Cell-cell junctions of dermal microvascular endothelial cells contain tight and adherens junction proteins in spatial proximity

Endothelial cell-cell contacts control the vascular permeability, thereby regulating the flow of solutes, macromolecules, and leukocytes between blood vessels and interstitial space. Because of specific needs, the endothelial permeability differs significantly between the tight blood-brain barrier endothelium and the more permeable endothelial lining of the non-brain microvasculature. Most likely, such differences are due to a differing architecture of the respective interendothelial cell contacts. However, while the molecules and junctional complexes of macrovascular endothelial cells and the blood-brain barrier endothelium are fairly well characterized, much less is known about the organization of intercellular contacts of microvascular endothelium. Toward this end, we developed a combined cross-linking and immunoprecipitation protocol which enabled us to map nearest neighbor interactions of junctional proteins in the human dermal microvascular endothelial cell line HMEC-1. We show that proteins typically located in tight or adherens junctions of epithelial cells are in the proximity in HMEC-1 cells. This contrasts with the separation of the different types of junctions observed in polarized epithelial cells and "tight" endothelial layers of the blood-brain barrier and argues for a need of the specific junctional contacts in microvascular endothelium possibly required to support an efficient transendothelial migration of leukocytes.     

Amino acid 324 in the simian immunodeficiency virus SIVmac V3 loop can confer CD4 independence and modulate the interaction with CCR5 and alternative coreceptors

The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4. The tested amino acid substitutions had highly differential effects on viral infectivity. Eleven of sixteen substitutions disrupted entry via CCR5 or the alternative coreceptor GPR15. Nevertheless, most of these variants replicated in the macaque T-cell line 221-89 and some also replicated in rhesus macaque peripheral blood monocytes, suggesting that efficient usage of CCR5 and GPR15 on cell lines is not a prerequisite for SIV replication in primary cells. Four variants showed enhanced entry into the macaque sMagi reporter cell line. However, sMagi cells did not express appreciable amounts of CCR5 and GPR15 mRNA, and entry into these cells was not efficiently blocked by a small-molecule CCR5 antagonist, suggesting that sMagi cells express as-yet-unidentified entry cofactors. In summary, we found that a single amino acid at position 324 in the SIV Env V3 loop can modulate both the efficiency and the types of coreceptors engaged by Env and allow for CD4-independent fusion in some cases.     

Prey-capture in the African clawed toad (Xenopus laevis): comparison of turning to visual and lateral line stimuli

Separately delivered visual and lateral line stimuli elicit similar but not identical orientation and approach by intact, sighted Xenopus. Response frequencies for visual stimuli declined sharply for distant or caudal stimuli while those for lateral line stimuli changed little. Turn angles correlated highly with stimulus angles but were smaller on average, so regression slopes were less than one. Regression slopes were smaller for visual than for lateral line stimuli, but this apparent difference was due to different distributions of stimulus distance interacting with the toad’s rotation center. Errors in final headings, most often under-rotations, did not differ by modality. Frequencies of lunges and arm capture movements were higher for visual stimuli both overall and especially for rostral proximal stimuli. The results demonstrate accurate orientation by sighted Xenopus to visual and lateral line stimuli; they are consistent with expectations based on in-register tectal maps. Orientation to lateral line stimuli is similar to previous results with blinded animals, revealing no heightened acuity in the latter. Modality differences indicate that the lateral line system is better for omnidirectional orientation and approach to distant stimuli whereas the visual system is more attuned to nearby rostral stimuli and more apt to mediate strikes.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Novel indeno[1,2-b]indoloquinones as inhibitors of the human protein kinase CK2 with antiproliferative activity towards a broad panel of cancer cell lines

We previously reported indeno[1,2-b]indoles as a novel class of potent inhibitors of the human protein kinase CK2. In the present study we prepared two novel quinoid derivatives, the indeno[1,2-b]indoloquinones 6b and 6c, and demonstrated inhibition of the human CK2 by the compounds. Furthermore, we showed substantial antiproliferative activity of both compounds towards a broad panel of human cancer cell lines in the low micromolar range. Whereas the earlier indeno[1,2-b]indoles have been shown to be selective for CK2, the indeno[1,2-b]indoloquinones 6b and 6c also inhibited the AMPK activated protein kinase ARK5, potentially contributing to the anti-cancer effects of the compounds. In addition, with compound 6b we found a very potent inhibitor of the leukemia-associated receptor tyrosine kinase FLT3, with an IC(50) of 0.18 μM.     

