Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type–specific RNA splicing was discovered and analyzed across tissues within an individual.

SUMMARYChanges in neuronal activity create local and transient changes in energy demands at synapses. Here we discover a metabolic compartment that forms in vivo near synapses to meet local energy demands and support synaptic function in Caenorhabditis elegans neurons. Under conditions of energy stress, glycolytic enzymes redistribute from a diffuse localization in the cytoplasm to a punctate localization adjacent to synapses. Glycolytic enzymes colocalize, suggesting the ad hoc formation of a glycolysis compartment, or a 'glycolytic metabolon', that can maintain local levels of ATP. Local formation of the glycolytic metabolon is dependent on presynaptic scaffolding proteins, and disruption of the glycolytic metabolon blocks the synaptic vesicle cycle, impairs synaptic recovery, and affects locomotion. Our studies indicate that under energy stress conditions, energy demands in C. elegans synapses are met locally through the assembly of a glycolytic metabolon to sustain synaptic function and behavior. †

ObjectivesRecent observations in systemic Juvenile Idiopathic Arthritis (sJIA) suggest an increasing incidence of high-mortality interstitial lung disease (ILD), characterized pathologically by a variant of pulmonary alveolar proteinosis (PAP). The co-occurrence of macrophage activation syndrome (MAS) and PAP in sJIA suggested a shared pathology, but features of drug reaction such as anaphylaxis, rashes, and eosinophilia are also common in these patients. We sought to investigate immunopathology and identify biomarkers of this lung disease.MethodsWe used SOMAscan to measure >1300 analytes in serum samples from healthy controls and patients with sJIA-PAP, sJIA, sJIA-MAS, and other associated diseases, and verified selected findings by ELISA and lung immunostaining. Because a sample’s proteome may reflect multiple states (SJIA, MAS, SJIA-PAP), we used linear regression modeling to identify subsets of altered proteins associated with each state.ResultsMarkers identified for sJIA, including SAA, S100A9, and S100A12, were consistent with previous reports. Proteome alterations in sJIA and MAS overlapped substantially, including new findings of heat shock proteins and glycolytic enzymes. sJIA, MAS, and sJIA-PAP shared elevation of IL-18. Importantly, we identified a unique sJIA-PAP signature whose expression was independent of sJIA-MAS activity. Key proteins were ICAM5 and MMP7, previously observed markers of fibrotic ILD. Immunohistochemistry showed expression of these proteins in sJIA-PAP lung, supporting a pulmonary source. The eosinophil chemoattractant CCL11 was elevated in sJIA-PAP, but not sJIA/MAS or other PAP.ConclusionsWe found novel circulating proteins associated specifically with sJIA, sJIA/MAS and sJIA-PAP. Select biomarkers, such as ICAM5, could aid in early detection and management of sJIA-PAP.Key MessagesPulmonary Alveolar Proteinosis (PAP) occurring in the setting of Systemic Juvenile Idiopathic Arthritis (sJIA) is an increasingly-noted, dangerous condition that has been associated with Macrophage Activation Syndrome (MAS).We evaluated >1300 serum proteins by aptamer array, verifying and localizing key proteins, and identified novel pathways associated with MAS (HSPs, glycolysis), candidate pathways/proteins associated with PAP in sJIA (e.g., ICAM5, MMP7, and type 2 chemokines), and divergence of the sJIA/MAS and sJIA-PAP proteomes.This analysis supports the evaluation of novel pathways in MAS, the validation of screening/monitoring biomarkers in sJIA-PAP, and the management of PAP as a disease process enmeshed with, but distinct from, sJIA and MAS.

