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During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Intracellular communications between breast cancer and fibroblast cells were reported to be involved in cancer proliferation, growth, and therapy resistance. The hallmarks of cancer-fibroblast interactions, consisting of caveolin 1 (Cav1) and mono-carboxylate transporter 4 (MCT4) (metabolic coupling markers), along with IL-6, TGFβ, and lactate secretion, are considered robust biomarkers predicting recurrence and metastasis. In order to promote a novel phenotype in normal fibroblasts, we predicted that breast cancer cells could be able to cause loss of Cav1 and increase of MCT4, as well as elevate IL-6 and TGFβ in nearby normal fibroblasts. We created a co-culture model using breast cancer (4T1) and normal fibroblast (NIH3T3) cell lines cultured under specific experimental conditions in order to directly test our theory. Moreover, we show that long-term co-culture of breast cancer cells and normal fibroblasts promotes loss of Cav1 and gain of MCT4 in adjacent fibroblasts and increase lactate secretion. These results were validated using the monoculture of each group separately as a control. In this system, we show that metformin inhibits IL-6 and TGFβ secretion and re-expresses Cav1 in both cells. However, MCT4 and lactate stayed high after treatment with metformin. In conclusion, our work shows that co-culture with breast cancer cells may cause significant alterations in the phenotype and secretion of normal fibroblasts. Metformin, however, may change this state and affect fibroblasts' acquired phenotypes. Moreover, mitochondrial inhibition by metformin after 8 days of treatment, significantly hinders tumor growth in mouse model of breast cancer. [ABSTRACT FROM AUTHOR]

The serine/threonine protein kinase Akt integrates diverse upstream inputs to regulate cell survival, growth, metabolism, migration, and differentiation. Mounting evidence suggests that Akt activity is differentially regulated depending on its subcellular location, which can include the plasma membrane, endomembrane, and nuclear compartment. This spatial control of Akt activity is critical for achieving signaling specificity and proper physiological functions, and deregulation of compartment-specific Akt signaling is implicated in various diseases, including cancer and diabetes. Understanding the spatial coordination of the signaling network centered around this key kinase and the underlying regulatory mechanisms requires precise tracking of Akt activity at distinct subcellular compartments within its native biological contexts. To address this challenge, new molecular tools are being developed, enabling us to directly interrogate the spatiotemporal regulation of Akt in living cells. These include, for instance, the newly developed genetically encodable fluorescent-protein-based Akt kinase activity reporter (AktAR2), which serves as a substrate surrogate of Akt kinase and translates Akt-specific phosphorylation into a quantifiable change in Förster resonance energy transfer (FRET). In addition, we developed the Akt substrate tandem occupancy peptide sponge (Akt-STOPS), which allows biochemical perturbation of subcellular Akt activity. Both molecular tools can be readily targeted to distinct subcellular localizations. Here, we describe a workflow to study Akt kinase activity at different subcellular locations in living cells. We provide a protocol for using genetically targeted AktAR2 and Akt-STOPS, along with fluorescence imaging in living NIH3T3 cells, to visualize and perturb, respectively, the activity of endogenous Akt kinase at different subcellular compartments. We further describe a protocol for using chemically inducible dimerization (CID) to control the plasma membrane-specific inhibition of Akt activity in real time. Lastly, we describe a protocol for maintaining NIH3T3 cells in culture, a cell line known to exhibit robust Akt activity. In all, this approach enables interrogation of spatiotemporal regulation and functions of Akt, as well as the intricate signaling networks in which it is embedded, at specific subcellular locations. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualizing and perturbing subcellular Akt kinase activity using AktAR and Akt-STOPS Basic Protocol 2: Using chemically inducible dimerization (CID) to control inhibition of Akt at the plasma membrane Support Protocol: Maintaining NIH3T3 cells in culture.

The salivary glands are a crucial site of replication for human cytomegalovirus (HCMV) and its murine counterpart, murine cytomegalovirus (MCMV). Studies of MCMV often involve the use of BALB/c strain mice, but most in vitro assays are carried out in the NIH 3T3 cell line, which is derived from Swiss Albino mice. This report describes a BALB/c-derived mouse salivary gland cell line immortalized using the SV40 large T antigen. Cells stained positive for PDGFR1 and negative for E-cadherin and PECAM-1, indicating mesenchymal origin. This cell line, which has been named murine salivary gland mesenchymal (mSGM), shows promise as a tool for ex vivo immunological assays due to its MHC haplotype match with the BALB/c mouse strain. In addition, plaque assays using mSGM rather than NIH 3T3 cells are significantly more sensitive for detecting low concentrations of MCMV particles. Finally, it is demonstrated that mSGM cells express all 3 BALB/c MHC class I isotypes and are susceptible to T cell-mediated ex vivo cytotoxicity assays, leading to many possible uses in immunological studies of MCMV.

The overuse of antibiotics in clinical and livestock settings is accelerating the selection of multidrug resistant bacterial pathogens. Antibiotic resistant bacteria result in increased mortality and financial strain on the health care and livestock industry. The development of new antibiotics has stalled, and novel strategies are needed as we enter the age of antibiotic resistance. Certain naturally occurring clays have been shown to have antimicrobial properties and kill antibiotic resistant bacteria. Harnessing the activity of compounds within these clays that harbor antibiotic properties offers new therapeutic opportunities for fighting the potentially devastating effects of the post antibiotic era. However, natural samples are highly heterogenous and exhibit variable antibacterial effectiveness, therefore synthesizing minerals of high purity with reproducible antibacterial activity is needed. Here we describe for the first time synthetic smectite clay minerals and Fe-sulfide microspheres that reproduce the geochemical antibacterial properties observed in natural occurring clays. We show that these mineral formulations are effective at killing the ESKAPE pathogens (Enterococcus sp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter sp., Pseudomonas aeruginosa and Enterobacter sp.) by maintaining Fe2+ solubility and reactive oxygen species (ROS) production while buffering solution pH, unlike the application of metals alone. Our results represent the first step in utilizing a geochemical process to treat antibiotic resistant topical or gastrointestinal infections in the age of antibiotic resistance.

