Baculovirus actin-rearrangement-inducing factor ARIF-1 induces the formation of dynamic invadosome clusters

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a pathogen of lepidopteran insects, has a striking dependence on the host cell actin cytoskeleton. During the delayed-early stage of infection, AcMNPV was shown to induce the accumulation of actin at the cortex of infected cells. However, the dynamics and molecular mechanism of cortical actin assembly remained unknown. Here, we show that AcMNPV induces dynamic cortical clusters of dot-like actin structures that mediate degradation of underlying extracellular matrix and therefore function similarly to clusters of invadosomes in mammalian cells. Furthermore, we find that the AcMNPV protein actin-rearrangement-inducing factor-1 (ARIF-1), which was previously shown to be necessary and sufficient for cortical actin assembly and efficient viral infection in insect hosts, is both necessary and sufficient for invadosome formation. We mapped the sequences within the C-terminal cytoplasmic region of ARIF-1 that are required for invadosome formation and identified individual tyrosine and proline residues that are required for organizing these structures. Additionally, we found that ARIF-1 and the invadosome-associated proteins cortactin and the Arp2/3 complex localize to invadosomes, and Arp2/3 complex is required for their formation. These ARIF-1-induced invadosomes may be important for the function of ARIF-1 in systemic virus spread. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Piezo1 activation using Yoda1 inhibits macropinocytosis and proliferation of cancer cells

Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by macropinosome formation. A certain number of mammalian mechanosensors are sensitive to membrane deformation and tension. However, it remains unclear whether macropinocytosis is regulated by mechanosensors. Focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). Although studies with Piezo1 knockout cells suggest that Piezo1 itself is not physiologically indispensable for macropinocytosis regulation, Yoda1 inhibited ruffle formation depending on the extracellular Ca2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca2+ ions can regulate EGF-stimulated macropinocytosis. Moreover, Yoda1 impaired cancer cell proliferation, suggesting the impact of macropinocytosis inhibition. We propose the potential for cancer therapy by macropinocytosis inhibition through the regulation of a mechanosensitive channel activity.

127 Plasma from Patients with Burn Injury Increases Endothelial Permeability In-Vitro

Introduction The contribution of endothelial injury to the pathogenesis of burn shock is not well characterized. Human umbilical endothelial cells (HUVECs) have been used to study endotheliopathy in myriad shock states. This work investigates the impact of burn patient plasma on the vascular endothelium and its barrier function. Methods HUVECs were seeded into the apical chambers of transwell plates and cultured over 5–7 days to a confluent monolayer which was confirmed by a transendothelial electrical resistance (TEER) of ≥30Ω. After IRB approval, plasma was collected from burn-injured patients 4 hours after admission. Demographic and injury characteristics were collected from the medical record. Plasma Syndecan-1 (SDC-1) was quantified by ELISA. HUVEC monolayers were exposed to 10% multi-donor pooled healthy human plasma (HHP) or burn patient plasma. Monolayers were subsequently incubated with FIT-C Dextran (40,000 kD). FIT-C diffusion through monolayers was measured in basal chamber supernatants. Monolayer permeability was measured with indices calculated by normalizing values to blank (transwell inserts) and HHP-treated monolayer FIT-C diffusion. HUVECs were also cultured on glass slides and exposed to HHP or burn patient plasma. Cells were fixed with 4% Paraformaldehyde and F-Actin was stained with Texas Red-Phalloidin. Intercellular gap area was calculated using imaging software. Differences between treatment conditions were analyzed with Welch’s t-test and one-way ANOVA, simple linear regression was used to characterize the relationship between plasma SDC-1 and permeability indices, significance was set at p < 0.05. Results Eight burn patient plasma samples were tested. Patients were mostly male (75%) with a mean age of 50±20 years and mean %TBSA burn of 37±34%. Five burn plasma samples significantly increased monolayer permeability. There were no significant differences between patient samples that increased permeability in age, TBSA, gender, or in-hospital mortality. Monolayer permeability indices increased between 7–15% (p< 0.05) among burn plasma treatment conditions (n=6) that increased permeability. There was a strong relationship between monolayer permeability index (%) and plasma SDC-1 (µg/mL) (p=0.03, R2=0.93). Morphological F-actin rearrangement was apparent on microscopy and intercellular gap area was increased in burn plasma treatment conditions (12% vs. 49%, p≤0.0007, n=6). Conclusions Plasma from burn patients induces endothelial damage that increases endothelial cell monolayer permeability. The endothelial biomarker SDC-1 is a reliable indicator of endothelial damage. F-actin rearrangement and an increase in intercellular gap area likely contributes to burn endotheliopathy.

