Identifying gene expression programs underlying both cell-type identity and cellular activities (e.g. life-cycle processes, responses to environmental cues) is crucial for understanding the organization of cells and tissues. Although single-cell RNA-Seq (scRNA-Seq) can quantify transcripts in individual cells, each cell's expression profile may be a mixture of both types of programs, making them difficult to disentangle. Here, we benchmark and enhance the use of matrix factorization to solve this problem. We show with simulations that a method we call consensus non-negative matrix factorization (cNMF) accurately infers identity and activity programs, including their relative contributions in each cell. To illustrate the insights this approach enables, we apply it to published brain organoid and visual cortex scRNA-Seq datasets; cNMF refines cell types and identifies both expected (e.g. cell cycle and hypoxia) and novel activity programs, including programs that may underlie a neurosecretory phenotype and synaptogenesis.

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. We reported previously that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling (Freedman et al., 2015). We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic, biochemical, and quantitative phosphopeptide analyses, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via a phosphorylated tyrosine (Y32) in its unique region. This distinct mode of c-Cbl recognition depresses steady-state expression of LynA in macrophages derived from mice. Mast cells, however, express little c-Cbl and have correspondingly high LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression thus builds cell specificity into the LynA checkpoint.

The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize Bacillus subtilis sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.

Patients with hippocampal amnesia play a central role in memory neuroscience but the neural underpinnings of amnesia are hotly debated. We hypothesized that focal hippocampal damage is associated with changes across the extended hippocampal system and that these, rather than hippocampal atrophy per se, would explain variability in memory between patients. We assessed this hypothesis in a uniquely large cohort of patients (n = 38) after autoimmune limbic encephalitis, a syndrome associated with focal structural hippocampal pathology. These patients showed impaired recall, recognition and maintenance of new information, and remote autobiographical amnesia. Besides hippocampal atrophy, we observed correlatively reduced thalamic and entorhinal cortical volume, resting-state inter-hippocampal connectivity and activity in posteromedial cortex. Associations of hippocampal volume with recall, recognition, and remote memory were fully mediated by wider network abnormalities, and were only direct in forgetting. Network abnormalities may explain the variability across studies of amnesia and speak to debates in memory neuroscience.

RIPK1, RIPK3, ZBP1 and TRIF, the four mammalian proteins harboring RIP homotypic interaction motif (RHIM) domains, are key components of inflammatory signaling and programmed cell death. RHIM-domain protein activation is mediated by their oligomerization; however, mechanisms that promote a return to homeostasis remain unknown. Here we show that autophagy is critical for the turnover of all RHIM-domain proteins. Macrophages lacking the autophagy gene Atg16l1accumulated highly insoluble forms of RIPK1, RIPK3, TRIF and ZBP1. Defective autophagy enhanced necroptosis by Tumor necrosis factor (TNF) and Toll-like receptor (TLR) ligands. TNF-mediated necroptosis was mediated by RIPK1 kinase activity, whereas TLR3- or TLR4-mediated death was dependent on TRIF and RIPK3. Unexpectedly, combined deletion of Atg16l1 and Zbp1 accelerated LPS-mediated necroptosis and sepsis in mice. Thus, ZBP1 drives necroptosis in the absence of the RIPK1-RHIM, but suppresses this process when multiple RHIM-domain containing proteins accumulate. These findings identify autophagy as a central regulator of innate inflammation governed by RHIM-domain proteins.

During CNS development, there is prominent expansion of the anterior region, the brain. In Drosophila, anterior CNS expansion emerges from three rostral features: (1) increased progenitor cell generation, (2) extended progenitor cell proliferation, (3) more proliferative daughters. We find that tailless (mouse Nr2E1/Tlx), otp/Rx/hbn (Otp/Arx/Rax) and Doc1/2/3 (Tbx2/3/6) are important for brain progenitor generation. These genes, and earmuff (FezF1/2), are also important for subsequent progenitor and/or daughter cell proliferation in the brain. Brain TF co-misexpression can drive brain-profile proliferation in the nerve cord, and can reprogram developing wing discs into brain neural progenitors. Brain TF expression is promoted by the PRC2 complex, acting to keep the brain free of anti-proliferative and repressive action of Hox homeotic genes. Hence, anterior expansion of the Drosophila CNS is mediated by brain TF driven 'super-generation' of progenitors, as well as 'hyper-proliferation' of progenitor and daughter cells, promoted by PRC2-mediated repression of Hox activity.

In silico and in vitro studies have made progress in understanding protein-protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of LAT, Grb2, Sos1, SLP-76, Nck, and WASP. These condensates move radially at the IS, traversing successive radially-oriented and concentric actin networks. To understand this movement, we biochemically reconstituted LAT condensates with actomyosin filaments. We found that basic regions of Nck and N-WASP/WASP promote association and co-movement of LAT condensates with actin, indicating conversion of weak individual affinities to high collective affinity upon phase separation. Condensates lacking these components were propelled differently, without strong actin adhesion. In cells, LAT condensates lost Nck as radial actin transitioned to the concentric network, and engineered condensates constitutively binding actin moved aberrantly. Our data show that Nck and WASP form a clutch between LAT condensates and actin in vitro and suggest that compositional changes may enable condensate movement by distinct actin networks in different regions of the IS.

