Actin Reorganization Research Articles

Hepatitis B virus X protein enhances liver cancer cell migration by regulating calmodulin-associated actin polymerization

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), which is a highly aggressive cancer. HBV X protein (HBx), one of four HBV gene products, plays pivotal roles in the development and metastasis of HCC. It has been reported that HBx induces liver cancer cell migration and reorganizes actin cytoskeleton, however the molecular basis for actin cytoskeleton reorganization remains obscure. In this study, we for the first time report that HBx promotes actin polymerization and liver cancer cell migration by regulating calcium modulated protein, calmodulin (CaM). HBx physically interacts with CaM to control the level of phosphorylated cofilin, an actin depolymerizing factor. Mechanistically, HBx interacts with CaM, liberates Hsp90 from its inhibitory partner CaM, and increases the activity of Hsp90, thus activating LIMK1/cofilin pathway. Interestingly, the interaction between HBx and CaM is calcium-dependent and requires the CaM binding motif on HBx. These results indicate that HBx modulates CaM which plays a regulatory role in Hsp90/LIMK1/cofilin pathway of actin reorganization, suggesting a new mechanism of HBV-induced HCC metastasis specifically derived by HBx.

Deletion of gmfA induces keratocyte-like migration in Dictyostelium.

Glia maturation factor (GMF) has been established as an inactivating factor of the actin‐related protein 2/3 (Arp2/3) complex, which regulates actin assembly. Regulation of actin assembly and reorganization is crucial for various cellular events, such as cell migration, cell division, and development. Here, to examine the roles of ADF‐H domain‐containing protein (also known as glia maturation factor; GmfA), the product of a single GMF homologous gene in Dictyostelium, gmfA‐null cells were generated. They had moderate defects in cell growth and cytokinesis. Interestingly, they showed a keratocyte‐like fan shape with a broader pseudopod, where Arp3 accumulated at higher levels than in wild‐type cells. They migrated with higher persistence, but their velocities were comparable to those of wild‐type cells. The polar pseudopods during cell division were also broader than those in wild‐type cells. However, GmfA did not localize at the pseudopods in migrating cells or the polar pseudopods in dividing cells. Adhesions of mutant cells to the substratum were much stronger than that of wild‐type cells. Although the mutant cells showed chemotaxis comparable to that of wild‐type cells, they formed disconnected streams during the aggregation stage; however, they finally formed normal fruiting bodies. These results suggest that GmfA plays a crucial role in cell migration.

Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P2 and PI4P in Human Platelets.

Phosphoinositides (PIs) are phosphorylated membrane lipids that have a plethora of roles in the cell, including vesicle trafficking, signaling, and actin reorganization. The most abundant PIs in the cell are phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-4-monophosphate (PI4P). The localization and roles of both PI(4,5)P2 and PI4P are well established, is the broadly accepted methodological approach for their immunocytochemical visualization in different cell compartments in several cell lines. However, not much is known about these PIs in platelets (PLTs), the smallest blood cells that detect vessel wall injury, activate, and stop the bleeding. Therefore, we sought to investigate the localization of PI(4,5)P2 and PI4P in resting and activated PLTs by antibody staining. Here, we show that the intracellular pools of PI(4,5)P2 and PI4P can be detected by the established staining protocol, and these pools can be modulated by inhibitors of OCRL phosphatase and PI4KIIIα kinase. However, although resting PLTs readily stain for the plasma membrane (PM) pools of PI(4,5)P2 and PI4P, just a few activated cells were stained with the established protocol. We show that optimized protocol allows for the visualization of PI(4,5)P2 and PI4P at PM in activated PLTs, which could also be modulated by OCRL and PI4KIIIα inhibitors. We conclude that PI(4,5)P2 and PI4P are more sensitive to lipid extraction by permeabilizing agents in activated than in resting human PLTs, which suggests their different roles during PLT activation.

