AlCl3-Promoted Intramolecular Indolinone-Quinolone Rearrangement of Spiro[indoline-3,2'-quinoxaline]-2,3'-diones: Easy Access to Quinolino[3,4-b]quinoxalin-6-ones.

A facile and direct intramolecular indolinone-quinolone rearrangement was developed for the synthesis of quinolino[3,4-b]quinoxalin-6-ones from spiro[indoline-3,2'-quinoxaline]-2,3'-diones, which are readily available with use of isatines, malononitrile, and 1,2-phenylenediamines under quite mild conditions. This efficient approach provides excellent yields and could potentially be used for the construction of a diverse library of quinolino[3,4-b]quinoxalin-6-ones for high-throughput screening in medicinal chemistry. The reaction mechanism is explored by extensive DFT calculations.

HIF1α-dependent hypoxia response in myeloid cells requires IRE1α.

Cellular adaptation to low oxygen tension triggers primitive pathways that ensure proper cell function. Conditions of hypoxia and low glucose are characteristic of injured tissues and hence successive waves of inflammatory cells must be suited to function under low oxygen tension and metabolic stress. While Hypoxia-Inducible Factor (HIF)-1α has been shown to be essential for the inflammatory response of myeloid cells by regulating the metabolic switch to glycolysis, less is known about how HIF1α is triggered in inflammation. Here, we demonstrate that cells of the innate immune system require activity of the inositol-requiring enzyme 1α (IRE1α/XBP1) axis in order to initiate HIF1α-dependent production of cytokines such as IL1ß, IL6 and VEGF-A. Knockout of either HIF1α or IRE1α in myeloid cells ameliorates vascular phenotypes in a model of retinal pathological angiogenesis driven by sterile inflammation. Thus, pathways associated with ER stress, in partnership with HIF1α, may co-regulate immune adaptation to low oxygen.

Starvation-induced proteasome assemblies in the nucleus link amino acid supply to apoptosis.

Eukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions.

Application of Autologous Peripheral Blood Mononuclear Cells into the Area of Spinal Cord Injury in a Subacute Period: A Feasibility Study in Pigs.

Peripheral blood presents an available source of cells for both fundamental research and clinical use. In our study, we have evaluated the therapeutic potential of peripheral blood mononuclear cells (PBMCs) excluding the preliminary sorting or mobilization of peripheral blood stem cells. We have evaluated the regenerative potential of PBMCs embedded into a fibrin matrix (FM) in a model of pig spinal cord injury. The distribution of transplanted PBMCs in the injured spinal cord was evaluated; PBMCs were shown to penetrate into the deep layers of the spinal cord and concentrate mainly in the grey matter. The results of the current study revealed an increase in the tissue integrity in the area adjacent to the epicenter of injury and the partially restored conduction along posterior columns of the spinal cord in animals after FM+PBMC application. The multiplex analysis of blood serum and cerebrospinal fluid showed the cytokine imbalance to occur without significantly shifting toward pro-inflammatory or anti-inflammatory cytokine cascades.

A potent nuclear export mechanism imposes USP16 cytoplasmic localization during interphase.

USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here, we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double-strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.This article has an associated First Person interview with the first author of the paper.

Improving Culture Conditions, Proliferation, and Migration of Porcine Mesenchymal Stem Cells on Spinal Cord Contusion Injury Model in vitro.

Adipose tissue-derived mesenchymal stem cells (AD-MSCs) are promising for cell therapy in spinal cord injury (SCI). The pig is one of the most approximate models of many human diseases, including SCI. In our study, we selected the optimal conditions for the culture of porcine AD-MSCs and developed an in vitro SCI model based on the culture of cells in injured spinal cord extracts (SCE) 3 days and 6 weeks after SCI. We show that Dulbecco's Modified Eagle Medium (DMEM) with 20% serum content, supplemented with a combination of 5 mM L-ascorbate-2-phosphate and nonessential amino acids, stimulated a typical fibroblast-like morphology and high proliferation of porcine AD-MSCs. SCE caused a higher proliferation of porcine AD-MSCs compared with extracts from an intact spinal cord. The optimal proliferating effect was achieved using rostral 3 days SCE, and proliferation was lower in caudal and central SCE. Porcine AD-MSCs migration to the 3 days and 6 weeks SCE was higher than to an intact one and preferred the rostral SCE, avoiding central and caudal SCE. We also studied 13 cytokines contained in SCE but did not observe any definite relationship between some analyte concentrations and a change in the behavior of AD-MSCs.

