Biomolecular condensates in cell biology and virology: Phase-separated membraneless organelles (MLOs).

Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of 2D and 3D phase-separated biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions. These functions include cell signaling, immune synapse function, nuclear transcription, RNA splicing and processing, mRNA storage and translation, virus replication and maturation, antiviral mechanisms, DNA sensing, synaptic transmission, protein turnover and mitosis. Components comprising MLOs often associate with low affinity; thus cell integrity can be critical to the maintenance of the full complement of respective MLO components. Phase-separated condensates are typically metastable (shape-changing) and can undergo dramatic, rapid and reversible assembly and disassembly in response to cell signaling events, cell stress, during mitosis, and after changes in cytoplasmic "crowding" (as observed with condensates of the human myxovirus resistance protein MxA). Increasing evidence suggests that neuron-specific aberrations in phase-separation properties of RNA-binding proteins (e.g. FUS and TDP-43) and others (such as the microtubule-binding protein tau) contribute to the development of degenerative neurological diseases (e.g. amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease). Thus, studies of liquid-like phase separation (LLPS) and the formation, structure and function of MLOs are of considerable importance in understanding basic cell biology and the pathogenesis of human diseases.

Platelets are relevant mediators of renal injury induced by primary endothelial lesions.

Several studies have suggested a prominent (pro)inflammatory and harmful role of platelets in renal disease, and newer work has also demonstrated platelet release of proangiogenic factors. In the present study, we investigated the role of platelets in a mouse model of selective endothelial cell injury using either platelet depletion or the pharmacological P2Y12 receptor blocker clopidogrel as an interventional strategy. The concanavalin A/anti-concanavalin A model was induced in left kidneys of C57bl/6J wild-type mice after initial platelet depletion or platelet-inhibiting therapy using clopidogrel. FACS analysis of glycoprotein IIb/IIIa/P-selectin double-positive platelets and platelet-derived microparticles demonstrated relevant platelet activation after the induction of selective endothelial injury in mice. Enhanced platelet activation persisted for 5 days after disease induction and was accompanied by increased amounts of circulating platelet-derived microparticles as potential mediators of a prolonged procoagulant state. By immunohistochemistry, we detected significantly reduced glomerular injury in platelet-depleted mice compared with control mice. In parallel, we also saw reduced endothelial loss and a consequently reduced repair response as indicated by diminished proliferative activity. The P2Y12 receptor blocker clopidogrel demonstrated efficacy in limiting platelet activation and subsequent endothelial injury in this mouse model of renal microvascular injury. In conclusion, platelets are relevant mediators of renal injury induced by primary endothelial lesions early on, as demonstrated by platelet depletion as well as platelet inhibition via the P2Y12 receptor. While strategies to prevent platelet-endothelial interactions have shown protective effects, the contribution of platelets during renal regeneration remains unknown.

A regulatory network of two galectins mediates the earliest steps of avian limb skeletal morphogenesis.

BACKGROUND: The skeletal elements of vertebrate embryonic limbs are prefigured by rod- and spot-like condensations of precartilage mesenchymal cells. The formation of these condensations depends on cell-matrix and cell-cell interactions, but how they are initiated and patterned is as yet unresolved. RESULTS: Here we provide evidence that galectins, ß-galactoside-binding lectins with ß-sandwich folding, play fundamental roles in these processes. We show that among the five chicken galectin (CG) genes, two, CG-1A, and CG-8, are markedly elevated in expression at prospective sites of condensation in vitro and in vivo, with their protein products appearing earlier in development than any previously described marker. The two molecules enhance one another's gene expression but have opposite effects on condensation formation and cartilage development in vivo and in vitro: CG-1A, a non-covalent homodimer, promotes this process, while the tandem-repeat-type CG-8 antagonizes it. Correspondingly, knockdown of CG-1A inhibits the formation of skeletal elements while knockdown of CG-8 enhances it. The apparent paradox of mutual activation at the gene expression level coupled with antagonistic roles in skeletogenesis is resolved by analysis of the direct effect of the proteins on precartilage cells. Specifically, CG-1A causes their aggregation, whereas CG-8, which has no adhesive function of its own, blocks this effect. The developmental appearance and regulation of the unknown cell surface moieties ("ligands") to which CG-1A and CG-8 bind were indicative of specific cognate- and cross-regulatory interactions. CONCLUSION: Our findings indicate that CG-1A and CG-8 constitute a multiscale network that is a major mediator, earlier-acting than any previously described, of the formation and patterning of precartilage mesenchymal condensations in the developing limb. This network functions autonomously of limb bud signaling centers or other limb bud positional cues.

