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Being able to visualize protein localizations within cells and tissues by means of immuno-fluorescence microscopy has been key to developments in cell biology and beyond. Gut et al. present a high-throughput method that achieves the detection of more than 40 different proteins in biological samples across multiple spatial scales. This allows the simultaneous quantification of their expression levels in thousands of single cells; captures their detailed subcellular distribution to various compartments, organelles, and cellular structures within each of these single cells; and places all this information within a multicellular context. Such a scale-crossing dataset empowers artificial intelligence–based computer vision algorithms to achieve a comprehensive profiling of intracellular protein maps to measure their responses to different multicellular, cellular, and pharmacological contexts, and to reveal new cellular states. Science , this issue p. eaar7042 Multiplexed (40-plex) indirect immunofluorescence quantifies subcellular protein organization on cellular and multicellular scales. Obtaining highly multiplexed protein measurements across multiple length scales has enormous potential for biomedicine. Here, we measured, by iterative indirect immunofluorescence imaging (4i), 40-plex protein readouts from biological samples at high-throughput from the millimeter to the nanometer scale. This approach simultaneously captures properties apparent at the population, cellular, and subcellular levels, including microenvironment, cell shape, and cell cycle state. It also captures the detailed morphology of organelles, cytoskeletal structures, nuclear subcompartments, and the fate of signaling receptors in thousands of single cells in situ. We used computer vision and systems biology approaches to achieve unsupervised comprehensive quantification of protein subcompartmentalization within various multicellular, cellular, and pharmacological contexts. Thus, highly multiplexed subcellular protein maps can be used to identify functionally relevant single-cell states.

Studying the phenotypic differences between genetically identical cells rather than their general features can reveal novel regulatory mechanisms for diverse cellular processes. Every cell biologist who has used a microscope knows that single cells in a population display variable behavior ( 1 ). Although heterogeneity between single cells is obvious in tissues and organisms, it can also be observed in populations of monoclonal cells that have been cultured under identical conditions. Besides having stochastic sources ( 2 , 3 ), phenotypic cell-to-cell variability among genetically identical cells can be deterministic and regulated, in both prokaryotic and mammalian cells ( 4 , 5 ). Molecular and cell biologists have traditionally ignored this phenomenon, in part because of technical limitations, but also because, historically, research has focused on mechanisms and processes that are common between cells. However, the mechanisms that make them different are likely to be equally important. Embracing this cell-to-cell variability as a fact in our scientific understanding requires a paradigm shift, but it will be necessary. In fact, it may be a strong boost for the field by uncovering novel and previously overlooked mechanisms of regulation at the cell population level.

Advancing age causes reduced hippocampal neurogenesis, associated with age-related cognitive decline. The spatial relationship of age-induced alterations in neural stem cells (NSCs) and surrounding cells within the hippocampal niche remains poorly understood due to limitations of antibody-based cellular phenotyping. We established iterative indirect immunofluorescence imaging (4i) in tissue sections, allowing for simultaneous detection of 18 proteins to characterize NSCs and surrounding cells in 2-, 6-, and 12-month-old mice. We show that reorganization of the dentate gyrus (DG) niche already occurs in middle-aged mice, paralleling the decline in neurogenesis. 4i-based tissue analysis of the DG identifies changes in cell-type contributions to the blood-brain barrier and microenvironments surrounding NSCs to play a pivotal role to preserve neurogenic permissiveness. The data provided represent a resource to characterize the principles causing alterations of stem cell-associated plasticity within the aging DG and provide a blueprint to analyze somatic stem cell niches across lifespan in complex tissues.

