities as measured in vitro may be necessary for selectivity in vivo, where competition exists between multiple binding partners.

Proteins and nucleic acids fold and behave in a highly occupied matrix of macromolecules, the cellular environment. Although this environment is filled up to a volume of 40% with macromolecules, the effect of crowding on biochemical reactions as well as biophysical properties of proteins has been rarely considered. Therefore, studies on macromolecular crowding are mainly conducted using artificially created polymer based substances as mimics of the in cell environment. Still, a common concept how the excluded volume effect in the cell affects protein folding, function and structure is lacking. Understanding these properties will be important to unravel the underlying mechanism of protein misfolding and aggregation as well as the behavior of intrinsically disordered proteins in a living cell.We introduce a FRET based random coil polymer to characterize crowding both in vitro and inside a living cell. We find different compression of the polymer in artificial crowding substances compared to protein crowders such as BSA or oocyte lysate. Injection of the probe inside cells confirms this result and reveals a heterogeneous environment which, on average, shows comparable polymer conformations as in diluted buffer. The polymer conformation is used to quantify crowding differences in the cytosol and the nucleus as well as to identify crowding in cells influenced by different extrinsic conditions. Severe osmotic stress is used to probe compression of the polymer in the highly concentrated cytosolic environment.We conclude that the FRET labelled polymer provides a new approach to investigate and characterize the cellular solvation properties with high spatio-temporal resolution in a variety of systems and identify crowding differences due to the architecture of the cellular matrix. Therefore, it will help to understand how the cell, as the native environment in which proteins evolved, might modulate and tune biomolecule properties and functions.