Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

We introduce a novel statistical approach that quantifies, for the first time, the amount of colocalization of two fluorescent-labeled proteins in an image automatically, removing the bias of visual interpretation. This is done by estimating simultaneously the maximum threshold of intensity for each color below which pixels do not show any statistical correlation. The sensitivity of the method was illustrated on simulated data by statistically confirming the existence of true colocalization in images with as little as 3% colocalization. This method was then tested on a large three-dimensional set of fixed cells cotransfected with CFP/YFP pairs of proteins that either co-compartmentalized, interacted, or were just randomly localized in the nucleolus. In this test, the algorithm successfully distinguished random color overlap from colocalization due to either co-compartmentalization or interaction, and results were verified by fluorescence resonance energy transfer. The accuracy and consistency of our algorithm was further illustrated by measuring, for the first time in live cells, the dissociation rate (k(d)) of the HIV-1 Rev/CRM1 export complex induced by the cytotoxin leptomycin B. Rev/CRM1 colocalization in nucleoli dropped exponentially after addition of leptomycin B at a rate of 1.25 x 10(-3) s(-1). More generally, this algorithm can be used to answer a variety of biological questions involving protein-protein interactions or co-compartmentalization and can be generalized to colocalization of more than two colors.

SummaryLiquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

Here we provide evidence for a critical role of the transient receptor potential cation channel, subfamily V, member 4 (TRPV4) in normal bladder function. Immunofluorescence demonstrated TRPV4 expression in mouse and rat urothelium and vascular endothelium, but not in other cell types of the bladder. Intracellular Ca 2+ measurements on urothelial cells isolated from mice revealed a TRPV4-dependent response to the selective TRPV4 agonist 4α-phorbol 12,13-didecanoate and to hypotonic cell swelling. Behavioral studies demonstrated that TRPV4 -/-mice manifest an incontinent phenotype but show normal exploratory activity and anxietyrelated behavior. Cystometric experiments revealed that TRPV4 -/-mice exhibit a lower frequency of voiding contractions as well as a higher frequency of nonvoiding contractions. Additionally, the amplitude of the spontaneous contractions in explanted bladder strips from TRPV4 -/-mice was significantly reduced. Finally, a decreased intravesical stretch-evoked ATP release was found in isolated whole bladders from TRPV4 -/-mice. These data demonstrate a previously unrecognized role for TRPV4 in voiding behavior, raising the possibility that TRPV4 plays a critical role in urothelium-mediated transduction of intravesical mechanical pressure.

The nuclear export protein XPO1 is overexpressed in cancer, leading to the cytoplasmic mislocalization of multiple tumor suppressor proteins. Existing XPO1-targeting agents lack selectivity and have been associated with significant toxicity. Small molecule selective inhibitors of nuclear export (SINEs) were designed that specifically inhibit XPO1. Genetic experiments and X-ray structures demonstrate that SINE covalently bind to a cysteine residue in the cargo-binding groove of

Since its first description 20 years ago, the tetrazolium-based colorimetric (MTT) assay using MT-4 cells for the detection of anti-HIV compounds has been widely used. This method, which remains popular, provides more information than more recently developed methods and, therefore, represents a useful methodology on its own or in combination with other screening systems. The replication of HIV in MT-4 cells is usually monitored 5 d after infection; therefore, this protocol can be divided into three steps: the infection (at day 0), an incubation period (5 d) and the evaluation (at day 5). The long-standing and intensive use of the MTT method has taught users of the limitations and, equally importantly, the unexpected advantages of the MT-4/MTT assay. The use of this method can be extended to antiviral testing of compounds against other cyto-destructive viruses.

he COVID-19 pandemic, caused by SARS-CoV-2, has resulted in a worldwide health crisis 1 and few effective drugs are available to treat patients with COVID-19. Although remdesivir initially seemed promising for severe cases 2 , the World Health Organization's Solidarity trial showed that it has no definite impact on mortality 3. Dexamethasone can reduce mortality by a third among critically ill patients with COVID-19, by suppressing the hyperactive immune response 4. However, as treatment benefits severe cases only to a limited extent, efficient and safe therapeutics are urgently required while awaiting the worldwide implementation of vaccines. Coronaviruses cause respiratory and intestinal infections in a broad range of mammals and birds. Seven human coronaviruses (HCoVs) are known, which probably all emerged as zoonoses from bats, mice or domestic animals 5. The four so-called 'common cold HCoVs'-229E, NL63, OC43 and HKU1-cause mild upper respiratory tract illnesses 6. In contrast, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV) and the recently emerged SARS-CoV-2 are highly pathogenic and cause severe, potentially lethal respiratory infections. As numerous coronaviruses reside in animal reservoirs and interspecies transmission frequently occurs 5,7,8 , there is a constant risk of new pathogenic coronaviruses spreading into the human population, as exemplified by the recent SARS-CoV-2 pandemic. Nevertheless, our options to prevent or treat coronavirus infections remain limited. Hence, the development of broad-spectrum anti-coronavirus drugs could help not only to address the current high medical need, but also to quickly contain zoonotic events in the future. Common host factors essential for replication of multiple coronaviruses represent attractive targets for broad-spectrum antiviral drugs. To develop such drugs, it is crucial to understand which host factors coronaviruses require to infect a cell, because each step of the coronavirus replication cycle (receptor binding, endocytosis, fusion, viral protein translation, genome replication, virion assembly and release) may serve as a target for intervention. Although the entry step of coronaviruses has been relatively well characterized, the host-virus interplay in later steps of the viral life cycle remains largely elusive. For SARS-CoV-2, previous studies have shown that the protein angiotensin-converting enzyme 2 (ACE2) can serve as a receptor in Vero E6 cells 9 or in human cells overexpressing ACE2 (refs. 10-12). In addition, it was shown that the SARS-CoV-2 spike (S) can be primed for fusion by cellular proteases such as furin, transmembrane serine protease 2 (TMPRSS2) or cathepsin B or L, depending on the target cell type 10,13. In the present study, we performed a series of genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-based genetic screens to identify host factors required for SARS-CoV-2 and HCoV-229E infection. We identified phosphoinositide 3-kinase (PI3K) type 3 as a common host factor for SARS-CoV-2,...

The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity1. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5–Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1–TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.