While much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. Here we built a hybrid microfluidic-hydrogel device for use in Caenorhabditis elegans to systematically examine and quantify the dynamic subcellular localization of the rate-limiting enzyme of glycolysis, phosphofructokinase-1/PFK-1.1. We determine that endogenous PFK-1.1 localizes to distinct, tissue-specific subcellular compartments in vivo. In neurons, PFK-1.1 is diffusely localized in the cytosol, but capable of dynamically forming phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in the dispersion of PFK-1.1 in the cytosol, indicating that PFK-1.1 reversibly organizes into biomolecular condensates in response to cues within the cellular environment. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Prolonged conditions of energy stress during sustained hypoxia alter the biophysical properties of PFK-1.1 in vivo, affecting its viscosity and mobility within phase-separated condensates. PFK-1.1's ability to form tetramers is critical for its capacity to form condensates in vivo, and heterologous self-association domain such as cryptochrome 2 (CRY2) is sufficient to constitutively induce the formation of PFK-1.1 condensates. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically compartmentalize in vivo to specific subcellular compartments in response to acute energy stress via multivalency as phase-separated condensates.

More than 95% of human genes are alternatively spliced. Yet, the extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach that is agnostic to transcript annotation, to detect cell-type-specific regulated splicing in > 110K carefully annotated single cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type specifically spliced. These results are validated with RNA FISH, single cell PCR, and in high throughput with Smart-seq2. Regulated splicing is found in ubiquitously expressed genes such as actin light chain subunit MYL6 and ribosomal protein RPS24, which has an epithelial-specific microexon. 13% of the statistically most variable splice sites in cell-type specifically regulated genes are also most variable in mouse lemur or mouse. SpliZ analysis further reveals 170 genes with regulated splicing during sperm development using, 10 of which are conserved in mouse and mouse lemur. The statistical properties of the SpliZ allow model-based identification of subpopulations within otherwise indistinguishable cells based on gene expression, illustrated by subpopulations of classical monocytes with stereotyped splicing, including an un-annotated exon, in SAT1, a Diamine acetyltransferase. Together, this unsupervised and annotation-free analysis of differential splicing in ultra high throughput droplet-based sequencing of human cells across multiple organs establishes splicing is regulated cell-type-specifically independent of gene expression.

The rapid emergence of large-scale atlas-level single-cell RNA-seq datasets presents remarkable opportunities for broad and deep biological investigations through integrative analyses. However, harmonizing such datasets requires integration approaches to be not only computationally scalable, but also capable of preserving a wide range of fine-grained cell populations. We created Portal, a unified framework of adversarial domain translation to learn harmonized representations of datasets. With innovation in model and algorithm designs, Portal achieves superior performance in preserving biological variation during integration, while achieving integration of millions of cells in minutes with low memory consumption. We show that Portal

Early stages of deadly respiratory diseases such as COVID-19 have been challenging to elucidate due to lack of an experimental system that recapitulates the cellular and structural complexity of the human lung, while allowing precise control over disease initiation and systematic interrogation of molecular events at cellular resolution. Here we show healthy human lung slices cultured ex vivo can be productively infected with SARS-CoV-2, and the cellular tropism of the virus and its distinct and dynamic effects on host cell gene expression can be determined by single cell RNA sequencing and reconstruction of “infection pseudotime” for individual lung cell types. This revealed the prominent SARS-CoV-2 target is a population of activated interstitial macrophages, which as infection proceeds accumulate thousands of viral RNA molecules per cell, comprising up to 60% of the cellular transcriptome and including canonical and novel subgenomic RNAs. During viral takeover, there is cell-autonomous induction of a specific host interferon program and seven chemokines (CCL2, 7, 8, 13, CXCL10) and cytokines (IL6, IL10), distinct from the response of alveolar macrophages in which neither viral takeover nor induction of a substantial inflammatory response occurs. Using a recombinant SARS-CoV-2 Spike-pseudotyped lentivirus, we show that entry into purified human lung macrophages depends on Spike but is not blocked by cytochalasin D or by an ACE2-competing monoclonal antibody, indicating a phagocytosis- and ACE2-independent route of entry. These results provide a molecular characterization of the initiation of COVID-19 in human lung tissue, identify activated interstitial macrophages as a prominent site of viral takeover and focus of inflammation, and suggest targeting of these macrophages and their signals as a new therapeutic modality for COVID-19 pneumonia and progression to ARDS. Our approach can be generalized to define the initiation program and evaluate therapeutics for any human lung infection at cellular resolution.

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