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.

Lipid Raft is required for PSGL-1 ligation induced HL-60 cell adhesion on ICAM-1.

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 ß2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the ß2 integrin activation and ß2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-ß-cyclodextrin (MßCD), we confirm the role of lipid raft in regulating the activation of ß2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MßCD 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 ß2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates ß2 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.

Identification of nucleolin as a lipid-raft-dependent ß1-integrin-interacting protein in A375 cell migration.

Lipid rafts are related to cell surface receptor function. Integrin is a major surface receptor protein in cell adhesion and migration on the extracellular matrix (ECM). Here, we showed that lipid rafts played a critical role in human melanoma A375 cell spreading and migration on fibronectin; an important component of the ECM that interacts with ß1 integrin. We found that the disruption of lipid rafts did not markedly inhibit the expression and activation of ß1 integrin. By coimmunoprecipitation and mass spectrometry, we investigated the influence of lipid rafts on the ß1 integrin complex and identified nucleolin as a potential lipid-raft-dependent ß1-integrin-interacting protein. Upon confirmation of the interaction between ß1 integrin and nucleolin, further studies revealed that nucleolin colocalized with ß1 integrin in lipid rafts and raft disruption interrupted their association. In addition, knockdown of nucleolin markedly attenuated A375 cell spreading and migration on fibronectin. Taken together, we demonstrated that nucleolin is a critical lipid-raft-dependent ß1-integrin-interacting protein in A375 cell spreading and migration on fibronectin.

Lipid rafts control human melanoma cell migration by regulating focal adhesion disassembly.

Tumor cell migration is a crucial step in the metastatic cascade, and interruption of this step is considered to be logically effective in preventing tumor metastasis. Lipid rafts, distinct liquid ordered plasma membrane microdomains, have been shown to influence cancer cell migration, but the underlying mechanisms are still not well understood. Here, we report that lipid rafts regulate the dynamics of actin cytoskeleton and focal adhesion in human melanoma cell migration. Disrupting the integrity of lipid rafts with methyl-ß cyclodextrin enhances actin stress fiber formation and inhibits focal adhesion disassembly, accompanied with alterations in cell morphology. Furthermore, actin cytoskeleton, rather than microtubules, mediates the lipid raft-dependent focal adhesion disassembly by regulating the dephosphorylation of focal adhesion proteins and the internalization of ß3 integrin. We also show that Src-RhoA-Rho kinase signaling pathway is responsible for lipid raft disruption-induced stress fiber formation. Taken together, these observations provide a new mechanism to further explain how lipid rafts regulate the migration of melanoma cell and suggest that lipid rafts may be novel and attractive targets for cancer therapy.

c-Abl Kinase Is a Regulator of αvß3 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 αvß3 integrin in response to PDGF stimulation. c-Abl kinase colocalizes with αvß3 integrin dynamically and affects αvß3 integrin affinity by regulating its cluster. The interaction between c-Abl kinase and αvß3 integrin was dependent on the activity of c-Abl kinase induced by PDGF stimulation, but was not dependent on the binding of αvß3 integrin with its ligands, suggesting that c-Abl kinase is not involved in the outside-in signaling of αvß3 integrin. Talin head domain was required for the interaction between c-Abl kinase and αvß3 integrin, and the SH3 domain of c-Abl kinase was involved in its interaction with talin and αvß3 integrin. Taken together, we have uncovered a novel and critical role of c-Abl kinase in αvß3 integrin mediated melanoma cell migration.

Lipid raft regulates the initial spreading of melanoma A375 cells by modulating ß1 integrin clustering.

Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for ß1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-ß-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing ß1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates ß1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to ß1 integrin, which may be crucial for ß1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins.

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.