Most eukaryotic genes express alternative polyadenylation (APA) isoforms. A growing number of RNA sequencing methods, especially those used for single-cell transcriptome analysis, generate reads close to the polyadenylation site (PAS), termed nearSite reads, hence inherently containing information about APA isoform abundance. Here, we present a probabilistic model-based method named MAAPER to utilize nearSite reads for APA analysis. MAAPER predicts PASs with high accuracy and sensitivity and examines different types of APA events with robust statistics. We show MAAPER's performance with both bulk and single-cell data and its applicability in unpaired or paired experimental designs.

During oropharyngeal candidiasis, Candida albicans activates the epidermal growth factor receptor (EGFR), which induces oral epithelial cells to endocytose the fungus and synthesize proinflammatory mediators. To elucidate EGFR signaling pathways that are stimulated by C. albicans, we used proteomics to identify 1,214 proteins that were associated with EGFR in C. albicans-infected cells. Seven of these proteins were selected for additional study. Among these proteins, WW domain-binding protein 2, Toll-interacting protein, interferon-induced transmembrane protein 3 (IFITM3), and the globular C1q receptor (gC1qR) were found to associate with EGFR in viable oral epithelial cells. Each of these proteins was required for maximal endocytosis of C. albicans, and all regulated fungus-induced production of interleukin-1β (IL-1β) and/or IL-8, either positively or negatively. gC1qR was found to function as a key coreceptor with EGFR. Interacting with the C. albicans Als3 invasin, gC1qR was required for the fungus to induce autophosphorylation of both EGFR and the ephrin type A receptor 2. The combination of gC1qR and EGFR was necessary for maximal endocytosis of C. albicans and secretion of IL-1β, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by human oral epithelial cells. In mouse oral epithelial cells, inhibition of gC1qR failed to block C. albicans-induced phosphorylation, and knockdown of IFITM3 did not inhibit C. albicans endocytosis, indicating that gC1qR and IFITM3 function differently in mouse versus human oral epithelial cells. Thus, this work provides an atlas of proteins that associate with EGFR and identifies several that play a central role in the response of human oral epithelial cells to C. albicans infection. IMPORTANCE Oral epithelial cells play a key role in the pathogenesis of oropharyngeal candidiasis. In addition to being target host cells for C. albicans adherence and invasion, they secrete proinflammatory cytokines and chemokines that recruit T cells and activated phagocytes to foci of infection. It is known that C. albicans activates EGFR on oral epithelial cells, which induces these cells to endocytose the organism and stimulates them to secrete proinflammatory mediators. To elucidate the EGFR signaling pathways that govern these responses, we analyzed the epithelial cell proteins that associate with EGFR in C. albicans-infected epithelial cells. We identified four proteins that physically associate with EGFR and that regulate different aspects of the epithelial response to C. albicans. One of these is gC1qR, which is required for C. albicans to activate EGFR, induce endocytosis, and stimulate the secretion of proinflammatory mediators, indicating that gC1qR functions as a key coreceptor with EGFR.

The homeodomain transcription factors sine oculis homeobox 3 (Six3) and ventral anterior homeobox 1 (Vax1) are required for brain development. Their expression in specific brain areas is maintained in adulthood, where their functions are poorly understood. To identify the roles of Six3 and Vax1 in neurons, we conditionally deleted each gene using Synapsincre , a promoter targeting maturing neurons, and generated Six3syn and Vax1syn mice. Six3syn and Vax1syn females, but not males, had reduced fertility, due to impairment of the luteinizing hormone (LH) surge driving ovulation. In nocturnal rodents, the LH surge requires a precise timing signal from the brain's circadian pacemaker, the suprachiasmatic nucleus (SCN), near the time of activity onset. Indeed, both Six3syn and Vax1syn females had impaired rhythmic SCN output, which was associated with weakened Period 2 molecular clock function in both Six3syn and Vax1syn mice. These impairments were associated with a reduction of the SCN neuropeptide vasoactive intestinal peptide in Vax1syn mice and a modest weakening of SCN timekeeping function in both Six3syn and Vax1syn mice. Changes in SCN function were associated with mistimed peak PER2::LUC expression in the SCN and pituitary in both Six3syn and Vax1syn females. Interestingly, Six3syn ovaries presented reduced sensitivity to LH, causing reduced ovulation during superovulation. In conclusion, we have identified novel roles of the homeodomain transcription factors SIX3 and VAX1 in neurons, where they are required for proper molecular circadian clock function, SCN rhythmic output, and female fertility.

Mutations in the hedgehog (Hh) signaling are implicated in birth defects and cancers, including medulloblastoma (MB), one of the most malignant pediatric brain tumors. Current Hh inhibitors face the challenge of drug resistance and tumor relapse, urging new insights in the Hh pathway regulation. Our previous study revealed how PDE4D controls global levels of cAMP in the cytoplasm to positively regulate Hh signaling; in the present study, we found that a specific isoform PDE4D3 is tethered to the centrosome by Myomegalin (Mmg), a centrosome/Golgi-associated protein. Mmg loss dislocates PDE4D3 from the centrosome, leading to local PKA overactivation and inhibition of the Hh signaling, leaving other PKA-related pathways unaffected. Mmg loss suppresses the proliferation of granule neuron precursors and blocks the growth of MB in mouse model. Our findings specify a new regulatory mechanism of the Hh pathway and highlight an exciting therapeutic avenue for Hh-related cancers with reduced side effects.