Prieurianin/endosidin 1 is an actin-stabilizing small molecule identified from a chemical genetic screen for circadian clock effectors in Arabidopsis thaliana

Chemical modulators are powerful tools to investigate biological processes. To identify circadian clock effectors, we screened a natural product library in the model plant Arabidopsis thaliana. Two compounds, prieurianin (Pri) and prieurianin acetate, were identified as causing a shorter circadian period. Recently, Pri was independently identified as a vesicle trafficking inhibitor and re-named endosidin 1 (ES1). Here we show that Pri primarily affects actin filament flexibility in vivo, later resulting in reduced severing and filament depolymerization. This stabilization of the actin cytoskeleton subsequently causes changes in vesicle trafficking. Pri also affected microfilaments in mammalian cells, indicating that its target is highly conserved; however, it did not alter actin dynamics in vitro, suggesting that its activity requires the presence of actin-associated proteins. Furthermore, well-characterized actin inhibitors shortened the period length of the Arabidopsis clock in a similar way to Pri, supporting the idea that Pri affects rhythms by altering the actin network. We conclude that actin-associated processes influence the circadian system in a light-dependent manner, but their disruption does not abolish rhythmicity. In summary, we propose that the primary effect of Pri is to stabilize the actin cytoskeleton system, thereby affecting endosome trafficking. Pri appears to stabilize actin filaments by a different mechanism from previously described inhibitors, and will be a useful tool to study actin-related cellular processes.     

Live-cell assays to identify regulators of ER-to-Golgi trafficking

We applied fluorescence microscopy-based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)-to-Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi-to-ER relocalization of GalT-CFP (β1,4-galactosyltransferase I-cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash-out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER-to-Golgi trafficking. Nine of them interfered with the rate of BFA-induced redistribution of GalT-CFP from the Golgi complex to the ER, six of them interfered with GalT-CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash-out and six of them were positive effectors in both assays. Notably, our live-cell approach captures regulator function in ER-to-Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens.     

miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus

miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes – endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.     

Evidence for a selective migration of fetus-specific CD4+CD25bright regulatory T cells from the peripheral blood to the decidua in human pregnancy

During pregnancy, the maternal immune system has to tolerate the persistence of fetal alloantigens. Many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. Murine studies suggest that CD4(+)CD25(+) T cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. Previous studies by our group demonstrate that a significantly higher percentage of activated T cells and CD4(+)CD25(bright) T cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy. In this study, we examined the phenotypic and functional properties of CD4(+)CD25(bright) T cells derived from maternal peripheral blood and decidual tissue. Depletion of CD4(+)CD25(bright) T cells from maternal peripheral blood demonstrates regulation to third party umbilical cord blood cells comparable to nonpregnant controls, whereas the suppressive capacity to umbilical cord blood cells of her own child is absent. Furthermore, maternal peripheral blood shows a reduced percentage of CD4(+)CD25(bright)FOXP3(+) and CD4(+)CD25(bright)HLA-DR(+) cells compared with peripheral blood of nonpregnant controls. In contrast, decidual lymphocyte isolates contain high percentages of CD4(+)CD25(bright) T cells with a regulatory phenotype that is able to down-regulate fetus-specific and fetus-nonspecific immune responses. These data suggest a preferential recruitment of fetus-specific regulatory T cells from maternal peripheral blood to the fetal-maternal interface, where they may contribute to the local regulation of fetus-specific responses.