Lower vertebrate and neonatal mammalian hearts exhibit the remarkable capacity to regenerate through the reprogramming of pre-existing cardiomyocytes. However, how cardiac injury initiates signaling pathways controlling this regenerative reprogramming remains to be defined. Here, we utilize in vivo biophysical and genetic fate mapping zebrafish studies to reveal that altered hemodynamic forces due to cardiac injury activate a sequential endocardial-myocardial signaling cascade to direct cardiomyocyte reprogramming and heart regeneration. Specifically, these altered forces are sensed by the endocardium through the mechanosensitive channel Trpv4 to control Klf2a transcription factor expression. Consequently, Klf2a then activates endocardial Notch signaling which results in the non-cell autonomous initiation of myocardial Erbb2 and BMP signaling to promote cardiomyocyte reprogramming and heart regeneration. Overall, these findings not only reveal how the heart senses and adaptively responds to environmental changes due to cardiac injury, but also provide insight into how flow-mediated mechanisms may regulate cardiomyocyte reprogramming and heart regeneration.

The number of ions required to drive substrate transport through a secondary active transporter determines the protein's ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter's mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na+-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures.

The ascidian embryo is an ideal system to investigate how cell position is determined during embryogenesis. Using 3D timelapse imaging and computational methods we analyzed the planar cell divisions in ascidian early embryos and found that spindles in every cell tend to align at metaphase in the long length of the apical surface except in cells undergoing unequal cleavage. Furthermore, the invariant and conserved cleavage pattern of ascidian embryos was found to consist in alternate planar cell divisions between ectoderm and endomesoderm. In order to test the importance of alternate cell divisions we manipulated zygotic transcription induced by β-catenin or downregulated wee1 activity, both of which abolish this cell cycle asynchrony. Crucially, abolishing cell cycle asynchrony consistently disrupted the spindle orienting mechanism underpinning the invariant cleavage pattern. Our results demonstrate how an evolutionary conserved cell cycle asynchrony maintains the invariant cleavage pattern driving morphogenesis of the ascidian blastula.

Biological systems are increasingly being studied by high throughput profiling of molecular data over time. Determining the set of time points to sample in studies that profile several different types of molecular data is still challenging. Here we present the Time Point Selection (TPS) method that solves this combinatorial problem in a principled and practical way. TPS utilizes expression data from a small set of genes sampled at a high rate. As we show by applying TPS to study mouse lung development, the points selected by TPS can be used to reconstruct an accurate representation for the expression values of the non selected points. Further, even though the selection is only based on gene expression, these points are also appropriate for representing a much larger set of protein, miRNA and DNA methylation changes over time. TPS can thus serve as a key design strategy for high throughput time series experiments. Supporting Website: www.sb.cs.cmu.edu/TPS.

Protein polarization underlies differentiation in metazoans and in bacteria. How symmetric polarization can instate functional asymmetry remains elusive. Here, we show by super-resolution photo-activated localization microscopy and edgetic mutations that the bitopic zinc-finger protein ZitP implements specialized developmental functions - pilus biogenesis and multifactorial swarming motility - while shaping distinct nanoscale (bi)polar architectures in the asymmetric model bacterium Caulobacter crescentus. Polar assemblage and accumulation of ZitP and its effector protein CpaM are orchestrated in time and space by conserved components of the cell cycle circuitry that coordinate polar morphogenesis with cell cycle progression, and also act on the master cell cycle regulator CtrA. Thus, this novel class of potentially widespread multifunctional polarity regulators is deeply embedded in the cell cycle circuitry.

Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm.

The cell membrane is a heterogeneously organized composite with lipid-protein micro-domains. The contractile actin cortex may govern the lateral organization of these domains in the cell membrane, yet the underlying mechanisms are not known. We recently reconstituted minimal actin cortices (MACs) (Vogel et al., 2013b) and here advanced our assay to investigate effects of rearranging actin filaments on the lateral membrane organization by introducing various phase-separated lipid mono- and bilayers to the MACs. The addition of actin filaments reorganized membrane domains. We found that the process reached a steady state where line tension and lateral crowding balanced. Moreover, the phase boundary allowed myosin driven actin filament rearrangements to actively move individual lipid domains, often accompanied by their shape change, fusion or splitting. Our findings illustrate how actin cortex remodeling in cells may control dynamic rearrangements of lipids and other molecules inside domains without directly binding to actin filaments.

