Pervasive mislocalization of pathogenic coding variants underlying human disorders.

Widespread sequencing has yielded thousands of missense variants predicted or confirmed as disease causing. This creates a new bottleneck: determining the functional impact of each variant-typically a painstaking, customized process undertaken one or a few genes and variants at a time. Here, we established a high-throughput imaging platform to assay the impact of coding variation on protein localization, evaluating 3,448 missense variants of over 1,000 genes and phenotypes. We discovered that mislocalization is a common consequence of coding variation, affecting about one-sixth of all pathogenic missense variants, all cellular compartments, and recessive and dominant disorders alike. Mislocalization is primarily driven by effects on protein stability and membrane insertion rather than disruptions of trafficking signals or specific interactions. Furthermore, mislocalization patterns help explain pleiotropy and disease severity and provide insights on variants of uncertain significance. Our publicly available resource extends our understanding of coding variation in human diseases.

Three million images and morphological profiles of cells treated with matched chemical and genetic perturbations.

The identification of genetic and chemical perturbations with similar impacts on cell morphology can elucidate compounds' mechanisms of action or novel regulators of genetic pathways. Research on methods for identifying such similarities has lagged due to a lack of carefully designed and well-annotated image sets of cells treated with chemical and genetic perturbations. Here we create such a Resource dataset, CPJUMP1, in which each perturbed gene's product is a known target of at least two chemical compounds in the dataset. We systematically explore the directionality of correlations among perturbations that target the same protein encoded by a given gene, and we find that identifying matches between chemical and genetic perturbations is a challenging task. Our dataset and baseline analyses provide a benchmark for evaluating methods that measure perturbation similarities and impact, and more generally, learn effective representations of cellular state from microscopy images. Such advancements would accelerate the applications of image-based profiling of cellular states, such as uncovering drug mode of action or probing functional genomics.

Pervasive mislocalization of pathogenic coding variants underlying human disorders.

Widespread sequencing has yielded thousands of missense variants predicted or confirmed as disease-causing. This creates a new bottleneck: determining the functional impact of each variant - largely a painstaking, customized process undertaken one or a few genes or variants at a time. Here, we established a high-throughput imaging platform to assay the impact of coding variation on protein localization, evaluating 3,547 missense variants of over 1,000 genes and phenotypes. We discovered that mislocalization is a common consequence of coding variation, affecting about one-sixth of all pathogenic missense variants, all cellular compartments, and recessive and dominant disorders alike. Mislocalization is primarily driven by effects on protein stability and membrane insertion rather than disruptions of trafficking signals or specific interactions. Furthermore, mislocalization patterns help explain pleiotropy and disease severity and provide insights on variants of unknown significance. Our publicly available resource will likely accelerate the understanding of coding variation in human diseases.

Optimizing the Cell Painting assay for image-based profiling.

In 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 on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by 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 on the basis of 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 Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay's ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and 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 typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1-2 weeks.This protocol is an update to Nat. Protoc. 11, 1757-1774 (2016): https://doi.org/10.1038/nprot.2016.105.

Predicting cell health phenotypes using image-based morphology profiling.

Genetic and chemical perturbations impact diverse cellular phenotypes, including multiple indicators of cell health. These readouts reveal toxicity and antitumorigenic effects relevant to drug discovery and personalized medicine. We developed two customized microscopy assays, one using four targeted reagents and the other three targeted reagents, to collectively measure 70 specific cell health phenotypes including proliferation, apoptosis, reactive oxygen species, DNA damage, and cell cycle stage. We then tested an approach to predict multiple cell health phenotypes using Cell Painting, an inexpensive and scalable image-based morphology assay. In matched CRISPR perturbations of three cancer cell lines, we collected both Cell Painting and cell health data. We found that simple machine learning algorithms can predict many cell health readouts directly from Cell Painting images, at less than half the cost. We hypothesized that these models can be applied to accurately predict cell health assay outcomes for any future or existing Cell Painting dataset. For Cell Painting images from a set of 1500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts. We provide a web app to browse predictions: http://broad.io/cell-health-app. Our approach can be used to add cell health annotations to Cell Painting datasets.

An analysis toolbox to explore mesenchymal migration heterogeneity reveals adaptive switching between distinct modes.