Liquid–liquid phase separation has been shown to underlie the formation and disassembly of membraneless organelles in cells, but the cellular mechanisms that control this phenomenon are poorly understood. A prominent example of regulated and reversible segregation of liquid phases may occur during mitosis, when membraneless organelles disappear upon nuclear-envelope breakdown and reappear as mitosis is completed. Here we show that the dual-specificity kinase DYRK3 acts as a central dissolvase of several types of membraneless organelle during mitosis. DYRK3 kinase activity is essential to prevent the unmixing of the mitotic cytoplasm into aberrant liquid-like hybrid organelles and the over-nucleation of spindle bodies. Our work supports a mechanism in which the dilution of phase-separating proteins during nuclear-envelope breakdown and the DYRK3-dependent degree of their solubility combine to allow cells to dissolve and condense several membraneless organelles during mitosis. The dual-specificity kinase DYRK3 acts as a central ‘dissolvase’, mediating the phase transitions of several types of membraneless organelles during mitosis.

An automated experimental and software pipeline for large-scale FISH enables spatial transcriptomics in thousands of single human cells at single-molecule resolution. Fluorescence in situ hybridization (FISH) is widely used to obtain information about transcript copy number and subcellular localization in single cells. However, current approaches do not readily scale to the analysis of whole transcriptomes. Here we show that branched DNA technology combined with automated liquid handling, high-content imaging and quantitative image analysis allows highly reproducible quantification of transcript abundance in thousands of single cells at single-molecule resolution. In addition, it allows extraction of a multivariate feature set quantifying subcellular patterning and spatial properties of transcripts and their cell-to-cell variability. This has multiple implications for the functional interpretation of cell-to-cell variability in gene expression and enables the unbiased identification of functionally relevant in situ signatures of the transcriptome without the need for perturbations. Because this method can be incorporated in a wide variety of high-throughput image-based approaches, we expect it to be broadly applicable.

Coordination of RNA abundance and production rate with cell size has been observed in diverse organisms and cell populations. However, how cells achieve such ‘scaling’ of transcription with size is unknown. Here we describe a genome-wide siRNA screen to identify regulators of global RNA production rates in HeLa cells. We quantify the single-cell RNA production rate using metabolic pulse-labelling of RNA and subsequent high-content imaging. Our quantitative, single-cell measurements of DNA, nascent RNA, proliferating cell nuclear antigen (PCNA), and total protein, as well as cell morphology and population-context, capture a detailed cellular phenotype. This allows us to account for changes in cell size and cell-cycle distribution (G1/S/G2) in perturbation conditions, which indirectly affect global RNA production. We also take advantage of the subcellular information to distinguish between nascent RNA localised in the nucleolus and nucleoplasm, to approximate ribosomal and non-ribosomal RNA contributions to perturbation phenotypes. Perturbations uncovered through this screen provide a resource for exploring the mechanisms of regulation of global RNA metabolism and its coordination with cellular states. Measurement(s) nascent RNA • Image • S phase • nucleolus organization • Cellular Morphology • Cell Cycle Phase Technology Type(s) metabolic labelling: 5-ethynyl uridine • spinning-disk confocal microscope • supervised machine learning • Image Processing Factor Type(s) gene expression Sample Characteristic - Organism HeLa cell Sample Characteristic - Environment cell culture Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.14332916

Kinome truths A genome-wide analysis of the human kinome — the sum total of protein, lipid and carbohydrate kinases — shows that the cell's signalling functions are intimately linked to endocytosis, the process by which material is imported into the cell inside a membrane vesicle. Many of the kinases involved in endocytosis are known to function in mitogenic signalling, stimulating cell division and lymphocyte transformation: this adds to growing evidence that endocytic transport and signal transduction are integrated processes. An accompanying manuscript looks at the role of some of the kinases identified in the kinome screen, revealing novel dynamic properties of caveola/raft-mediated endocytosis that are distinct from the current picture of membrane transport. A functional genomics approach has revealed that caveolae/raft-mediated endocytosis is subject to regulation by a large number of kinases 1 . Here we explore the role of some of these kinases in caveolae dynamics. We discover that caveolae operate using principles different from classical membrane trafficking. First, each caveolar coat contains a set number (one ‘quantum’) of caveolin-1 molecules. Second, caveolae are either stored as in stationary multi-caveolar structures at the plasma membrane, or undergo continuous cycles of fission and fusion with the plasma membrane in a small volume beneath the surface, without disassembling the caveolar coat. Third, a switch mechanism shifts caveolae from this localized cycle to long-range cytoplasmic transport. We have identified six kinases that regulate different steps of the caveolar cycle. Our observations reveal new principles in caveolae trafficking and suggest that the dynamic properties of caveolae and their transport competence are regulated by different kinases operating at several levels.