Isocitrate dehydrogenase 1 (IDH1) is a key metabolic enzyme for maintaining cytosolic levels of α-ketoglutarate (AKG) and preserving the redox environment of the cytosol. Wild-type (WT) IDH1 converts isocitrate to AKG; however, mutant IDH1-R132H that is recurrent in human cancers catalyzes the neomorphic production of the oncometabolite d-2-hydroxyglutrate (D-2HG) from AKG. Recent work suggests that production of l-2-hydroxyglutarte in cancer cells can be regulated by environmental changes, including hypoxia and intracellular pH (pHi). However, it is unknown whether and how pHi affects the activity of IDH1-R132H. Here, we show that in cells IDH1-R132H can produce D-2HG in a pH-dependent manner with increased production at lower pHi. We also identify a molecular mechanism by which this pH sensitivity is achieved. We show that pH-dependent production of D-2HG is mediated by pH-dependent heterodimer formation between IDH1-WT and IDH1-R132H. In contrast, neither IDH1-WT nor IDH1-R132H homodimer formation is affected by pH. Our results demonstrate that robust production of D-2HG by IDH1-R132H relies on the coincidence of (1) the ability to form heterodimers with IDH1-WT and (2) low pHi or highly abundant AKG substrate. These data suggest cancer-associated IDH1-R132H may be sensitive to physiological or microenvironmental cues that lower pH, such as hypoxia or metabolic reprogramming. This work reveals new molecular considerations for targeted therapeutics and suggests potential synergistic effects of using catalytic IDH1 inhibitors targeting D-2HG production in combination with drugs targeting the tumor microenvironment.

Rapid death of infected cells is an important antiviral strategy. However, fast decisions that are based on limited evidence can be erroneous and cause unnecessary cell death and subsequent tissue damage. How cells optimize their death decision making strategy to maximize both speed and accuracy is unclear. Here, we show that exposure to TNF, which is secreted by macrophages during viral infection, causes cells to change their decision strategy from "slow and accurate" to "fast and error-prone". Mathematical modeling combined with experiments in cell culture and whole organ culture show that the regulation of the cell death decision strategy is critical to prevent HSV-1 spread. These findings demonstrate that immune regulation of cellular cognitive processes dynamically changes a tissues' tolerance for self-damage, which is required to protect against viral spread.

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

Any chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In biological bilayers, heterogeneity is considered in the context of small (10-200 nm) sterol and sphingolipid-enriched heterogeneous and highly dynamic domains. Several techniques can be used to assess membrane heterogeneity in living systems. Our approach is to use a fluorescent reporter molecule immersed in the bilayer, which, by changes in its spectroscopic properties, senses physical-chemistry aspects of the membrane. This dye in combination with microscopy and fluctuation techniques can give information about membrane heterogeneity at different temporal and spatial levels: going from average fluidity to number and diffusion coefficient of nanodomains. LAURDAN (6-dodecanoyl-2-(dimethylamino) naphthalene), is a fluorescent probe designed and synthesized in 1979 by Gregorio Weber with the purpose to study the phenomenon of dipolar relaxation. The spectral displacement observed when LAURDAN is either in fluid or gel phase permitted the use of the technique in the field of membrane dynamics. The quantitation of the spectral displacement was first addressed by the generalized polarization (GP) function in the cuvette, a ratio of the difference in intensity at two wavelengths divided by their sum. In 1997, GP measurements were done for the first time in the microscope, adding to the technique the spatial resolution and allowing the visualization of lipid segregation both in liposomes and cells. A new prospective to the membrane heterogeneity was obtained when LAURDAN fluorescent lifetime measurements were done in the microscope. Two channel lifetime imaging provides information on membrane polarity and dipole relaxation (the two parameters responsible for the spectral shift of LAURDAN), and the application of phasor analysis allows pixel by pixel understanding of these two parameters in the membrane. To increase temporal resolution, LAURDAN GP was combined with fluctuation correlation spectroscopy (FCS) and the motility of nanometric highly packed structures in biological membranes was registered. Lately the application of phasor analysis to spectral images from membranes labeled with LAURDAN allows us to study the full spectra pixel by pixel in an image. All these methodologies, using LAURDAN, offer the possibility to address different properties of membranes depending on the question being asked. In this Account, we will focus on the principles, advantages, and limitations of different approaches to orient the reader to select the most appropriate technique for their research.

Acute myeloid leukemia (AML) is a high remission, high relapse fatal blood cancer. Although mTORC1 is a master regulator of cell proliferation and survival, its inhibitors have not performed well as AML treatments. To uncover the dynamics of mTORC1 activity in vivo, fluorescent probes are developed to track single cell proliferation, apoptosis and mTORC1 activity of AML cells in the bone marrow of live animals and to quantify these activities in the context of microanatomical localization and intra-tumoral heterogeneity. When chemotherapy drugs commonly used clinically are given to mice with AML, apoptosis is rapid, diffuse and not preferentially restricted to anatomic sites. Dynamic measurement of mTORC1 activity indicated a decline in mTORC1 activity with AML progression. However, at the time of maximal chemotherapy response, mTORC1 signaling is high and positively correlated with a leukemia stemness transcriptional profile. Cell barcoding reveals the induction of mTORC1 activity rather than selection of mTORC1 high cells and timed inhibition of mTORC1 improved the killing of AML cells. These data define the real-time dynamics of AML and the mTORC1 pathway in association with AML growth, response to and relapse after chemotherapy. They provide guidance for timed intervention with pathway-specific inhibitors.

Vertebrate Hedgehog signals are transduced through the primary cilium, a specialized lipid microdomain that is required for Smoothened activation. Cilia-associated sterol and oxysterol lipids bind to Smoothened to activate the Hedgehog pathway, but how ciliary lipids are regulated is incompletely understood. Here we identified DHCR7, an enzyme that produces cholesterol, activates the Hedgehog pathway, and localizes near the ciliary base. We found that Hedgehog stimulation negatively regulates DHCR7 activity and removes DHCR7 from the ciliary microenvironment, suggesting that DHCR7 primes cilia for Hedgehog pathway activation. In contrast, we found that Hedgehog stimulation positively regulates the oxysterol synthase CYP7A1, which accumulates near the ciliary base and produces oxysterols that promote Hedgehog signaling in response to pathway activation. Our results reveal that enzymes involved in lipid biosynthesis in the ciliary microenvironment promote Hedgehog signaling, shedding light on how ciliary lipids are established and regulated to transduce Hedgehog signals.