Identifying the hub genes for Duchenne muscular dystrophy and Becker muscular dystrophy by weighted correlation network analysis

BackgroundThe goal of this study is to identify the hub genes for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) via weighted correlation network analysis (WGCNA).MethodsThe gene expression profile of vastus lateralis biopsy samples obtained in 17 patients with DMD, 11 patients with BMD and 6 healthy individuals was downloaded from the Gene Expression Omnibus (GEO) database (GSE109178). After obtaining different expressed genes (DEGs) via GEO2R, WGCNA was conducted using R package, modules and genes that highly associated with DMD, BMD, and their age or pathology were screened. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analysis and protein–protein interaction (PPI) network analysis were also conducted. Hub genes and highly correlated clustered genes were identified using Search Tool for the Retrieval of Interacting Genes (STRING) and Cystoscape software.ResultsOne thousand four hundred seventy DEGs were identified between DMD and control, with 1281 upregulated and 189 downregulated DEGs. Four hundred and twenty DEGs were found between BMD and control, with 157 upregulated and 263 upregulated DEGs. Fourteen modules with different colors were identified for DMD vs control, and 7 modules with different colors were identified for BMD vs control. Ten hub genes were summarized for DMD and BMD respectively, 5 hub genes were summarized for BMD age, 5 and 3 highly correlated clustered genes were summarized for DMD age and BMD pathology, respectively. In addition, 20 GO enrichments were found to be involved in DMD, 3 GO enrichments were found to be involved in BMD, 3 GO enrichments were found to be involved in BMD age.ConclusionIn DMD, several hub genes were identified: C3AR1, TLR7, IRF8, FYB and CD33(immune and inflammation associated genes), TYROBP, PLEK, AIF1(actin reorganization associated genes), LAPTM5 and NT5E(cell death and arterial calcification associated genes, respectively). In BMD, a number of hub genes were identified: LOX, ELN, PLEK, IKZF1, CTSK, THBS2, ADAMTS2, COL5A1(extracellular matrix associated genes), BCL2L1 and CDK2(cell cycle associated genes).

The phosphoinositide coincidence detector Phafin2 promotes macropinocytosis by coordinating actin organisation at forming macropinosomes

Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and disassembly are coordinated around macropinosomes to allow them to form and subsequently pass through the dense actin network underlying the plasma membrane to move towards the cell center for maturation. Here we show that the PH and FYVE domain protein Phafin2 is recruited transiently to newly-formed macropinosomes by a mechanism that involves coincidence detection of PtdIns3P and PtdIns4P. Phafin2 also interacts with actin via its PH domain, and recruitment of Phafin2 coincides with actin reorganization around nascent macropinosomes. Moreover, forced relocalization of Phafin2 to the plasma membrane causes rearrangement of the subcortical actin cytoskeleton. Depletion of Phafin2 inhibits macropinosome internalization and maturation and prevents KRAS-transformed cancer cells from utilizing extracellular protein as an amino acid source. We conclude that Phafin2 promotes macropinocytosis by controlling timely delamination of actin from nascent macropinosomes for their navigation through the dense subcortical actin network.

Phagocytic 'teeth' and myosin-II 'jaw' power target constriction during phagocytosis.

Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely driven by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f ('teeth') that appear to be interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, supporting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings present a phagocytic cup shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

Atorvastatin Modulates the Efficacy of Electroporation and Calcium Electrochemotherapy