Reduction of ciliary length through pharmacologic or genetic inhibition of CDK5 attenuates polycystic kidney disease in a model of nephronophthisis.

Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.

Systematic proteomics of the VCP-UBXD adaptor network identifies a role for UBXN10 in regulating ciliogenesis.

The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to 'segregate' ubiquitylated proteins from their binding partners. VCP acts through UBX-domain-containing adaptors that provide target specificity, but the targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left-right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP-UBXN10 in ciliogenesis.

ANKS6 is the critical activator of NEK8 kinase in embryonic situs determination and organ patterning.

The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study, we identify ANKS6 as a target and activator of NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6(Streaker) and Nek8(Roc). Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6(Strkr) mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8(Roc) mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left-right patterning, cardiopulmonary development and renal morphogenesis.

The quantitative architecture of centromeric chromatin.

The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ∼400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ∼4% of all centromeric nucleosomes, forms a ∼50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation.DOI: http://dx.doi.org/10.7554/eLife.02137.001.

Loss of the ciliary kinase Nek8 causes left-right asymmetry defects.

A missense mutation in mouse Nek8, which encodes a ciliary kinase, produces the juvenile cystic kidneys (jck) model of polycystic kidney disease, but the functions of Nek8 are incompletely understood. Here, we generated a Nek8-null allele and found that homozygous mutant mice die at birth and exhibit randomization of left-right asymmetry, cardiac anomalies, and glomerular kidney cysts. The requirement for Nek8 in left-right patterning is conserved, as knockdown of the zebrafish ortholog caused randomized heart looping. Ciliogenesis was intact in Nek8-deficient embryos and cells, but we observed misexpression of left-sided marker genes early in development, suggesting that nodal ciliary signaling was perturbed. We also generated jck/Nek8 compound heterozygotes; these mutants developed less severe cystic disease than jck homozygotes and provided genetic evidence that the jck allele may encode a gain-of-function protein. Notably, NEK8 and polycystin-2 (PC2) proteins interact, and we found that Nek8(-/-) and Pkd2(-/-) embryonic phenotypes are strikingly similar. Nek8-deficient embryos and cells did express PC2 normally, which localized properly to the cilia. However, similar to cells lacking PC2, NEK8-depleted inner medullary collecting duct cells exhibited a defective response to fluid shear, suggesting that NEK8 may play a role in mediating PC2-dependent signaling.

Determination of membrane protein transporter oligomerization in native tissue using spatial fluorescence intensity fluctuation analysis.

Membrane transporter proteins exist in a complex dynamic equilibrium between various oligomeric states that include monomers, dimers, dimer of dimers and higher order oligomers. Given their sub-optical microscopic resolution size, the oligomerization state of membrane transporters is difficult to quantify without requiring tissue disruption and indirect biochemical methods. Here we present the application of a fluorescence measurement technique which combines fluorescence image moment analysis and spatial intensity distribution analysis (SpIDA) to determine the oligomerization state of membrane proteins in situ. As a model system we analyzed the oligomeric state(s) of the electrogenic sodium bicarbonate cotransporter NBCe1-A in cultured cells and in rat kidney. The approaches that we describe offer for the first time the ability to investigate the oligomeric state of membrane transporter proteins in their native state.

Ligand-induced clustering of EGF receptors: a quantitative study by fluorescence image moment analysis.

Fluorescence microscopy is widely used in the life sciences, but largely for qualitative imaging. Here we apply a bioanalytical technique, fluorescence image moment analysis, to demonstrate how the distribution of the fluorescent molecules can be measured directly from confocal microscopy images. We measured the oligomerization state of EGF-eGPF receptors expressed in CHO-K1 cells in situ.

Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis.

Measuring protein interactions is key to understanding cell signaling mechanisms, but quantitative analysis of these interactions in situ has remained a major challenge. Here, we present spatial intensity distribution analysis (SpIDA), an analysis technique for image data obtained using standard fluorescence microscopy. SpIDA directly measures fluorescent macromolecule densities and oligomerization states sampled within single images. The method is based on fitting intensity histograms calculated from images to obtain density maps of fluorescent molecules and their quantal brightness. Because spatial distributions are acquired by imaging, SpIDA can be applied to the analysis of images of chemically fixed tissue as well as live cells. However, the technique does not rely on spatial correlations, freeing it from biases caused by subcellular compartmentalization and heterogeneity within tissue samples. Analysis of computer-based simulations and immunocytochemically stained GABA(B) receptors in spinal cord samples shows that the approach yields accurate measurements over a broader range of densities than established procedures. SpIDA is applicable to sampling within small areas (6 µm(2)) and reveals the presence of monomers and dimers with single-dye labeling. Finally, using GFP-tagged receptor subunits, we show that SpIDA can resolve dynamic changes in receptor oligomerization in live cells. The advantages and greater versatility of SpIDA over current techniques open the door to quantificative studies of protein interactions in native tissue using standard fluorescence microscopy.