The retinoblastoma (RB) tumor suppressor is functionally inactivated in a wide range of human tumors where this inactivation promotes tumorigenesis in part by allowing uncontrolled proliferation. RB has been extensively studied, but its mechanisms of action in normal and cancer cells remain only partly understood. Here, we describe a new mouse model to investigate the consequences of RB depletion and its re-activation in vivo. In these mice, induction of shRNA molecules targeting RB for knock-down results in the development of phenotypes similar to Rb knock-out mice, including the development of pituitary and thyroid tumors. Re-expression of RB leads to cell cycle arrest in cancer cells and repression of transcriptional programs driven by E2F activity. Thus, continuous RB loss is required for the maintenance of tumor phenotypes initiated by loss of RB, and this new mouse model will provide a new platform to investigate RB function in vivo.

Macrophages are important immune cells that participate in both innate and adaptive immune responses, such as phagocytosis, recognition of molecular patterns, and activation of the immune response. In this study, murine peritoneal macrophages were isolated and then activated by LPS, HSV and VSV. Integrative proteomic and precision N-glycoproteomic profiling were conducted to assess the underlying macrophage activation. We identified a total of 587 glycoproteins, including 1239 glycopeptides, 526 monosaccharide components, and 8326 intact glycopeptides in glycoproteomics, as well as a total of 4496 proteins identified in proteomic analysis. These glycoproteins are widely involved in important biological processes, such as antigen presentation, cytokine production and glycosylation progression. Under the stimulation of the different pathogens, glycoproteins showed a dramatic change. We found that receptors in the Toll-like receptor pathway, such as Tlr2 and CD14, were increased under LPS and HSV stimulation. Glycosylation of those proteins was proven to influence their subcellular locations.

Hyperspectral imaging is highly sought after in many fields including mineralogy and geology, environment and agriculture, astronomy and, importantly, biomedical imaging and biological fluorescence. We developed ultrafast phasor-based hyperspectral snapshot microscopy based on sine/cosine interference filters for biomedical imaging not feasible with conventional hyperspectral detection methods. Current approaches rely on slow spatial or spectral scanning limiting their application in living biological tissues, while faster snapshot methods such as image mapping spectrometry and multispectral interferometry are limited in spatial and/or spectral resolution, are computationally demanding, and imaging devices are very expensive to manufacture. Leveraging light sheet microscopy, phasor-based hyperspectral snapshot microscopy improved imaging speed 10-100 fold which, combined with minimal light exposure and high detection efficiency, enabled hyperspectral metabolic imaging of live, three-dimensional mouse tissues not feasible with other methods. As a fit-free method that does not require any a priori information often unavailable in complex and evolving biological systems, the rule of linear combinations of the phasor could spectrally resolve subtle differences between cell types in the developing zebrafish retina and spectrally separate and track multiple organelles in 3D cultured cells over time. The sine/cosine snapshot method is adaptable to any microscope or imaging device thus making hyperspectral imaging and fit-free analysis based on linear combinations broadly available to researchers and the public.

The corneal epithelium is consistently regenerated by limbal stem/progenitor cells (LSCs), a very small population of adult stem cells residing in the limbus. Several Wnt ligands, including Wnt6, are preferentially expressed in the limbus. To investigate the role of Wnt6 in regulating proliferation and maintenance of human LSCs in an in vitro LSC expansion setting, we generated NIH-3T3 feeder cells to overexpress different levels of Wnt6. Characterization of LSCs cultured on Wnt6 expressing 3T3 cells showed that high level of Wnt6 increased proliferation of LSCs. Medium and high levels of Wnt6 also increased the percentage of small cells (diameter ≤ 12 µm), a feature of the stem cell population. Additionally, the percentage of cells expressing the differentiation marker K12 was significantly reduced in the presence of medium and high Wnt6 levels. Although Wnt6 is mostly known as a canonical Wnt ligand, our data showed that canonical and non-canonical Wnt signaling pathways were activated in the Wnt6-supplemented LSC cultures, a finding suggesting that interrelationships between both pathways are required for LSC regulation.

The Hippo pathway plays critical roles in cell growth, differentiation, organ development and tissue homeostasis, whereas its dysregulation can lead to tumorigenesis. YAP and TAZ are transcription co-activators and represent the main downstream effectors of the Hippo pathway. Here, we show that heat stress induces a strong and rapid YAP dephosphorylation and activation. The effect of heat shock on YAP is dominant to other signals known to modulate the Hippo pathway. Heat shock inhibits LATS kinase by promoting HSP90-dependent LATS interaction with and inactivation by protein phosphatase 5. Heat shock also induces LATS ubiquitination and degradation. YAP and TAZ are crucial for cellular heat shock responses, including the heat shock transcriptome and cell viability. This study uncovers previously unknown mechanisms of Hippo regulation by heat shock, as well as physiological functions of YAP, in the heat stress response. Our observations also reveal a potential combinational therapy involving hyperthermia and targeting of the Hippo pathway.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth, nutrient and energy status cues to control cell growth and metabolism. While mTORC1 activation at the lysosome is well characterized, it is not clear how this complex is regulated at other subcellular locations. Here, we combine location-selective kinase inhibition, live-cell imaging and biochemical assays to probe the regulation of growth factor-induced mTORC1 activity in the nucleus. Using a nuclear targeted Akt Substrate-based Tandem Occupancy Peptide Sponge (Akt-STOPS) that we developed for specific inhibition of Akt, a critical upstream kinase, we show that growth factor-stimulated nuclear mTORC1 activity requires nuclear Akt activity. Further mechanistic dissection suggests that nuclear Akt activity mediates growth factor-induced nuclear translocation of Raptor, a regulatory scaffolding component in mTORC1, and localization of Raptor to the nucleus results in nuclear mTORC1 activity in the absence of growth factor stimulation. Taken together, these results reveal a mode of regulation of mTORC1 that is distinct from its lysosomal activation, which controls mTORC1 activity in the nuclear compartment.