Electroporation is influenced by the features of the targeted cell membranes, e.g., the cholesterol content and the surface tension of the membrane. The latter is eventually affected by the organization of actin fibers. Atorvastatin is a statin known to influence both the cholesterol content and the organization of actin. This work analyzes the effects of the latter on the efficacy of electroporation of cancer cells. In addition, herein, electroporation was combined with calcium chloride (CaEP) to assess as well the effects of the statin on the efficacy of electrochemotherapy. Cholesterol-rich cell lines MDA-MB231, DU 145, and A375 underwent (1) 48 h preincubation or (2) direct treatment with 50 nM atorvastatin. We studied the impact of the statin on cholesterol and actin fiber organization and analyzed the cells’ membrane permeability. The viability of cells subjected to PEF (pulsed electric field) treatments and CaEP with 5 mM CaCl2 was examined. Finally, to assess the safety of the therapy, we analyzed the N-and E-cadherin localization using confocal laser microscopy. The results of our investigation revealed that depending on the cell line, atorvastatin preincubation decreases the total cholesterol in the steroidogenic cells and induces reorganization of actin nearby the cell membrane. Under low voltage PEFs, actin reorganization is responsible for the increase in the electroporation threshold. However, when subject to high voltage PEF, the lipid composition of the cell membrane becomes the regulatory factor. Namely, preincubation with atorvastatin reduces the cytotoxic effect of low voltage pulses and enhances the cytotoxicity and cellular changes induced by high voltage pulses. The study confirms that the surface tension regulates of membrane permeability under low voltage PEF treatment. Accordingly, to reduce the unfavorable effects of preincubation with atorvastatin, electroporation of steroidogenic cells should be performed at high voltage and combined with a calcium supply.

Cereblon regulates NK cell cytotoxicity and migration via Rac1 activation.

Rearrangement of the actin cytoskeleton is critical for cytotoxic and immunoregulatory functions as well as migration of natural killer (NK) cells. However, dynamic reorganization of actin is a complex process, which remains largely unknown. Here, we investigated the role of the protein Cereblon (CRBN), an E3 ubiquitin ligase complex co‐receptor and the primary target of the immunomodulatory drugs, in NK cells. We observed that CRBN partially colocalizes with F‐actin in chemokine‐treated NK cells and is recruited to the immunological synapse, thus suggesting a role for this protein in cytoskeleton reorganization. Accordingly, silencing of CRBN in NK cells results in a reduced cytotoxicity that correlates with a defect in conjugate and lytic synapse formation. Moreover, CRBN depletion significantly impairs the ability of NK cells to migrate and reduces the enhancing effect of lenalidomide on NK cell migration. Finally, we provided evidence that CRBN is required for activation of the small GTPase Rac1, a critical mediator of cytoskeleton dynamics. Indeed, in CRBN‐depleted NK cells, chemokine‐mediated or target cell–mediated Rac1 activation is significantly reduced. Altogether our data identify a critical role for CRBN in regulating NK cell functions and suggest that this protein may mediate the stimulatory effect of lenalidomide on NK cells.

PIP2 and septin control STIM1/Orai1 assembly by regulating cytoskeletal remodeling via a CDC42-WASP/WAVE-ARP2/3 protein complex

Store-operated calcium entry (SOCE) is triggered by assembly of Orai1 with STIM proteins in ER-PM junctions. Plasma membrane PIP2 as well as PIP2-binding protein, SEPT4, significantly impact Orai1-STIM1 interaction. While septins and PIP2 can organize the actin cytoskeleton, it is unclear whether the status of actin within the junctions contributes to SOCE. We report herein that actin remodeling modulates STIM1 clustering. Our findings show that a PIP2- and SEPT4-dependent mechanism involving CDC42, WASP/WAVE, and ARP2 regulates actin remodeling into a ring-like structure around STIM1 puncta. CDC42 localization in the ER-plasma membrane region is enhanced following ER-Ca2+ store depletion. PIP2 depletion or knockdown of SEPT4 attenuate the recruitment of CDC42 to the ER-PM region. Importantly, knockdown of SEPT4, or CDC42+ARP2, disrupts the organization of actin as well as STIM1 clustering. Consequently, Orai1 recruitment to STIM1 puncta, SOCE, and NFAT translocation to the nucleus are all attenuated. Ca2+ influx induced by STIM1-C terminus is not affected by CDC42 knockdown. In aggregate, our findings reveal that PIP2 and SEPT4 affect Orai1/STIM1 clustering by coordinating actin remodeling within ER-PM junctions. This dynamic reorganization of actin has an important role in regulation of SOCE and downstream Ca2+-dependent effector functions.