Angiotensin II type 2 receptor-dependent increases in nitric oxide synthase expression in the pulmonary endothelium is mediated via a G alpha i3/Ras/Raf/MAPK pathway.

We have previously reported that angiotensin II (ANG II) stimulated Src tyrosine kinase via a pertussis toxin-sensitive type 2 receptor, which, in turn, activates MAPK, resulting in an increase in nitric oxide synthase (NOS) expression in pulmonary artery endothelial cells (PAECs). The present study was designed to investigate the pathway by which ANG II activates Src leading to an increase in ERK1/ERK2 phosphorylation and an increase in NOS protein in PAECs. Transfection of PAECs with G alpha(i3) dominant negative (DN) cDNA blocked the ANG II-dependent activation of Src, ERK1/ERK2 phosphorylation, and increase in NOS expression. ANG II stimulated an increase in tyrosine phosphorylation of sequence homology of collagen (Shc; 15 min) that was prevented when PAECs were pretreated with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-d]pyrimidine (PP2), a Src inhibitor. ANG II induced a Src-dependent association between Shc and growth factor receptor-bound protein 2 (Grb2) and between Grb2 and son of sevenless (Sos), both of which were maximal at 15 min. The ANG II-dependent increase in Ras GTP binding was prevented when PAECs were pretreated with the AT(2) antagonist PD-123319 or with PP2 or were transfected with Src DN cDNA. ANG II-dependent activation of MAPK and the increase in endothelial NOS (eNOS) were prevented when PAECs were transfected with Ras DN cDNA or treated with FTI-277, a farnesyl transferase inhibitor. ANG II induction of Raf-1 phosphorylation was prevented when PAECs were pretreated with PD-123319 and PP2. Raf kinase inhibitor 1 prevented the ANG II-dependent increase in eNOS expression. Collectively, these data suggest that G alpha(i3), Shc, Grb2, Ras, and Raf-1 link Src to activation of MAPK and to the AT(2)-dependent increase in eNOS expression in PAECs.

Threonine phosphorylation of integrin beta3 in calyculin A-treated platelets is selectively sensitive to 5'-iodotubercidin.

Exposure of platelets to toxins (calyculin A or okadaic acid) that inhibit protein serine/threonine phosphatases types 1 and 2A, at concentrations that block aggregatory and secretory responses, results in the phosphorylation of several platelet proteins including integrin beta(3). Since protein phosphorylation represents a balance between kinase and phosphatase activities, this increase in phosphorylation reflects either the removal of phosphatases that oppose constitutively active kinases known to reside in the platelet (e.g., casein kinase 2) or the activation of endogenous kinases. In this study, we demonstrate that the addition of calyculin A promotes the activation of several endogenous platelet protein kinases, including p42/44(mapk), p38(mapk), Akt/PKB, and LKB1. Using a pharmacologic approach, we assessed whether inhibition of these and other enzymes block phosphorylation of beta(3). Inhibitors of p38(mapk), casein kinase, AMP kinase, protein kinase C, and calcium-calmodulin-dependent kinases did not block phosphorylation of beta(3) on thr(753). In contrast, 5'-iodotubercidin, at 50 muM, blocks beta(3) phosphorylation without affecting the efficacy of calyculin A to inhibit platelet aggregation and spreading. These data dissociate threonine phosphorylation of beta(3) molecules and inhibition of platelet responses by protein phosphatase inhibitors.

Cyclic arginine-glycine-aspartic acid peptide inhibits macrophage infiltration of the kidney and carotid artery lesions in apo-E-deficient mice.

