Quantitative comparison of nuclear transport inhibition by SARS coronavirus ORF6 reveals the importance of oligomerization.

Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia et al., Cell Rep. 33, 108234 (2020); L. Miorin et al., Proc. Natl. Acad. Sci. U.S.A. 117, 28344-28354 (2020); and M. Frieman et al., J. Virol. 81, 9812-9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98-RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98-RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.

Screen for Modulation of Nucleocapsid Protein Condensation Identifies Small Molecules with Anti-Coronavirus Activity.

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase-separated condensates that are hypothesized to play critical roles in viral replication, transcription, and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43, and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

Dorsal lip maturation and initial archenteron extension depend on Wnt11 family ligands.

Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes during the tailbud period. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues by imaging intact fixed embryos stained for anillin and microtubules. We found that the initial extension of the archenteron is correlated with blastopore lip maturation, and archenteron extension is dramatically disrupted by decreased Wnt11 family signaling. We were aided in our interpretation of the immunofluorescence by the novel, membrane proximal location of the cleavage furrow protein anillin in the epithelium of the blastopore lip and early archenteron.

High-Content Screening and Computational Prediction Reveal Viral Genes That Suppress the Innate Immune Response.

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single-virus or -gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human virus genes. We found that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-κB and interferon regulatory factor 3 (IRF3). We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins. IMPORTANCE Infectious diseases caused by viral pathogens exacerbate health care and economic burdens. Numerous viral biomolecules suppress the human innate immune system, enabling viruses to evade an immune response from the host. Despite our current understanding of viral replications and immune evasion, new viral proteins, including those encoded by uncultivated viruses or emerging viruses, are being unearthed at a rapid pace from large-scale sequencing and surveillance projects. The use of medium- and high-throughput functional assays to characterize immunosuppressive functions of viral proteins can advance our understanding of viral replication and possibly treatment of infections. In this study, we assembled a large viral-gene library from diverse viral families and developed a high-content assay to test for inhibition of innate immunity pathways. Our work expands the tools that can rapidly link sequence and protein function, representing a practical step toward early-stage evaluation of emerging and understudied viruses.

Extracellular hyaluronate pressure shaped by cellular tethers drives tissue morphogenesis.

How tissues acquire complex shapes is a fundamental question in biology and regenerative medicine. Zebrafish semicircular canals form from invaginations in the otic epithelium (buds) that extend and fuse to form the hubs of each canal. We find that conventional actomyosin-driven behaviors are not required. Instead, local secretion of hyaluronan, made by the enzymes uridine 5'-diphosphate dehydrogenase (ugdh) and hyaluronan synthase 3 (has3), drives canal morphogenesis. Charged hyaluronate polymers osmotically swell with water and generate isotropic extracellular pressure to deform the overlying epithelium into buds. The mechanical anisotropy needed to shape buds into tubes is conferred by a polarized distribution of actomyosin and E-cadherin-rich membrane tethers, which we term cytocinches. Most work on tissue morphogenesis ascribes actomyosin contractility as the driving force, while the extracellular matrix shapes tissues through differential stiffness. Our work inverts this expectation. Hyaluronate pressure shaped by anisotropic tissue stiffness may be a widespread mechanism for powering morphological change in organogenesis and tissue engineering.

Chromatin bridges, not micronuclei, activate cGAS after drug-induced mitotic errors in human cells.

Mitotic errors can activate cyclic GMP-AMP synthase (cGAS) and induce type I interferon (IFN) signaling. Current models propose that chromosome segregation errors generate micronuclei whose rupture activates cGAS. We used a panel of antimitotic drugs to perturb mitosis in human fibroblasts and measured abnormal nuclear morphologies, cGAS localization, and IFN signaling in the subsequent interphase. Micronuclei consistently recruited cGAS without activating it. Instead, IFN signaling correlated with formation of cGAS-coated chromatin bridges that were selectively generated by microtubule stabilizers and MPS1 inhibitors. cGAS activation by chromatin bridges was suppressed by drugs that prevented cytokinesis. We confirmed cGAS activation by chromatin bridges in cancer lines that are unable to secrete IFN by measuring paracrine transfer of 2'3'-cGAMP to fibroblasts, and in mouse cells. We propose that cGAS is selectively activated by self-chromatin when it is stretched in chromatin bridges. Immunosurveillance of cells that fail mitosis, and antitumor actions of taxanes and MPS1 inhibitors, may depend on this effect.

