Evaluating single-cell cluster stability using the Jaccard similarity index.

MOTIVATION: One major goal of single-cell RNA sequencing (scRNAseq) experiments is to identify novel cell types. With increasingly large scRNAseq datasets, unsupervised clustering methods can now produce detailed catalogues of transcriptionally distinct groups of cells in a sample. However, the interpretation of these clusters is challenging for both technical and biological reasons. Popular clustering algorithms are sensitive to parameter choices, and can produce different clustering solutions with even small changes in the number of principal components used, the k nearest neighbor and the resolution parameters, among others. RESULTS: Here, we present a set of tools to evaluate cluster stability by subsampling, which can guide parameter choice and aid in biological interpretation. The R package scclusteval and the accompanying Snakemake workflow implement all steps of the pipeline: subsampling the cells, repeating the clustering with Seurat and estimation of cluster stability using the Jaccard similarity index and providing rich visualizations. AVAILABILITYAND IMPLEMENTATION: R package scclusteval: https://github.com/crazyhottommy/scclusteval Snakemake workflow: https://github.com/crazyhottommy/pyflow_seuratv3_parameter Tutorial: https://crazyhottommy.github.io/EvaluateSingleCellClustering/.

Copper Acquisition and Utilization in Fungi.

Fungal cells colonize and proliferate in distinct niches, from soil and plants to diverse tissues in human hosts. Consequently, fungi are challenged with the goal of obtaining nutrients while simultaneously elaborating robust regulatory mechanisms to cope with a range of availability of nutrients, from scarcity to excess. Copper is essential for life but also potentially toxic. In this review we describe the sophisticated homeostatic mechanisms by which fungi acquire, utilize, and control this biochemically versatile trace element. Fungal pathogens, which can occupy distinct host tissues that have their own intrinsic requirements for copper homeostasis, have evolved mechanisms to acquire copper to successfully colonize the host, disseminate to other tissues, and combat host copper bombardment mechanisms that would otherwise mitigate virulence.

Reconstitution of a thermophilic Cu+ importer in vitro reveals intrinsic high-affinity slow transport driving accumulation of an essential metal ion.

Acquisition of the trace element copper (Cu) is critical to drive essential eukaryotic processes such as oxidative phosphorylation, iron mobilization, peptide hormone biogenesis, and connective tissue maturation. The Ctr1/Ctr3 family of Cu importers, first discovered in fungi and conserved in mammals, are critical for Cu+ movement across the plasma membrane or mobilization from endosomal compartments. Whereas ablation of Ctr1 in mammals is embryonic lethal, and Ctr1 is critical for dietary Cu absorption, cardiac function, and systemic iron distribution, little is known about the intrinsic contribution of Ctr1 for Cu+ permeation through membranes or its mechanism of action. Here, we identify three members of a Cu+ importer family from the thermophilic fungus Chaetomium thermophilum: Ctr3a and Ctr3b, which function on the plasma membrane, and Ctr2, which likely functions in endosomal Cu mobilization. All three proteins drive Cu and isoelectronic silver (Ag) uptake in cells devoid of Cu+ importers. Transport activity depends on signature amino acid motifs that are conserved and essential for all Ctr1/3 transporters. Ctr3a is stable and amenable to purification and was incorporated into liposomes to reconstitute an in vitro Ag+ transport assay characterized by stopped-flow spectroscopy. Ctr3a has intrinsic high-affinity metal ion transport activity that closely reflects values determined in vivo, with slow turnover kinetics. Given structural models for mammalian Ctr1, Ctr3a likely functions as a low-efficiency Cu+ ion channel. The Ctr1/Ctr3 family may be tuned to import essential yet potentially toxic Cu+ ions at a slow rate to meet cellular needs, while minimizing labile intracellular Cu+ pools.

Gene duplication and neo-functionalization in the evolutionary and functional divergence of the metazoan copper transporters Ctr1 and Ctr2.

Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu+ across both the plasma membrane and endomembranes. Although mammalian Ctr1 functions as a Cu+ transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr2, has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr1 activity, but its functional and evolutionary relationship to Ctr1 is unclear. Here we report a biochemical, genetic, and phylogenetic comparison of metazoan Ctr1 and Ctr2, suggesting that Ctr2 arose over 550 million years ago as a result of a gene duplication event followed by loss of Cu+ transport activity. Using a random mutagenesis and growth selection approach, we identified amino acid substitutions in human and mouse Ctr2 proteins that support copper-dependent growth in yeast and enhance copper accumulation in Ctr1-/- mouse embryonic fibroblasts. These mutations revert Ctr2 to a more ancestral Ctr1-like state while maintaining endogenous functions, such as stimulating Ctr1 cleavage. We suggest key structural aspects of metazoan Ctr1 and Ctr2 that discriminate between their biological roles, providing mechanistic insights into the evolutionary, biochemical, and functional relationships between these two related proteins.