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.

Many cell- and tissue-level functions are coordinated by intracellular signaling pathways that trigger the expression of context-specific target genes. Yet the input-output relationships that link pathways to the genes they activate are incompletely understood. Mapping the pathway-decoding logic of natural target genes could also provide a basis for engineering novel signal-decoding circuits. Here we report the construction of synthetic immediate-early genes (SynIEGs), target genes of Erk signaling that implement complex, user-defined regulation and can be monitored by using live-cell biosensors to track their transcription and translation. We demonstrate the power of this approach by confirming Erk duration-sensing by FOS, elucidating how the BTG2 gene is differentially regulated by external stimuli, and designing a synthetic immediate-early gene that selectively responds to the combination of growth factor and DNA damage stimuli. SynIEGs pave the way toward engineering molecular circuits that decode signaling dynamics and combinations across a broad range of cellular contexts.

A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.

Spatial transcriptomics seeks to integrate single cell transcriptomic data within the three-dimensional space of multicellular biology. Current methods to correlate a cell's position with its transcriptome in living tissues have various limitations. We developed an approach, called 'ZipSeq', that uses patterned illumination and photocaged oligonucleotides to serially print barcodes ('zipcodes') onto live cells in intact tissues, in real time and with an on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in vitro wound healing, live lymph node sections and a live tumor microenvironment. In all cases, we discovered new gene expression patterns associated with histological structures. In the tumor microenvironment, this demonstrated a trajectory of myeloid and T cell differentiation from the periphery inward. A combinatorial variation of ZipSeq efficiently scales in the number of regions defined, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.

G-protein coupled receptors (GPCRs) and their associated heterotrimeric G proteins impinge on pathways that control epithelial cell self-renewal and differentiation. Although it is known that Gαs protein signaling regulates skin homeostasis in vivo, the role of GPCR-coupled Gαi proteins in the skin is unclear. Here, by using a chemogenetic approach, we demonstrate that GPCR-Gαi activation can regulate keratinocyte proliferation and differentiation and that overactivation of Gαi-signaling in the basal compartment of the mouse skin can lead to epidermal hyperplasia. Our results expand our understanding of the role of GPCR-cAMP signaling in skin homeostasis and reveal overlapping and divergent roles of the cAMP-regulating heterotrimeric Gαs and Gαi proteins in keratinocytes.

Transduction of extracellular matrix mechanics affects cell migration, proliferation, and differentiation. While this mechanotransduction is known to depend on the regulation of focal adhesion kinase phosphorylation on Y397 (FAKpY397), the mechanism remains elusive. To address this, we developed a mathematical model to test the hypothesis that FAKpY397-based mechanosensing arises from the dynamics of nanoscale integrin clustering, stiffness-dependent disassembly of integrin clusters, and FAKY397 phosphorylation within integrin clusters. Modeling results predicted that integrin clustering dynamics governs how cells convert substrate stiffness to FAKpY397, and hence governs how different cell types transduce mechanical signals. Existing experiments on MDCK cells and HT1080 cells, as well as our new experiments on 3T3 fibroblasts, confirmed our predictions and supported our model. Our results suggest a new pathway by which integrin clusters enable cells to calibrate responses to their mechanical microenvironment.

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.

Alphaviruses are emerging, mosquito-transmitted RNA viruses with poorly understood cellular tropism and species selectivity. Mxra8 is a receptor for multiple alphaviruses including chikungunya virus (CHIKV). We discovered that while expression of mouse, rat, chimpanzee, dog, horse, goat, sheep, and human Mxra8 enables alphavirus infection in cell culture, cattle Mxra8 does not. Cattle Mxra8 encodes a 15-amino acid insertion in its ectodomain that prevents Mxra8 binding to CHIKV. Identical insertions are present in zebu, yak, and the extinct auroch. As other Bovinae lineages contain related Mxra8 sequences, this insertion likely occurred at least 5 million years ago. Removing the Mxra8 insertion in Bovinae enhances alphavirus binding and infection, while introducing the insertion into mouse Mxra8 blocks CHIKV binding, prevents infection by multiple alphaviruses in cells, and mitigates CHIKV-induced pathogenesis in mice. Our studies on how this insertion provides resistance to CHIKV infection could facilitate countermeasures that disrupt Mxra8 interactions with alphaviruses.

Tissues and organs are composed of diverse cell types, which poses a major challenge for cell-type-specific profiling of gene expression. Current metabolic labeling methods rely on exogenous pyrimidine analogs that are only incorporated into RNA in cells expressing an exogenous enzyme. This approach assumes that off-target cells cannot incorporate these analogs. We disprove this assumption and identify and characterize the enzymatic pathways responsible for high background incorporation. We demonstrate that mammalian cells can incorporate uracil analogs and characterize the enzymatic pathways responsible for high background incorporation. To overcome these limitations, we developed a new small molecule-enzyme pair consisting of uridine/cytidine kinase 2 and 2'-azidouridine. We demonstrate that 2'-azidouridine is only incorporated in cells expressing uridine/cytidine kinase 2 and characterize selectivity mechanisms using molecular dynamics and X-ray crystallography. Furthermore, this pair can be used to purify and track RNA from specific cellular populations, making it ideal for high-resolution cell-specific RNA labeling. Overall, these results reveal new aspects of mammalian salvage pathways and serve as a new benchmark for designing, characterizing and evaluating methodologies for cell-specific labeling of biomolecules.

