Role of NS2 specific RNA binding and phosphorylation in liquid-liquid phase separation and virus assembly.

Liquid-liquid phase separation (LLPS) has assumed a prominent role in biological cell systems, where it underpins the formation of subcellular compartments necessary for cell function. We investigated the underlying mechanism of LLPS in virus infected cells, where virus inclusion bodies are formed by an RNA-binding phosphoprotein (NS2) of Bluetongue virus to serve as sites for subviral particle assembly and virus maturation. We show that NS2 undergoes LLPS that is dependent on protein phosphorylation and RNA-binding and that LLPS occurrence is accompanied by a change in protein secondary structure. Site-directed mutagenesis identified two critical arginine residues in NS2 responsible for specific RNA binding and thus for NS2-RNA complex driven LLPS. Reverse genetics identified the same residues as essential for VIB assembly in infected cells and virus viability. Our findings suggest that a specific arginine-RNA interaction in the context of a phosphorylated state drives LLPS in this, and possibly other, virus infections

c-Abl Kinase Is a Regulator of αvβ3 Integrin Mediated Melanoma A375 Cell Migration.

Integrins are heterodimeric transmembrane receptors that physically link the extracellular matrix (ECM) to the intracellular actin cytoskeleton, and are also signaling molecules that transduce signals bi-directionally across the plasma membrane. Integrin regulation is essential for tumor cell migration in response to growth factors. c-Abl kinase is a nonreceptor tyrosine kinase and is critical for signaling transduction from various receptors. Here we show that c-Abl kinase is involved in A375 cell migration mediated by αvβ3 integrin in response to PDGF stimulation. c-Abl kinase colocalizes with αvβ3 integrin dynamically and affects αvβ3 integrin affinity by regulating its cluster. The interaction between c-Abl kinase and αvβ3 integrin was dependent on the activity of c-Abl kinase induced by PDGF stimulation, but was not dependent on the binding of αvβ3 integrin with its ligands, suggesting that c-Abl kinase is not involved in the outside-in signaling of αvβ3 integrin. Talin head domain was required for the interaction between c-Abl kinase and αvβ3 integrin, and the SH3 domain of c-Abl kinase was involved in its interaction with talin and αvβ3 integrin. Taken together, we have uncovered a novel and critical role of c-Abl kinase in αvβ3 integrin mediated melanoma cell migration

Actin polymerization negatively regulates p53 function by impairing its nuclear import in response to DNA damage.

Actin, one of the most evolutionarily conservative proteins in eukaryotes, is distributed both in the cytoplasm and the nucleus, and its dynamics plays important roles in numerous cellular processes. Previous evidence has shown that actin interacts with p53 and this interaction increases in the process of p53 responding to DNA damage, but the physiological significance of their interaction remains elusive. Here, we show that DNA damage induces both actin polymerization and p53 accumulation. To further understand the implication of actin polymerization in p53 function, cells were treated with actin aggregation agent. We find that the protein level of p53 decrease. The change in p53 is a consequence of the polymeric actin anchoring p53 in the cytoplasm, thus impairing p53 nuclear import. Analysis of phosphorylation and ubiquitination of p53 reveals that actin polymerization promotes the p53 phosphorylation at Ser315 and reduces the stabilization of p53 by recruiting Aurora kinase A. Taken together, our results suggest that the actin polymerization serves as a negative modulator leading to the impairment of nuclear import and destabilization of p53. On the basis of our results, we propose that actin polymerization might be a factor participating in the process of orchestrating p53 function in response to DNA damage

c-Abl kinase directly associates with talin head domain and regulates β₃ integrin ligation concomitantly after PDGF stimulation.

<p>(A) Overnight serum-starved A375 cells were either untreated or treated with 20 ng/ml PDGF for the indicated times. Thereafter, the cells were lysed, and the lysates were immunoblotted with indicated antibodies. In inhibition experiments, cells were preincubated with STI571 for 10 min, and the inhibitor remained in the medium throughout the assay period. (B) Serum-starved A375 cells were stimulated as above. The lysates were immunoprecipitated with anti-talin antibody and immunoblotted with indicated antibodies. (C) Serum-starved A375 cells were treated as above. The lysates were incubated with GST or GST fusion proteins, and the bound proteins were separated by SDS-PAGE and immunoblotted with indicated antibodies. (D) GST or GST fusion proteins were incubated with Ni²⁺Sepharose coated with His-talin2-head. Bound proteins were separated by SDS-PAGE and immunoblotted with anti-GST antibody. (E) c-Abl kinase immunoprecipitates from PDGF treated or untreated A375 cells were separated by SDS-PAGE and transferred onto nitrocellulose membrane. After incubation with GST or GST-talin2-head, the membrane was immunoblotted with anti-GST antibody. Blots were immunoblotted with c-Abl kinase antibody to show equal loading. The immunoblotting bands were quantified by densitometry, and data were normalized with respect to the controls. Data are representative of three independent experiments.</p

c-Abl kinase affects PDGF induced A375 migration.