Interactions of leukocytes with the vascular endothelium culminating in their diapedesis represent not only a crucial event in immune surveillance and defense but are also critically involved in the pathogenesis of many inflammatory diseases, including atherosclerosis. Our previous in vitro studies using atomic force microscopy measurement of monocyte-endothelial cell interaction have demonstrated that a cyclic arginine-glycine-aspartic acid peptide (cRGD) inhibited their adhesion through very late antigen (alpha4beta1-integrin; VLA4)-vascular cell adhesion molecule-1 by 60% with the IC50 = 100 nM. To elucidate the potential efficacy of this peptide in vivo in preventing atherogenesis, experiments were performed in apolipoprotein E (ApoE)-deficient (-/-) mice fed a Western diet and receiving chronic treatment with cRGD peptide for 2-4 wk. In addition, some animals were subjected to a temporary carotid artery ligation while receiving the above treatment. Formation of fatty streaks and infiltration of the vascular wall with macrophages were not affected by cRGD treatment. Infiltration of the carotid artery postligation was significantly reduced in the cRGD-treated animals, as was the lipid accumulation. Furthermore, cRGD-treated ApoE-/- mice exhibited significantly lesser macrophage infiltration and lipid accumulation in the kidneys, the site of the highest expression of VLA4. These data demonstrated that cRGD peptide is a potent inhibitor of monocyte/macrophage infiltration of the injured macrovasculature and of the renal microvasculature, where it results in the attenuation of lipid accumulation. Formation of fatty streaks in the aortic root was not inhibitable by this treatment.

Src kinase mediates angiotensin II-dependent increase in pulmonary endothelial nitric oxide synthase.

We have previously demonstrated that angiotensin II (Ang II) stimulates nitric oxide (NO) production in bovine pulmonary artery endothelial cells (BPAECs) by increasing NO synthase (NOS) expression via the type 2 receptor. The purpose of this study was to identify the Ang II-dependent signaling pathway that mediates this increase in endothelial NOS (eNOS). The Ang II-dependent increase in eNOS expression is prevented when BPAECs are pretreated with the tyrosine kinase inhibitors, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine, which also blocked Ang II-dependent mitogen-activated protein kinase (MAPK) kinase/extracellular-regulated protein kinase (MEK)-1 and MAPK phosphorylation, suggesting that Src is upstream of MAPK in this pathway. Transfection of BPAECs with an Src dominant negative mutant cDNA prevented the Ang II-dependent Src activation and increase in eNOS protein expression. PD98059, a MEK-1 inhibitor, prevented the Ang II-dependent phosphorylation of extracellular-regulated protein kinases 1 and 2 and increase in eNOS expression. Neither AG1478, an epidermal growth factor receptor kinase inhibitor, nor AG1295, a platelet derived growth factor receptor kinase inhibitor, had any effect on Ang II-stimulated Src activity, MAPK activation, or eNOS expression. Pertussis toxin prevented the Ang II-dependent increase in Src activity, MAPK activation, and eNOS expression. These data suggest that Ang II stimulates Src tyrosine kinase via a pertussis toxin-sensitive pathway, which in turn activates the MAPK pathway, resulting in increased eNOS protein expression in BPAECs.

Identification of 5,6-trans-epoxyeicosatrienoic acid in the phospholipids of red blood cells.

A novel eicosanoid, 5,6-trans-epoxy-8Z,11Z,14Z-eicosatrienoic acid (5,6-trans-EET), was identified in rat red blood cells. Characterization of 5,6-trans-EET in the sn-2 position of the phospholipids was accomplished by hydrolysis with phospholipase A(2) followed by gas chromatography/mass spectrometry as well as electrospray ionization-tandem mass spectrometry analyses. The electron ionization spectrum of 5,6-erythro-dihydroxyeicosatrienoic acid (5,6-erythro-DHET), converted from 5,6-trans-EET in the samples, matches that of the authentic standard. Hydrogenation of the extracted 5,6-erythro-DHET with platinum(IV) oxide/hydrogen resulted in an increase of the molecular mass by 6 daltons and the same retention time shift as an authentic standard in gas chromatography, suggesting the existence of three olefins as well as the 5,6-erythro-dihydroxyl structure in the metabolite. Match of retention times by chromatography indicated identity of the stereochemistry of the red blood cell 5,6-erythro-DHET vis à vis the synthetic standard. High pressure liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of the phospholipase A(2)-hydrolyzed lipid extracts from red blood cells revealed match of the mass spectrum and retention time of the compound with the authentic 5,6-trans-EET standard, providing direct evidence of the existence of 5,6-trans-EET in red blood cells. The presence of other trans-EETs was also demonstrated. The ability of both 5,6-trans-EET and its product 5,6-erythro-DHET to relax preconstricted renal interlobar arteries was significantly greater than that of 5,6-cis-EET. In contrast, 5,6-cis-EET and 5,6-trans-EET were equipotent in their capacity to inhibit collagen-induced rat platelet aggregation, whereas 5,6-erythro-DHET was without effect. We propose that the red blood cells serve as a reservoir for epoxides which on release may act in a vasoregulatory capacity.