We synthesized an anthracene derivative with solvatochromic properties to be used as a molecular probe for membrane dynamics and supramolecular organization. A nine carbon atom acyl chain and a dimethylamino substitution were introduced at positions 2 and 6 of the anthracene ring, respectively. This derivative, 2-nonanoyl-6-(dimethylamino)anthracene (termed CAPRYDAA), is a molecular probe designed to mimic the well-known membrane probe LAURDAN's location and response in the lipid membranes. Due to the larger distance between the electron donor and acceptor groups, its absorption and emission bands are red-shifted according to the polarity of the media. The photophysical behavior of CAPRYDAA was measured in homogeneous media, synthetic bilayer and cells, both in a cuvette and in a fluorescence microscope, using one and two-photon excitation. Our results show a comparable physicochemical behavior of CAPRYDAA with LAURDAN, but with the advantage of using visible light (488 nm) as an excitation source. CAPRYDAA was also excitable by two-photon laser sources, making it easy to combine CAPRYDAA with either blue or red emission probes. In GUVs or cells, CAPRYDAA can discriminate the lipid phases and liquid-liquid phase heterogeneity. This new membrane probe shows the bathochromic properties of the PRODAN-based probes designed by Weber, overcoming the need for UV or two-photon excitation and facilitating the studies on the membrane properties using regular confocal microscopes.

In mammalian cells, widespread acceleration of cytoplasmic mRNA degradation is linked to impaired RNA polymerase II (Pol II) transcription. This mRNA decay-induced transcriptional repression occurs during infection with gammaherpesviruses including Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), which encode an mRNA endonuclease that initiates widespread RNA decay. Here, we show that MHV68-induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. This reduced Pol II occupancy is accompanied by down-regulation of multiple Pol II subunits and TFIIB in the nucleus of infected cells, as revealed by mass spectrometry-based global measurements of protein abundance. Viral genes, despite the fact that they require Pol II for transcription, escape transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We propose a model in which the ability to escape from transcriptional repression is linked to the localization of viral DNA within replication compartments, providing a means for these viruses to counteract decay-induced transcript loss.

LINC complexes (Linker of Nucleoskeleton and Cytoskeleton), consisting of inner nuclear membrane SUN (Sad1, UNC-84) proteins and outer nuclear membrane KASH (Klarsicht, ANC-1, and Syne Homology) proteins, are essential for nuclear positioning, cell migration and chromosome dynamics. To test the in vivo functions of conserved interfaces revealed by crystal structures, Cain et al used a combination of Caenorhabditis elegans genetics, imaging in cultured NIH 3T3 fibroblasts, and Molecular Dynamic simulations, to study SUN-KASH interactions. Conserved aromatic residues at the -7 position of the C-termini of KASH proteins and conserved disulfide bonds in LINC complexes play important roles in force transmission across the nuclear envelope. Other properties of LINC complexes, such as the helices preceding the SUN domain, the longer coiled-coils spanning the perinuclear space and higher-order organization may also function to transmit mechanical forces generated by the cytoskeleton across the nuclear envelope.

Simultaneous profiling of transcriptome and chromatin accessibility within single cells is a powerful approach to dissect gene regulatory programs in complex tissues. However, current tools are limited by modest throughput. We now describe an ultra high-throughput method, Paired-seq, for parallel analysis of transcriptome and accessible chromatin in millions of single cells. We demonstrate the utility of Paired-seq for analyzing the dynamic and cell-type-specific gene regulatory programs in complex tissues by applying it to mouse adult cerebral cortex and fetal forebrain. The joint profiles of a large number of single cells allowed us to deconvolute the transcriptome and open chromatin landscapes in the major cell types within these brain tissues, infer putative target genes of candidate enhancers, and reconstruct the trajectory of cellular lineages within the developing forebrain.

Abnormal retinal neovascularization associated with various retinopathies can result in irreversible vision loss. Although the mechanisms involved in this occurrence is unclear, increasing evidence suggests that aberrant Wnt signaling participates in the pathogenesis of abnormal neovascularization. Because Wnt signaling upregulation can be induced by oxidative stress through the activation of disheveled (DVL), a key molecule in the Wnt signaling pathway, we investigated whether oxidative stress can activate Wnt signaling and induce angiogenic phenotypes in human retinal microvascular endothelial cells (HRMECs). We found that increased Wnt signaling activity, as well as enhanced angiogenic phenotypes, such as tube formation and cell migration, were detected in the hydrogen peroxide-treated HRMECs. Moreover, these effects were effectively suppressed by a small-molecule Wnt inhibitor targeting the PDZ domain of DVL. Therefore, we propose that targeting abnormal Wnt signaling at the DVL level with a small-molecule inhibitor may represent a novel approach in retinal neovascularization treatment and prevention.

Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)-only found in higher eukaryotes-regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA-domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p but enhanced K7ac, indicating that RPRD proteins recruit K7 deacetylases, including HDAC1. We also report autoregulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Wnt treatment markedly increased the endocytosis and degradation in lysosomes of BSA. In this study, we report that in addition to receptor-mediated endocytosis, Wnt also triggers the intake of large amounts of extracellular fluid by macropinocytosis, a nonreceptor-mediated actin-driven process. Macropinocytosis induction is rapid and independent of protein synthesis. In the presence of Wnt, large amounts of nutrient-rich packages such as proteins and glycoproteins were channeled into lysosomes after fusing with smaller receptor-mediated vesicles containing glycogen synthase kinase 3 (GSK3) and protein arginine ethyltransferase 1 (PRMT1), an enzyme required for canonical Wnt signaling. Addition of Wnt3a, as well as overexpression of Disheveled (Dvl), Frizzled (Fz8), or dominant-negative Axin induced endocytosis. Depletion of the tumor suppressors adenomatous polyposis coli (APC) or Axin dramatically increased macropinocytosis, defined by incorporation of the high molecular weight marker tetramethylrhodamine (TMR)-dextran and its blockage by the Na+/H+ exchanger ethylisopropyl amiloride (EIPA). Macropinocytosis was blocked by dominant-negative vacuolar protein sorting 4 (Vps4), indicating that the Wnt pathway is dependent on multivesicular body formation, a process called microautophagy. SW480 colorectal cancer cells displayed constitutive macropinocytosis and increased extracellular protein degradation in lysosomes, which were suppressed by restoring full-length APC. Accumulation of the transcriptional activator β-catenin in the nucleus of SW480 cells was inhibited by methyltransferase inhibition, EIPA, or the diuretic amiloride. The results indicate that Wnt signaling switches metabolism toward nutrient acquisition by engulfment of extracellular fluids and suggest possible treatments for Wnt-driven cancer progression.