GhADF6-mediated actin reorganization is associated with defence against Verticillium dahliae infection in cotton.

Several studies have revealed that actin depolymerizing factors (ADFs) participate in plant defence responses; however, the functional mechanisms appear intricate and need further exploration. In this study, we identified an ADF6 gene in upland cotton (designated as GhADF6) that is evidently involved in cotton's response to the fungal pathogen Verticillium dahliae. GhADF6 binds to actin filaments and possesses actin severing and depolymerizing activities in vitro and in vivo. When cotton root (the site of the fungus invasion) was inoculated with the pathogen, the expression of GhADF6 was markedly down‐regulated in the epidermal cells. By virus‐induced gene silencing analysis, the down‐regulation of GhADF6 expression rendered the cotton plants tolerant to V. dahliae infection. Accordingly, the abundance of actin filaments and bundles in the root cells was significantly higher than that in the control plant, which phenocopied that of the V. dahliae‐challenged wild‐type cotton plant. Altogether, our results provide evidence that an increase in filament actin (F‐actin) abundance as well as dynamic actin remodelling are required for plant defence against the invading pathogen, which are likely to be fulfilled by the coordinated expressional regulation of the actin‐binding proteins, including ADF.

A Framework of Paracellular Transport via Nanoparticles-Induced Endothelial Leakiness.

Nanomaterial‐induced endothelial leakiness (NanoEL) is an interfacial phenomenon denoting the paracellular transport of nanoparticles that is pertinent to nanotoxicology, nanomedicine and biomedical engineering. While the NanoEL phenomenon is complementary to the enhanced permeability and retention effect in terms of their common applicability to delineating the permeability and behavior of nanoparticles in tumoral environments, these two effects significantly differ in scope, origin, and manifestation. In the current study, the descriptors are fully examined of the NanoEL phenomenon elicited by generic citrate‐coated gold nanoparticles (AuNPs) of changing size and concentration, from microscopic gap formation and actin reorganization down to molecular signaling pathways and nanoscale interactions of AuNPs with VE‐cadherin and its intra/extracellular cofactors. Employing synergistic in silico methodologies, for the first time the molecular and statistical mechanics of cadherin pair disruption, especially in response to AuNPs of the smallest size and highest concentration are revealed. This study marks a major advancement toward establishing a comprehensive NanoEL framework for complementing the understanding of the transcytotic pathway and for guiding the design and application of future nanomedicines harnessing the myriad functions of the mammalian vasculature.

A kinase-independent function of PAK is crucial for pathogen-mediated actin remodelling.

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor β-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.

Mechanical competition alters the cellular interpretation of an endogenous genetic program.

The intrinsic genetic program of a cell is not sufficient to explain all of the cell's activities. External mechanical stimuli are increasingly recognized as determinants of cell behavior. In the epithelial folding event that constitutes the beginning of gastrulation in Drosophila, the genetic program of the future mesoderm leads to the establishment of a contractile actomyosin network that triggers apical constriction of cells and thereby tissue folding. However, some cells do not constrict but instead stretch, even though they share the same genetic program as their constricting neighbors. We show here that tissue-wide interactions force these cells to expand even when an otherwise sufficient amount of apical, active actomyosin is present. Models based on contractile forces and linear stress-strain responses do not reproduce experimental observations, but simulations in which cells behave as ductile materials with nonlinear mechanical properties do. Our models show that this behavior is a general emergent property of actomyosin networks in a supracellular context, in accordance with our experimental observations of actin reorganization within stretching cells.

BRAF Modulates Stretch-Induced Intercellular Gap Formation through Localized Actin Reorganization.