Immunomodulatory drug discovery from herbal medicines: Insights from organ-specific activity and xenobiotic defenses.

Traditional herbal medicines, which emphasize a holistic, patient-centric view of disease treatment, provide an exciting starting point for discovery of new immunomodulatory drugs. Progress on identification of herbal molecules with proven single agent activity has been slow, in part because of insufficient consideration of pharmacology fundamentals. Many molecules derived from medicinal plants exhibit low oral bioavailability and rapid clearance, leading to low systemic exposure. Recent research suggests that such molecules can act locally in the gut or liver to activate xenobiotic defense pathways that trigger beneficial systemic effects on the immune system. We discuss this hypothesis in the context of four plant-derived molecules with immunomodulatory activity: indigo, polysaccharides, colchicine, and ginsenosides. We end by proposing research strategies for identification of novel immunomodulatory drugs from herbal medicine sources that are informed by the possibility of local action in the gut or liver, leading to generation of systemic immune mediators.

PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition triggers antitumor immunity.

PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.

So many ways to naturally kill a cancer cell.

Natural killer (NK) cells participate in cancer immunosurveillance and cancer immunotherapy. Live cell imaging of cancer cells targeted by NK cells, published today in BMC Biology by Zhu et al., reveals a remarkable diversity of programmed cell death pathways induced in individual cells. Pathway choice depends on the state of the target cell actin cytoskeleton and a novel death pathway, granzyme-induced necroptosis, could be of broad importance in cancer immunotherapy.

Self-Organization of Cellular Units.

The purpose of this review is to explore self-organizing mechanisms that pattern microtubules (MTs) and spatially organize animal cell cytoplasm, inspired by recent experiments in frog egg extract. We start by reviewing conceptual distinctions between self-organizing and templating mechanisms for subcellular organization. We then discuss self-organizing mechanisms that generate radial MT arrays and cell centers in the absence of centrosomes. These include autocatalytic MT nucleation, transport of minus ends, and nucleation from organelles such as melanosomes and Golgi vesicles that are also dynein cargoes. We then discuss mechanisms that partition the cytoplasm in syncytia, in which multiple nuclei share a common cytoplasm, starting with cytokinesis, when all metazoan cells are transiently syncytial. The cytoplasm of frog eggs is partitioned prior to cytokinesis by two self-organizing modules, protein regulator of cytokinesis 1 (PRC1)-kinesin family member 4A (KIF4A) and chromosome passenger complex (CPC)-KIF20A. Similar modules may partition longer-lasting syncytia, such as early Drosophila embryos. We end by discussing shared mechanisms and principles for the MT-based self-organization of cellular units.

Publisher Correction: Colchicine acts selectively in the liver to induce hepatokines that inhibit myeloid cell activation.

A Correction to this paper has been published: https://doi.org/10.1038/s42255-021-00397-5.

O-GlcNAc modification of nuclear pore complexes accelerates bidirectional transport.

Macromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG-repeat domains in NPCs are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated transport of proteins in both directions, and decreasing modification slowed transport. Superresolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the nonspecific permeability of the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.

Colchicine acts selectively in the liver to induce hepatokines that inhibit myeloid cell activation.

Colchicine has served as a traditional medicine for millennia and remains widely used to treat inflammatory and other disorders. Colchicine binds tubulin and depolymerizes microtubules, but it remains unclear how this mechanism blocks myeloid cell recruitment to inflamed tissues. Here we show that colchicine inhibits myeloid cell activation via an indirect mechanism involving the release of hepatokines. We find that a safe dose of colchicine depolymerizes microtubules selectively in hepatocytes but not in circulating myeloid cells. Mechanistically, colchicine triggers Nrf2 activation in hepatocytes, leading to secretion of anti-inflammatory hepatokines, including growth differentiation factor 15 (GDF15). Nrf2 and GDF15 are required for the anti-inflammatory action of colchicine in vivo. Plasma from colchicine-treated mice inhibits inflammatory signalling in myeloid cells in a GDF15-dependent manner, by positive regulation of SHP-1 (PTPN6) phosphatase, although the precise molecular identities of colchicine-induced GDF15 and its receptor require further characterization. Our work shows that the efficacy and safety of colchicine depend on its selective action on hepatocytes, and reveals a new axis of liver-myeloid cell communication. Plasma GDF15 levels and myeloid cell SHP-1 activity may be useful pharmacodynamic biomarkers of colchicine action.

shinyDepMap, a tool to identify targetable cancer genes and their functional connections from Cancer Dependency Map data.