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

Studies of the accelerated aging disorder Hutchinson-Gilford progeria syndrome (HGPS) can potentially reveal cellular defects associated with physiological aging. HGPS results from expression and abnormal nuclear envelope association of a farnesylated, truncated variant of prelamin A called progerin. We surveyed the diffusional mobilities of nuclear membrane proteins to identify proximal effects of progerin expression. The mobilities of three proteins-SUN2, nesprin-2G, and emerin-were reduced in fibroblasts from children with HGPS compared with those in normal fibroblasts. These proteins function together in nuclear movement and centrosome orientation in fibroblasts polarizing for migration. Both processes were impaired in fibroblasts from children with HGPS and in NIH 3T3 fibroblasts expressing progerin, but were restored by inhibiting protein farnesylation. Progerin affected both the coupling of the nucleus to actin cables and the oriented flow of the cables necessary for nuclear movement and centrosome orientation. Progerin overexpression increased levels of SUN1, which couples the nucleus to microtubules through nesprin-2G and dynein, and microtubule association with the nucleus. Reducing microtubule-nuclear connections through SUN1 depletion or dynein inhibition rescued the polarity defects. Nuclear movement and centrosome orientation were also defective in fibroblasts from normal individuals over 60 y, and both defects were rescued by reducing the increased level of SUN1 in these cells or inhibiting dynein. Our results identify imbalanced nuclear engagement of the cytoskeleton (microtubules: high; actin filaments: low) as the basis for intrinsic cell polarity defects in HGPS and physiological aging and suggest that rebalancing the connections can ameliorate the defects.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

Chromosomal rearrangements involving receptor tyrosine kinases (RTK) are a clinically relevant oncogenic mechanism in human cancers. These chimeric oncoproteins often contain the C-terminal kinase domain of the RTK joined in cis to various N-terminal, nonkinase fusion partners. The functional role of the N-terminal fusion partner in RTK fusion oncoproteins is poorly understood. Here, we show that distinct N-terminal fusion partners drive differential subcellular localization, which imparts distinct cell signaling and oncogenic properties of different, clinically relevant ROS1 RTK fusion oncoproteins. SDC4-ROS1 and SLC34A2-ROS1 fusion oncoproteins resided on endosomes and activated the MAPK pathway. CD74-ROS1 variants that localized instead to the endoplasmic reticulum (ER) showed compromised activation of MAPK. Forced relocalization of CD74-ROS1 from the ER to endosomes restored MAPK signaling. ROS1 fusion oncoproteins that better activate MAPK formed more aggressive tumors. Thus, differential subcellular localization controlled by the N-terminal fusion partner regulates the oncogenic mechanisms and output of certain RTK fusion oncoproteins. SIGNIFICANCE: ROS1 fusion oncoproteins exhibit differential activation of MAPK signaling according to subcellular localization, with ROS1 fusions localized to endosomes, the strongest activators of MAPK signaling.

Bipolar disorder (BD) is a serious mood disorder associated with circadian rhythm abnormalities. Risk for BD is genetically encoded and overlaps with systems that maintain circadian rhythms. Lithium is an effective mood stabilizer treatment for BD, but only a minority of patients fully respond to monotherapy. Presently, we hypothesized that lithium-responsive BD patients (Li-R) would show characteristic differences in chronotype and cellular circadian rhythms compared to lithium non-responders (Li-NR). Selecting patients from a prospective, multi-center, clinical trial of lithium monotherapy, we examined morning vs. evening preference (chronotype) as a dimension of circadian rhythm function in 193 Li-R and Li-NR BD patients. From a subset of 59 patient donors, we measured circadian rhythms in skin fibroblasts longitudinally over 5 days using a bioluminescent reporter (Per2-luc). We then estimated circadian rhythm parameters (amplitude, period, phase) and the pharmacological effects of lithium on rhythms in cells from Li-R and Li-NR donors. Compared to Li-NRs, Li-Rs showed a difference in chronotype, with higher levels of morningness. Evening chronotype was associated with increased mood symptoms at baseline, including depression, mania, and insomnia. Cells from Li-Rs were more likely to exhibit a short circadian period, a linear relationship between period and phase, and period shortening effects of lithium. Common genetic variation in the IP3 signaling pathway may account for some of the individual differences in the effects of lithium on cellular rhythms. We conclude that circadian rhythms may influence response to lithium in maintenance treatment of BD.

Breast cancers enduring treatment with chemotherapy may be enriched for cancer stem cells or tumor-initiating cells, which have an enhanced capacity for self-renewal, tumor initiation, and/or metastasis. Breast cancer cells that express the type I tyrosine kinaselike orphan receptor ROR1 also may have such features. Here we find that the expression of ROR1 increased in breast cancer cells following treatment with chemotherapy, which also enhanced expression of genes induced by the activation of Rho-GTPases, Hippo-YAP/TAZ, or B lymphoma Mo-MLV insertion region 1 homolog (BMI1). Expression of ROR1 also enhanced the capacity of breast cancer cells to invade Matrigel, form spheroids, engraft in Rag2-/-[Formula: see text] mice, or survive treatment with paclitaxel. Treatment of mice bearing breast cancer patient-derived xenografts (PDXs) with the humanized anti-ROR1 monoclonal antibody cirmtuzumab repressed expression of genes associated with breast cancer stemness, reduced activation of Rho-GTPases, Hippo-YAP/TAZ, or BMI1, and impaired the capacity of breast cancer PDXs to metastasize or reengraft Rag2-/-[Formula: see text] mice. Finally, treatment of PDX-bearing mice with cirmtuzumab and paclitaxel was more effective than treatment with either alone in eradicating breast cancer PDXs. These results indicate that targeting ROR1 may improve the response to chemotherapy of patients with breast cancer.