Protein kinase C (PKC)-induced phosphorylation of ROMK1 is essential for the surface expression of ROMK1 channels.

We carried out in vitro phosphorylation assays to determine whether ROMK1 is a substrate of protein kinase C (PKC) and used the two-electrode voltage clamp method to investigate the role of serine residues 4, 183, and 201, the three putative PKC phosphorylation sites, in the regulation of ROMK1 channel activity. Incubation of the purified His-tagged ROMK1 protein with PKC and radiolabeled ATP resulted in (32)P incorporation into ROMK1 detected by autoradiography. Moreover, the in vitro phosphorylation study of three synthesized peptides corresponding to amino acids 1-16, 174-189, and 196-211 of ROMK1 revealed that serine residues 4 and 201 of ROMK1 were the two main PKC phosphorylation sites. In contrast, (32)P incorporation of peptide 174-189 was absent. In vitro phosphorylation studies with ROMK1 mutants, R1S4/201A, R1S4/183A, and R1S183/201A, demonstrated that the phosphorylation levels of R1S4/201A were significantly lower than those of the other two mutants. Also, the Ba(2+)-sensitive K(+) current in oocytes injected with green fluorescent protein (GFP)-R1S4/201A was only 5% of that in oocytes injected with wild type GFP-ROMK1. In contrast, the K(+) current in oocytes injected with GFP-ROMK1 mutants containing either serine residue 4 or 201 was similar to those injected with wild type ROMK1. Confocal microscope imaging shows that the surface expression of the K(+) channels was significantly diminished in oocytes injected with R1S4/201A and completely absent in oocytes injected with R1S4/183/201A. Furthermore, the biotin labeling technique confirmed that the membrane fraction of ROMK channels was almost absent in HEK293 cells transfected with either R1S4/201A or R1S4/183/201A. However, when serine residues 4 and 201 were mutated to aspartate, the K(+) currents and the surface expression were completely restored. Finally, addition of calphostin C in the incubation medium significantly decreased the K(+) current in comparison with that under control conditions. Biotin labeling technique further indicated that inhibition of PKC decreases the surface ROMK1 expression in human embryonic kidney (HEK) cells transfected with ROMK1. We conclude that ROMK1 is a substrate of PKC and that serine residues 4 and 201 are the two main PKC phosphorylation sites that are essential for the expression of ROMK1 in the cell surface.

K depletion increases protein tyrosine kinase-mediated phosphorylation of ROMK.

We purified His-tagged ROMK1 and carried out in vitro phosphorylation assays with (32)P-radiolabeled ATP to determine whether ROMK1 protein is a substrate for PTK. Addition of active c-Src and [(32)P]ATP to the purified ROMK1 protein resulted in the phosphorylation of the ROMK1 protein. However, c-Src did not phosphorylate R1Y337A in which tyrosine residue 337 was mutated to alanine. Furthermore, phosphopeptide mapping identified two phosphopeptides from the trypsin-digested ROMK1 protein. In contrast, no phosphorylated peptide has been found in the trypsin-digested R1Y337A protein. This suggested that two phosphorylated peptides might contain the same tyrosine residue. Also, addition of c-Src and [(32)P]ATP phosphorylated the synthesized peptide corresponding to amino acid sequence 333-362 of the COOH terminus of ROMK1. We then examined the effect of dietary K intake on the tyrosine-phosphorylated ROMK level. Although the ROMK channels pulled down by immunoprecipitation with ROMK antibody were the same from rats on a K-deficient diet or on a high-K diet, more ROMK channels were phosphorylated by PTK in rats on a K-deficient diet than those on a high-K diet. We conclude that ROMK1 can be phosphorylated by PTK and that tyrosine residue 337 is the key site for the phosphorylation. Also, the tyrosine phosphorylation of ROMK is modulated by dietary K intake. This strongly suggests that PTK is an important member of the aldosterone-independent signal transduction pathway for regulating renal K secretion.