Baculovirus actin-rearrangement-inducing factor ARIF-1 induces the formation of dynamic clusters of invadosome-like structures

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a pathogen of lepidopteran insects, has a striking dependence on the host cell actin cytoskeleton. During the delayed-early stage of infection, AcMNPV was shown to induce the accumulation of actin at the cortex of infected cells. However, the dynamics and molecular mechanism of cortical actin assembly remained unknown. Here, we show that AcMNPV induces dynamic cortical clusters of dot-like actin structures that resemble clusters of invadosomes in mammalian cells. Furthermore, we find that the AcMNPV protein actin-rearrangement-inducing factor-1 (ARIF-1), which was previously shown to be necessary and sufficient for cortical actin assembly and efficient viral infection in insect hosts, is both necessary and sufficient for invadosome-like structure formation. We mapped the sequences within the C-terminal cytoplasmic region of ARIF-1 that are required for invadosome-like structure formation and identified individual tyrosine and proline residues that are required for organizing these structures. Additionally, we found that ARIF-1 and the invadosome-associated proteins cortactin and the Arp2/3 complex localize to invadosome-like structures, and Arp2/3 complex is required for their formation. These ARIF-1-induced invadosome-like structures may be important for the function of ARIF-1 in systemic virus spread.

Inflammasome Activation in Bovine Peripheral Blood-Derived Macrophages Is Associated with Actin Rearrangement

Inflammation is critical for infection control and acts as an arsenal defense mechanism against invading microbes through activation of the host immune system. It works via its inflammasome components to sense the dangerous invading microorganism and send messages to the immune system to destroy them. To date, the function of bovine macrophage inflammasome and its relationship with actin has not been identified. This study aimed to investigate the activation of bovine inflammasome by phase one flagellin from Salmonella typhimurium and its interaction with actin. Bovine monocyte-derived macrophages were prepared and challenged with S. typhimurium SL1344 phase one flagellin. The results demonstrated the relationship between the flagellin-based activation of inflammasome and actin rearrangement. The flagellin-based activation of inflammasome promoted the activation and co-localization of F-actin and the inflammasome complex. Actin was remodeled to different degrees according to the stage of inflammasome activation. The actin redistribution varied from polymerization to filopodia, while at the stage of pyroptotic cell death, actin was broken down and interacted with activated inflammasome complexes. In conclusion, flagellin-dependent inflammasome activation and actin localization to the inflammasome at the stage of pyroptotic cell death may be of importance for appropriate immune responses, pending further studies to explore the exact cross-linking between the inflammasome complex and actin.

Paeoniflorin Inhibits Hepatocyte Growth Factor- (HGF-) Induced Migration and Invasion and Actin Rearrangement via Suppression of c-Met-Mediated RhoA/ROCK Signaling in Glioblastoma

Paeoniflorin (PF), as one of the important valid natural compounds of the total glucosides of peony, has displayed a potential effect in cancer prevention and treatment. Aggressive migration and invasion, as an important process, can contribute to tumor progression through infiltrating the surround normal tissue. Actin cytoskeleton rearrangement plays a key role in cells migration and invasion, involving multiple signal pathways. HGF/c-Met signal, as an important couple of oncoprotein, has been demonstrated to regulate actin cytoskeleton rearrangement. In our study, we aim to explore whether paeoniflorin can inhibit migration and invasion and actin cytoskeleton rearrangement via regulation of HGF/c-Met/RhoA/ROCK signal. Various approaches were applied to demonstrate the mechanism of paeoniflorin-mediated anticancer effect, including cell wound healing assay, invasion assay, immunofluorescence staining and transfection, and western blotting. We observed that paeoniflorin inhibited HGF-induced migration and invasion and actin cytoskeleton rearrangement in glioblastoma cells. Furthermore, the inhibition of HGF-induced migration and invasion and actin cytoskeleton rearrangement involved c-Met-mediated RhoA/ROCK signaling in glioblastoma. Thus, our study proved that paeoniflorin could inhibit migration and invasion and actin cytoskeleton rearrangement through inhibition of HGF/c-Met/RhoA/ROCK signaling in glioblastoma, suggesting that paeoniflorin might be a candidate compound to treat glioblastoma.