Individual cancers rely on distinct essential genes for their survival. The Cancer Dependency Map (DepMap) is an ongoing project to uncover these gene dependencies in hundreds of cancer cell lines. To make this drug discovery resource more accessible to the scientific community, we built an easy-to-use browser, shinyDepMap (https://labsyspharm.shinyapps.io/depmap). shinyDepMap combines CRISPR and shRNA data to determine, for each gene, the growth reduction caused by knockout/knockdown and the selectivity of this effect across cell lines. The tool also clusters genes with similar dependencies, revealing functional relationships. shinyDepMap can be used to (1) predict the efficacy and selectivity of drugs targeting particular genes; (2) identify maximally sensitive cell lines for testing a drug; (3) target hop, that is, navigate from an undruggable protein with the desired selectivity profile, such as an activated oncogene, to more druggable targets with a similar profile; and (4) identify novel pathways driving cancer cell growth and survival.

Spatial variation of microtubule depolymerization in large asters.

Microtubule plus-end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared with the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and microtubule-associated proteins (MAPs) in the interior cytosol compared with that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density.

Co-movement of astral microtubules, organelles and F-actin by dynein and actomyosin forces in frog egg cytoplasm.

How bulk cytoplasm generates forces to separate post-anaphase microtubule (MT) asters in Xenopus laevis and other large eggs remains unclear. Previous models proposed that dynein-based, inward organelle transport generates length-dependent pulling forces that move centrosomes and MTs outwards, while other components of cytoplasm are static. We imaged aster movement by dynein and actomyosin forces in Xenopus egg extracts and observed outward co-movement of MTs, endoplasmic reticulum (ER), mitochondria, acidic organelles, F-actin, keratin, and soluble fluorescein. Organelles exhibited a burst of dynein-dependent inward movement at the growing aster periphery, then mostly halted inside the aster, while dynein-coated beads moved to the aster center at a constant rate, suggesting organelle movement is limited by brake proteins or other sources of drag. These observations call for new models in which all components of the cytoplasm comprise a mechanically integrated aster gel that moves collectively in response to dynein and actomyosin forces.

In vivo microscopy reveals macrophage polarization locally promotes coherent microtubule dynamics in migrating cancer cells.

Microtubules (MTs) mediate mitosis, directional signaling, and are therapeutic targets in cancer. Yet in vivo analysis of cancer cell MT behavior within the tumor microenvironment remains challenging. Here we developed an imaging pipeline using plus-end tip tracking and intravital microscopy to quantify MT dynamics in live xenograft tumor models. Among analyzed features, cancer cells in vivo displayed higher coherent orientation of MT dynamics along their cell major axes compared with 2D in vitro cultures, and distinct from 3D collagen gel cultures. This in vivo MT phenotype was reproduced in vitro when cells were co-cultured with IL4-polarized MΦ. MΦ depletion, MT disruption, targeted kinase inhibition, and altered MΦ polarization via IL10R blockade all reduced MT coherence and/or tumor cell elongation. We show that MT coherence is a defining feature for in vivo tumor cell dynamics and migration, modulated by local signaling from pro-tumor macrophages.

Microtubule Growth Rates Are Sensitive to Global and Local Changes in Microtubule Plus-End Density.

The microtubule cytoskeleton plays critically important roles in numerous cellular functions in eukaryotes, and it does so across a functionally diverse and morphologically disparate range of cell types [1]. In these roles, microtubule assemblies must adopt distinct morphologies and physical dimensions to perform specific functions [2-5]. As such, these macromolecular assemblies-as well as the dynamics of the individual microtubule polymers from which they are made-must scale and change in accordance with cell size, geometry, and function. Microtubules in cells typically assemble to a steady state in mass, leaving enough of their tubulin subunits soluble to allow rapid growth and turnover. This suggests some negative feedback that limits the extent of assembly, for example, decrease in growth rate, or increase in catastrophe rate, as the soluble subunit pool decreases. Although these ideas have informed the field for decades, they have not been observed experimentally. Here, we describe the application of an experimental approach that combines cell-free extracts with photo-patterned hydrogel micro-enclosures as a means to investigate microtubule dynamics in cytoplasmic volumes of defined size and shape. Our measurements reveal a negative correlation between microtubule plus-end density and microtubule growth rates and suggest that these rates are sensitive to the presence of nearby growing ends.