Sensory Input, Sex, and Function Shape Hypothalamic Cell Type Development.

Mammalian behavior and physiology undergo dramatic changes in early life. Young animals rely on conspecifics to meet their homeostatic needs, until weaning and puberty initiate nutritional independence and sex-specific social interactions, respectively. How neuronal populations regulating homeostatic functions and social behaviors develop and mature during these transitions remains unclear. We used paired transcriptomic and chromatin accessibility profiling to examine the developmental trajectories of neuronal populations in the hypothalamic preoptic region, where cell types with key roles in physiological and behavioral control have been identified1-6. These data reveal a remarkable diversity of developmental trajectories shaped by the sex of the animal, and the location and behavioral or physiological function of the corresponding cell types. We identify key stages of preoptic development, including the perinatal emergence of sex differences, postnatal maturation and subsequent refinement of signaling networks, and nonlinear transcriptional changes accelerating at the time of weaning and puberty. We assessed preoptic development in various sensory mutants and find a major role for vomeronasal sensing in the timing of preoptic cell type maturation. These results provide novel insights into the development of neurons controlling homeostatic functions and social behaviors and lay ground for examining the dynamics of these functions in early life.

A Hypothalamic Circuit Underlying the Dynamic Control of Social Homeostasis.

Social grouping increases survival in many species, including humans1,2. By contrast, social isolation generates an aversive state (loneliness) that motivates social seeking and heightens social interaction upon reunion3-5. The observed rebound in social interaction triggered by isolation suggests a homeostatic process underlying the control of social drive, similar to that observed for physiological needs such as hunger, thirst or sleep3,6. In this study, we assessed social responses in multiple mouse strains and identified the FVB/NJ line as exquisitely sensitive to social isolation. Using FVB/NJ mice, we uncovered two previously uncharacterized neuronal populations in the hypothalamic preoptic nucleus that are activated during social isolation and social rebound and that orchestrate the behavior display of social need and social satiety, respectively. We identified direct connectivity between these two populations of opposite function and with brain areas associated with social behavior, emotional state, reward, and physiological needs, and showed that animals require touch to assess the presence of others and fulfill their social need, thus revealing a brain-wide neural system underlying social homeostasis. These findings offer mechanistic insight into the nature and function of circuits controlling instinctive social need and for the understanding of healthy and diseased brain states associated with social context.

A synthetic biological approach to reconstitution of inositide signaling pathways in bacteria.

Inositide lipid (PIP) and soluble (IP) signaling pathways produce essential cellular codes conserved in eukaryotes. In many cases, deconvoluting metabolic and functional aspects of individual pathways are confounded by promiscuity and multiplicity of PIP and IP kinases and phosphatases. We report a molecular genetic approach that reconstitutes eukaryotic inositide lipid and soluble pathways in a prokaryotic cell which inherently lack inositide kinases and phosphatases in their genome. By expressing synthetic cassettes of eukaryotic genes, we have reconstructed the heterologous formation of a range of inositide lipids, including PI(3)P, PI(4,5)P2 and PIP3. In addition, we report the reconstruction of lipid-dependent production of inositol hexakisphosphate (IP6). Our synthetic system is scalable, reduces confounding metabolic issues, for example it is devoid of inositide phosphatases and orthologous kinases, and enables accurate characterization gene product enzymatic activity and substrate selectivity. This genetically engineered tool is designed to help interpret metabolic pathways and may facilitate in vivo testing of regulators and small molecule inhibitors. In summary, heterologous expression of inositide pathways in bacteria provide a malleable experimental platform for aiding signaling biologists and offers new insights into metabolism of these essential pathways.

X-ray structures of the high-affinity copper transporter Ctr1.

Copper (Cu) is an essential trace element for growth and development and abnormal Cu levels are associated with anemia, metabolic disease and cancer. Evolutionarily conserved from fungi to humans, the high-affinity Cu+ transporter Ctr1 is crucial for both dietary Cu uptake and peripheral distribution, yet the mechanisms for selective permeation of potentially toxic Cu+ ions across cell membranes are unknown. Here we present X-ray crystal structures of Ctr1 from Salmo salar in both Cu+-free and Cu+-bound states, revealing a homo-trimeric Cu+-selective ion channel-like architecture. Two layers of methionine triads form a selectivity filter, coordinating two bound Cu+ ions close to the extracellular entrance. These structures, together with Ctr1 functional characterization, provide a high resolution picture to understand Cu+ import across cellular membranes and suggest therapeutic opportunities for intervention in diseases characterized by inappropriate Cu accumulation.