Quantification of receptor tyrosine kinase transactivation through direct dimerization and surface density measurements in single cells.

Cell signaling involves dynamic changes in protein oligomerization leading to the formation of different signaling complexes and modulation of activity. Spatial intensity distribution analysis (SpIDA) is an image analysis method that can directly measure oligomerization and trafficking of endogenous proteins in single cells. Here, we show the use of SpIDA to quantify dimerization/activation and surface transport of receptor protein kinases--EGF receptor and TrkB--at early stages of their transactivation by several G protein-coupled receptors (GPCRs). Transactivation occurred on the same timescale and was directly limited by GPCR activation but independent of G-protein coupling types. Early receptor protein kinase transactivation and internalization were not interdependent for all receptor pairs tested, revealing heterogeneity between groups of GPCRs. SpIDA also detected transactivation of TrkB by dopamine receptors in intact neurons. By allowing for time and space resolved quantification of protein populations with heterogeneous oligomeric states, SpIDA provides a unique approach to undertake single cell multivariate quantification of signaling processes involving changes in protein interactions, trafficking, and activity.

Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT component.

HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at least two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for ∼35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release.

Fluorescence microscopy investigations of ligand propagation and accessibility under adherent cells.

Fluorescence microscopy methods including total internal reflection fluorescence and confocal laser scanning microscopy have played a major role in modern cell biology research by permitting imaging of fluorescently tagged macromolecules in living cells. These methods are often used to examine the initial events in signal transduction, which involve interactions occurring between membrane receptors and ligands such as antibodies and growth factors. Most quantitative biophysical applications using these fluorescence imaging methods, including ligand binding assays, are based on the assumption that the fluorophore label of interest has equal access to all areas of the membrane on the cell. Our findings suggest that there is limited accessibility of fluorophores (25±2%)(-) under the basal membrane of adherent CHO-K1 cells expressing epidermal growth factor receptor plated on a bare glass in standard two-dimensional tissue cultures. The authors present a detailed study of the extent to which a small fluorescent dye molecule (Alexa 647) is able to propagate under the basal membrane of cells plated on a variety of biologically compatible substrates: fibronectin, bovine serum albumin, poly-d-lysine, collagen I, collagen IV, Geltrex™, and fibronectin such as binding polymer. For nonspecific dye propagation the best overall accessibility was achieved using a thin layer preparation of a commercially available basement membrane matrix, Geltrex™ (67±8%). Coupling of a specific high affinity ligand (epidermal growth factor) to the dye did result in a moderate increase in propagation for most substrates examined. Despite the overall increase in propagation for most substrates (60%-80%), large areas under the central regions of the adherent cells still remained inaccessible to the fluorescently labeled ligand. More importantly, the presence of the specific ligand did not result in consistent increase in ligand propagation. Taken together these results suggest that the reduced accessibility is not exclusively due to steric effects, and the chemistry of both the ligand and the substrate may be important when working under conditions of reduced dimensionality.

Measurement of monomer-oligomer distributions via fluorescence moment image analysis.

We present higher-order moment analysis of fluorescence intensity fluctuations from individual laser scanning microscopy images applied to study monomer-oligomer distributions. We demonstrate that the number densities and brightness ratios of a mixed population of monomers and oligomers can be determined by analyzing higher-order moments of the fluorescence intensity fluctuations from individual images for specific ranges of densities and particle brightness ratios. Computer simulations and experiments with fluorescent microspheres and cells were performed to illustrate the detection limits and accuracy of this statistical approach. The simulation results show that the concentration of the dimer or oligomer population should be less than or equal to the monomeric concentration for the method to provide accurate results, and that the upper density detection limit of the population of monomers is one order-of-magnitude higher than the concentration of the oligomers. We implemented this technique to resolve two populations of fluorescent microspheres with different brightness ratios and we also applied the moment-analysis method to examine the distribution of aggregation states of PDGF-beta receptors in human fibroblast cells. The method was able to resolve a tetrameric population of the PDGF-beta receptors relative to the background distribution of nonspecifically bound fluorophore.