Drugs that modify the epigenome are powerful tools for treating cancer, but these drugs often have pleiotropic effects, and identifying patients who will benefit from them remains a major clinical challenge. Here we show that medulloblastomas driven by the transcription factor Gfi1 are exquisitely dependent on the enzyme lysine demethylase 1 (Kdm1a/Lsd1). We demonstrate that Lsd1 physically associates with Gfi1, and that these proteins cooperate to inhibit genes involved in neuronal commitment and differentiation. We also show that Lsd1 is essential for Gfi1-mediated transformation: Gfi1 proteins that cannot recruit Lsd1 are unable to drive tumorigenesis, and genetic ablation of Lsd1 markedly impairs tumor growth in vivo. Finally, pharmacological inhibitors of Lsd1 potently inhibit growth of Gfi1-driven tumors. These studies provide important insight into the mechanisms by which Gfi1 contributes to tumorigenesis, and identify Lsd1 inhibitors as promising therapeutic agents for Gfi1-driven medulloblastoma.

Previously, Aznar et al., showed that Daple/CCDC88C enables Wnt receptors to transactivate trimeric G-proteins during non-canonical Wnt signaling via a novel G-protein binding and activating (GBA) motif. By doing so, Daple serves two opposing roles; earlier during oncogenesis it suppresses neoplastic transformation and tumor growth, but later it triggers epithelial-to-mesenchymal-transition (EMT). We have identified and characterized two isoforms of the human Daple gene. While both isoforms cooperatively suppress tumor growth via their GBA motif, only the full-length transcript triggers EMT and invasion. Both isoforms are suppressed during colon cancer progression, and their reduced expression carries additive prognostic significance. These findings provide insights into the opposing roles of Daple during cancer progression and define the G-protein regulatory GBA motif as one of the minimal modules essential for Daple's role as a tumor suppressor.

Intracellular protein and proteomic studies using mass spectrometry, imaging microscopy, flow cytometry, or western blotting techniques require genetic manipulation, cell permeabilization, and/or cell lysis. We present a biophysical method that employs a nanoaspirator to 'fish' native cytoplasmic or nuclear proteins from single mammalian cells, without compromising cell viability, followed by ex cellulo quantitative detection. Our work paves the way for spatiotemporally-controlled, quantitative, live, single-cell proteomics.

Murine herpesvirus-68 (MHV-68) productively infects mouse lungs, exhibiting a complex pathology characteristic of both acute viral infections and chronic respiratory diseases. We sought to discover proteins differentially expressed in bronchoalveolar lavage (BAL) from mice infected with MHV-68. Mice were infected intranasally with MHV-68. After nine days, as the lytic phase of infection resolved, differential BAL proteins were identified by two-dimensional (2D) electrophoresis and mass spectrometry. Of 23 unique proteins, acute phase proteins, vitamin A transport, and oxidative stress response factors Pdx6 and EC-SOD (Sod3) were enriched. Correspondingly, iNOS2 was induced in lung tissue by seven days post-infection. Oxidative stress was partly a direct result of MHV-68 infection, as reactive oxygen species (ROS) were induced in cultured murine NIH3T3 fibroblasts and human lung A549 cells infected with MHV-68. Finally, mice infected with a recombinant MHV-68 co-expressing inflammatory cytokine murine interleukin 6 (IL6) showed exacerbated oxidative stress and soluble type I collagen characteristic of tissue recovery. Thus, oxidative stress appears to be a salient feature of MHV-68 pathogenesis, in part caused by lytic replication of the virus and IL6. Proteins and small molecules in lung oxidative stress networks therefore may provide new therapeutic targets to ameliorate respiratory virus infections.

Fluorescence spectroscopy, coupled with microscopy, opens new frontiers for the study of dynamic processes with high spatio-temporal resolution. The application of phasor plots to FLIM and hyperspectral imaging demonstrate unprecedented capabilities to study complex photophysics at the subcellular level. Using these approaches we studied the effects of an H2O2 bolus on NIH-3T3 membranes dynamics monitored by LAURDAN fluorescence. Exposure of NIH-3T3 cells to a bolus of H2O2 modifies the cell membranes and, in particular, the plasma membrane in a complex manner. The LAURDAN results reveal that the peroxide treatment decreases membrane fluidity but surprisingly increases dipolar relaxation around the excited probe. Using the Multidimensional-phasor approach we elucidated the complex photophysics of LAURDAN incorporated into cell membrane after H2O2 exposure. The results indicate the occurrence of LAURDAN fast-diffusion from gel↔ld phases in membranes exposed to a H2O2 bolus. An ad hoc hypothesis is presented to interpret the results in the context of H2O2 oxidative distress/eustress.

Particular brain regions and cell populations exhibit increased susceptibility to aging-related stresses. Here, we describe the age-specific and brain-region-specific accumulation of ribosome-associated 3' UTR RNAs that lack the 5' UTR and open reading frame. Our study reveals that this phenomenon impacts hundreds of genes in aged D1 spiny projection neurons of the mouse striatum and also occurs in the aging human brain. Isolated 3' UTR accumulation is tightly correlated with mitochondrial gene expression and oxidative stress, with full-length mRNA expression that is reduced but not eliminated, and with production of short 3' UTR-encoded peptides. Depletion of the oxidation-sensitive Fe-S cluster ribosome recycling factor ABCE1 induces the accumulation of 3' UTRs, consistent with a model in which ribosome stalling and mRNA cleavage by No-Go decay yields isolated 3' UTR RNAs protected by ribosomes. Isolated 3' UTR accumulation is a hallmark of brain aging, likely reflecting regional differences in metabolism and oxidative stress.