Animals have sophisticated homeostatic controls. While mammalian body temperature fluctuates throughout the day, small ectotherms, such as Drosophila achieve a body temperature rhythm (BTR) through their preference of environmental temperature. Here, we demonstrate that pigment dispersing factor (PDF) neurons play an important role in setting preferred temperature before dawn. We show that small lateral ventral neurons (sLNvs), a subset of PDF neurons, activate the dorsal neurons 2 (DN2s), the main circadian clock cells that regulate temperature preference rhythm (TPR). The number of temporal contacts between sLNvs and DN2s peak before dawn. Our data suggest that the thermosensory anterior cells (ACs) likely contact sLNvs via serotonin signaling. Together, the ACs-sLNs-DN2s neural circuit regulates the proper setting of temperature preference before dawn. Given that sLNvs are important for sleep and that BTR and sleep have a close temporal relationship, our data highlight a possible neuronal interaction between body temperature and sleep regulation.

The non-canonical Wnt/Ca2+ signaling pathway plays important roles in embryonic development, tissue formation and diseases. However, it is unclear how the Wnt ligand-stimulated, G protein-coupled receptor Frizzled activates phospholipases for calcium release. Here, we report that the zebrafish/human phosphatidylinositol transfer protein Sec14l3/SEC14L2 act as GTPase proteins to transduce Wnt signals from Frizzled to phospholipase C (PLC). Depletion of sec14l3 attenuates Wnt/Ca2+ responsive activity and causes convergent and extension (CE) defects in zebrafish embryos. Biochemical analyses in mammalian cells indicate that Sec14l3-GDP forms complex with Frizzled and Dishevelled; Wnt ligand binding of Frizzled induces translocation of Sec14l3 to the plasma membrane; and then Sec14l3-GTP binds to and activates phospholipase Cδ4a (Plcδ4a); subsequently, Plcδ4a initiates phosphatidylinositol-4,5-bisphosphate (PIP2) signaling, ultimately stimulating calcium release. Furthermore, Plcδ4a can act as a GTPase-activating protein to accelerate the hydrolysis of Sec14l3-bound GTP to GDP. Our data provide a new insight into GTPase protein-coupled Wnt/Ca2+ signaling transduction.

Spinal Muscular Atrophy (SMA) is caused by diminished Survival of Motor Neuron (SMN) protein, leading to neuromuscular junction (NMJ) dysfunction and spinal motor neuron (MN) loss. Here, we report that reduced SMN function impacts the action of a pertinent microRNA and its mRNA target in MNs. Loss of the C. elegans SMN ortholog, SMN-1, causes NMJ defects. We found that increased levels of the C. elegans Gemin3 ortholog, MEL-46, ameliorates these defects. Increased MEL-46 levels also restored perturbed microRNA (miR-2) function in smn-1(lf) animals. We determined that miR-2 regulates expression of the C. elegans M2 muscarinic receptor (m2R) ortholog, GAR-2. GAR-2 loss ameliorated smn-1(lf) and mel-46(lf) synaptic defects. In an SMA mouse model, m2R levels were increased and pharmacological inhibition of m2R rescued MN process defects. Collectively, these results suggest decreased SMN leads to defective microRNA function via MEL-46 misregulation, followed by increased m2R expression, and neuronal dysfunction in SMA.

The transcription factor TCF7L1 is an embryonic stem cell signature gene that is upregulated in multiple aggressive cancer types, but its role in skin tumorigenesis has not yet been defined. Here we document TCF7L1 upregulation in skin squamous cell carcinoma (SCC) and demonstrate that TCF7L1 overexpression increases tumor incidence, tumor multiplicity, and malignant progression in the chemically induced mouse model of skin SCC. Additionally, we show that downregulation of TCF7L1 and its paralogue TCF7L2 reduces tumor growth in a xenograft model of human skin SCC. Using separation-of-function mutants, we show that TCF7L1 promotes tumor growth, enhances cell migration, and overrides oncogenic RAS-induced senescence independently of its interaction with β-catenin. Through transcriptome profiling and combined gain- and loss-of-function studies, we identified LCN2 as a major downstream effector of TCF7L1 that drives tumor growth. Our findings establish a tumor-promoting role for TCF7L1 in skin and elucidate the mechanisms underlying its tumorigenic capacity.

We present an approach to study macromolecular assemblies by detecting component proteins' characteristic high-resolution projection patterns, calculated from their known 3D structures, in single electron cryo-micrographs. Our method detects single apoferritin molecules in vitreous ice with high specificity and determines their orientation and location precisely. Simulations show that high spatial-frequency information and-in the presence of protein background-a whitening filter are essential for optimal detection, in particular for images taken far from focus. Experimentally, we could detect small viral RNA polymerase molecules, distributed randomly among binding locations, inside rotavirus particles. Based on the currently attainable image quality, we estimate a threshold for detection that is 150 kDa in ice and 300 kDa in 100 nm thick samples of dense biological material.