<p>(A) a, the serum-starved cell suspension, with or without 10 µM STI571 (c-Abl kinase inhibitor), was seeded onto 0.8 μm diameter upper transwell chamber, which was coated with fibronectin. Free-serum medium with 50 ng/ml PDGF was put into the lower chamber to induce cell migration. b, migrations were determined by counting cells in randomly selected five microscope field per well. Bars represent mean ± S.D. (B) the effects of different concentration of STI571 on cell migration. The migration experiment of A375 cells out of agarose drop explants treated with or without STI571 was conducted. After 24 h, the distant of migration was measured using inverted microscope fitted with a rule in eyepiece. a, Cell migration rate was evaluated by the distance of the leading edge of migrating cells from the edge of agarose droplet. b, The distance of cell migration was measured, the extent of cell migration within the drop  =  [(total area/drop area) ×100] –100. (C) A375 cells were transfected with WT c-Abl expression vector or specific siRNA targeting c-Abl. The migration experiment of A375 cell or tansfected cells was performed in transwell plates (b was the statistic value of a). (D) A375 cells or cells transfected with WT c-Abl or c-Abl specific siRNA were lysed (For the transfected cells, cells were lysed after 24 h transfection). Lysates were blotted with c-Abl and actin antibodies. (E) a. A375 cells or the cells transfected with WT c-Abl or c-Abl specific siRNA were plated into 24-well plated and allowed to form a confluent monolayer. After overnight serum starvation, the cell monolayer was “wounded” with a pipette tip. The cells were incubated with DMEM containing 20 ng/ml PDGF and then imaged. b. Cell migration was determined as shrinkage of an average gap area. Bars represent mean ± S.D of three independent experiments. **, p<0.01, ***, p<0.001 with respect to control.</p

β₃ integrin engagement and F-actin do not affect the interaction of c-Abl kinase and α_vβ₃ integrin.

<p>(A) a. Serum-starved A375 cells were digested with EDTA and suspended in serum-free medium for 2 h to restore the state of the cells. After that the cells were incubated on fibronectin-coated coverslips with or without PDGF stimulation for indicated times. Then, the cells were fixed and stained for c-Abl kinase and α_vβ₃ integrin. In inhibition experiments, cells were preincubated with STI571 for 10 min, and the inhibitor remained in the medium throughout the assay period. b. A375 cells were counted in 5–10 fields, and the percentages were calculated by dividing the number of polarized or unpolarized cells by the total number. Data were obtained from >25 cells for each field. (B) Cells were allowed to adhere and spread for 24 h, and Cytochalasin D (0.5 µg/ml) was added into the medium for 5 min. Then the cells were fixed and stained with phalloidin. (C) Serum-starved A375 cells were treated with Cytochalasin D (0.5 µg/ml) for 5 min and washed for three times with PBS. After stimulation with PDGF for 1 h, the cells were fixed and labeled with the antibodies of c-Abl kinase and α_vβ₃ integrin. (D) Serum-starved A375 cells in suspension, as indicated, were stimulated with PDGF or allowed to adhere for 1 h, or treated with Cytochalasin D (0.5 µg/ml) for 5 min. Thereafter, the cells were lysed and the supernatants were incubated overnight at 4°C with α_vβ₃ integrin antibody for immunoprecipitation. Immunoprecipitated complexes were collected with protein A-Sepharose beads, then were washed and resolved by SDS-PAGE and immunoblotted with indicated antibodies. Data are representative of three independent experiments.</p

Lipid raft is involved in PSGL-1 ligation induced activation of β2 integrin.

<p>A, The PSGL-1 ligated HL-60 cells were pretreated with MβCD for 30 min at indicated concentrations, and then the cells were allowed to bind to the plates coated with ICAM-1. Cells adherent to rh-ICAM-1 were expressed as a percentage of the untreated control. Data are representative of three independent experiments and all Statistical differences were compared with control cells (without treatment). B, Cells were pretreated with MβCD (10 mM) or replenished with cholesterol (chol) after MβCD treatment, then ligated. C, HL-60 cells were treated as in B, and the treated cells were stained with IB4 for β2 integrin followed by TRITC-conjugated secondary antibody, then the cells were analysed under confocal microscope. D, The number of resting or clustered cells was counted and the percentages were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081807#pone-0081807-g003" target="_blank">Figure 3C</a>. E, Cells were ligated for indicated times, and then the cells were stained with Alexa Fluor 488 conjugated-Ctx-B and IB4 Plus TRITC-conjugated secondary antibody. The merged images demonstrate the colocalization (yellow). Original scale bar = 5 μm. F, HL-60 cells were ligated or not, then cells were lysed and lipid rafts were isolated by fractionation. β2 integrin in fractions (1 to 12) was detected by western blotting. Flotillin-2 was employed as lipid raft marker. Data are representative of three independent experiments. Bars represent mean ± SD of three independent experiments. All Statistical differences were determined by One-way ANOVA. **, P < 0.01, ***, P < 0.001.</p

Lipid raft affects PSGL-1 distribution.

<p>A, Cell lysate fractions, separated by the sucrose density gradient centrifugation, were evaluated by immunoblotting for PSGL-1. Fractions were isolated from resting HL-60 cells or the cell treated with 10 mM MβCD. GM1 was used as lipid raft marker. B, HL-60 cells were ligated or not, or the cells were pretreated with MβCD before ligation, or reincubated with chol (cholesterol). F-actin was visualized by staining with FITC-conjugated phalloidin. C, Cells were treated as in B, and then the treated cells were labeled with KPL-1 and TRITC-conjugated secondary antibodies. Pictures shown were typical ones under fluorescence microscope. D, The ratio of clustered HL-60 cells treated as in C was calculated. Data are representative of three independent experiments. Bars represent mean ± SD of three independent experiments. All statistical differences were determined by One-way ANOVA. **, P < 0.01.</p

Lipid Raft Is Required for PSGL-1 Ligation Induced HL-60 Cell Adhesion on ICAM-1

<div><p>P-selectin glycoprotein ligand-1 (PSGL-1) and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD), we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation) to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.</p> </div