Mesenchymal (lamellipodial) migration is heterogeneous, although whether this reflects progressive variability or discrete, 'switchable' migration modalities, remains unclear. We present an analytical toolbox, based on quantitative single-cell imaging data, to interrogate this heterogeneity. Integrating supervised behavioral classification with multivariate analyses of cell motion, membrane dynamics, cell-matrix adhesion status and F-actin organization, this toolbox here enables the detection and characterization of two quantitatively distinct mesenchymal migration modes, termed 'Continuous' and 'Discontinuous'. Quantitative mode comparisons reveal differences in cell motion, spatiotemporal coordination of membrane protrusion/retraction, and how cells within each mode reorganize with changed cell speed. These modes thus represent distinctive migratory strategies. Additional analyses illuminate the macromolecular- and cellular-scale effects of molecular targeting (fibronectin, talin, ROCK), including 'adaptive switching' between Continuous (favored at high adhesion/full contraction) and Discontinuous (low adhesion/inhibited contraction) modes. Overall, this analytical toolbox now facilitates the exploration of both spontaneous and adaptive heterogeneity in mesenchymal migration.

Non-monotonic cellular responses to heterogeneity in talin protein expression-level.

Talin is a key cell-matrix adhesion component with a central role in regulating adhesion complex maturation, and thereby various cellular properties including adhesion and migration. However, knockdown studies have produced inconsistent findings regarding the functional influence of talin in these processes. Such discrepancies may reflect non-monotonic responses to talin expression-level variation that are not detectable via canonical "binary" comparisons of aggregated control versus knockdown cell populations. Here, we deployed an "analogue" approach to map talin influence across a continuous expression-level spectrum, which we extended with sub-maximal RNAi-mediated talin depletion. Applying correlative imaging to link live cell and fixed immunofluorescence data on a single cell basis, we related per cell talin levels to per cell measures quantitatively defining an array of cellular properties. This revealed both linear and non-linear correspondences between talin expression and cellular properties, including non-monotonic influences over cell shape, adhesion complex-F-actin association and adhesion localization. Furthermore, we demonstrate talin level-dependent changes in networks of correlations among adhesion/migration properties, particularly in relation to cell migration speed. Importantly, these correlation networks were strongly affected by talin expression heterogeneity within the natural range, implying that this endogenous variation has a broad, quantitatively detectable influence. Overall, we present an accessible analogue method that reveals complex dependencies on talin expression-level, thereby establishing a framework for considering non-linear and non-monotonic effects of protein expression-level heterogeneity in cellular systems.

Disentangling Membrane Dynamics and Cell Migration; Differential Influences of F-actin and Cell-Matrix Adhesions.

Cell migration is heavily interconnected with plasma membrane protrusion and retraction (collectively termed "membrane dynamics"). This makes it difficult to distinguish regulatory mechanisms that differentially influence migration and membrane dynamics. Yet such distinctions may be valuable given evidence that cancer cell invasion in 3D may be better predicted by 2D membrane dynamics than by 2D cell migration, implying a degree of functional independence between these processes. Here, we applied multi-scale single cell imaging and a systematic statistical approach to disentangle regulatory associations underlying either migration or membrane dynamics. This revealed preferential correlations between membrane dynamics and F-actin features, contrasting with an enrichment of links between cell migration and adhesion complex properties. These correlative linkages were often non-linear and therefore context-dependent, strengthening or weakening with spontaneous heterogeneity in cell behavior. More broadly, we observed that slow moving cells tend to increase in area, while fast moving cells tend to shrink, and that the size of dynamic membrane domains is independent of cell area. Overall, we define macromolecular features preferentially associated with either cell migration or membrane dynamics, enabling more specific interrogation and targeting of these processes in future.

Plasticity in the macromolecular-scale causal networks of cell migration.

Heterogeneous and dynamic single cell migration behaviours arise from a complex multi-scale signalling network comprising both molecular components and macromolecular modules, among which cell-matrix adhesions and F-actin directly mediate migration. To date, the global wiring architecture characterizing this network remains poorly defined. It is also unclear whether such a wiring pattern may be stable and generalizable to different conditions, or plastic and context dependent. Here, synchronous imaging-based quantification of migration system organization, represented by 87 morphological and dynamic macromolecular module features, and migration system behaviour, i.e., migration speed, facilitated Granger causality analysis. We thereby leveraged natural cellular heterogeneity to begin mapping the directionally specific causal wiring between organizational and behavioural features of the cell migration system. This represents an important advance on commonly used correlative analyses that do not resolve causal directionality. We identified organizational features such as adhesion stability and adhesion F-actin content that, as anticipated, causally influenced cell migration speed. Strikingly, we also found that cell speed can exert causal influence over organizational features, including cell shape and adhesion complex location, thus revealing causality in directions contradictory to previous expectations. Importantly, by comparing unperturbed and signalling-modulated cells, we provide proof-of-principle that causal interaction patterns are in fact plastic and context dependent, rather than stable and generalizable.