Creating enough glue to protect the body may require phase separation in skin cells Liquid-liquid phase separation (LLPS), the unmixing of inhomogeneous fluids into two or more phases, is emerging as a paradigm for the formation of a myriad of membraneless compartments inside cells ( 1 , 2 ). This type of spatial organization, in contrast to membrane-bound compartmentalization, has long lacked unifying principles. However, the physiological relevance of compartmentalization through LLPS inside cells is still poorly understood and often speculative. Additionally, regulatory mechanisms through which cells control and exploit LLPS are still emerging. On page 1210 of this issue, Garcia Quiroz et al. ( 3 ) show that keratohyalin granules (KGs) that are formed during epidermal differentiation in the skin are pH-sensitive liquid-like protein condensates. Formation of KGs may be physiologically important because mutations that cause defects in this process are associated with the common skin barrier defect ichthyosis vulgaris.

Key Points There are a number of different methods and techniques for genetic perturbation screens. The phenomenon of cell-to-cell variability in mammalian cells has implications for the interpretation of gene function. We now have the ability to quantify, at a large scale, multiple parameters of genetic perturbation effects in thousands of single cells. Functional genetic interactions can be inferred from multivariate quantitative readouts. We present an outlook on the opportunities that the single-cell paradigm will bring to unravel the biological complexity of mammalian cells. Large-scale genetic perturbation screens have been central to many biological discoveries. This Review outlines the recent advances in the quantification of various perturbations across large numbers of single cells simultaneously and describes the use of genetic perturbation screens to infer functional interactions between genes and phenotypes. Large-scale genetic perturbation screens are a classical approach in biology and have been crucial for many discoveries. New technologies can now provide unbiased quantification of multiple molecular and phenotypic changes across tens of thousands of individual cells from large numbers of perturbed cell populations simultaneously. In this Review, we describe how these developments have enabled the discovery of new principles of intracellular and intercellular organization, novel interpretations of genetic perturbation effects and the inference of novel functional genetic interactions. These advances now allow more accurate and comprehensive analyses of gene function in cells using genetic perturbation screens.

Many membraneless organelles are liquid-like domains that form inside the active, viscoelastic environment of living cells through phase separation. To investigate the potential coupling of phase separation with the cytoskeleton, we quantify the structural correlations of membraneless organelles (stress granules) and cytoskeletal filaments (microtubules) in a human-derived epithelial cell line. We find that microtubule networks are substantially denser in the vicinity of stress granules. When microtubules are depolymerized, the sub-units localize near the surface of the stress granules. We interpret these data using a thermodynamic model of partitioning of particles to the surface and bulk of the droplets. In this framework, our data are consistent with a weak (≲ k B T ) affinity of the microtubule sub-units for stress granule interfaces. As microtubules polymerize, their interfacial affinity increases, providing sufficient adhesion to deform droplets and/or the network. Our work suggests that proteins and other objects in the cell have a non-specific affinity for droplet interfaces that increases with the contact area and becomes most apparent when they have no preference for the interior of a droplet over the rest of the cytoplasm. We validate this basic physical phenomenon in vitro through the interaction of a simple protein–RNA condensate with microtubules. Many organelles in the cell are not encapsulated in a membrane—they are liquid-like domains formed through phase separation. The liquid-like nature of such domains leads to adhesive interactions between the cytoskeleton filaments and organelles.