Mechanical forces acting on cell–cell adhesion modulate the barrier function of endothelial cells. The actively remodeled actin cytoskeleton impinges on cell–cell adhesion to counteract external forces. We applied stress on endothelial monolayers by mechanical stretch to uncover the role of BRAF in the stress-induced response. Control cells responded to external forces by organizing and stabilizing actin cables in the stretched cell junctions. This was accompanied by an increase in intercellular gap formation, which was prevented in BRAF knockdown monolayers. In the absence of BRAF, there was excess stress fiber formation due to the enhanced reorganization of actin fibers. Our findings suggest that stretch-induced intercellular gap formation, leading to a decrease in barrier function of blood vessels, can be reverted by BRAF RNAi. This is important when the endothelium experiences changes in external stresses caused by high blood pressure, leading to edema, or by immune or cancer cells in inflammation or metastasis.

Journey to the Center of the Cell: Cytoplasmic and Nuclear Actin in Immune Cell Functions.

Actin cytoskeletal dynamics drive cellular shape changes, linking numerous cell functions to physiological and pathological cues. Mutations in actin regulators that are differentially expressed or enriched in immune cells cause severe human diseases known as primary immunodeficiencies underscoring the importance of efficienct actin remodeling in immune cell homeostasis. Here we discuss recent findings on how immune cells sense the mechanical properties of their environement. Moreover, while the organization and biochemical regulation of cytoplasmic actin have been extensively studied, nuclear actin reorganization is a rapidly emerging field that has only begun to be explored in immune cells. Based on the critical and multifaceted contributions of cytoplasmic actin in immune cell functionality, nuclear actin regulation is anticipated to have a large impact on our understanding of immune cell development and functionality.

NGPF2 triggers synaptic scaling up through ALK-LIMK-cofilin-mediated mechanisms.

Synaptic scaling is an extensively studied form of homeostatic plasticity critically involved in various brain functions. Although it is accepted that synaptic scaling is expressed through the postsynaptic accumulation of AMPA receptors (AMPARs), the induction mechanism remains elusive. In this study, we show that TTX treatment induces rapid but transient release of the neurite growth-promoting factor 2 (NGPF2), and this release is necessary and sufficient for TTX-induced scaling up. In addition, we show that inhibition of the anaplastic lymphoma kinase (ALK)-LIMK-cofilin signaling pathway blocks TTX- and NGPF2-induced synaptic scaling up. Furthermore, we show that TTX-induced release of NGPF2 is protein synthesis dependent and requires fragile X mental retardation protein 1 (FMRP1). These results indicate that activity blockade induces NGPF2 synthesis and release to trigger synaptic scaling up through LIMK-cofilin-dependent actin reorganization, spine enlargement, and stabilization of AMPARs at the synapse.

Ultrasmall Molybdenum Disulfide Quantum Dots Cage Alzheimer's Amyloid Beta to Restore Membrane Fluidity.

Alzheimer's disease (AD) is a major cause of dementia characterized by the overexpression of transmembrane amyloid precursor protein and its neurotoxic byproduct amyloid beta (Aβ). A small peptide of considerable hydrophobicity, Aβ is aggregation prone catalyzed by the presence of cell membranes, among other environmental factors. Accordingly, current AD mitigation strategies often aim at breaking down the Aβ-membrane communication, yet no data is available concerning the cohesive interplay of the three key entities of the cell membrane, Aβ, and its inhibitor. Using a lipophilic Laurdan dye and confocal fluorescence microscopy, we observed cell membrane perturbation and actin reorganization induced by Aβ oligomers but not by Aβ monomers or amyloid fibrils. We further revealed recovery of membrane fluidity by ultrasmall MoS2 quantum dots, also shown in this study as a potent inhibitor of Aβ amyloid aggregation. Using discrete molecular dynamics simulations, we uncovered the binding of MoS2 and Aβ monomers as mediated by hydrophilic interactions between the quantum dots and the peptide N-terminus. In contrast, Aβ oligomers and fibrils were surface-coated by the ultrasmall quantum dots in distinct testudo-like, reverse protein-corona formations to prevent their further association with the cell membrane and adverse effects downstream. This study offers a crucial new insight and a viable strategy for regulating the amyloid aggregation and membrane-axis of AD pathology with multifunctional nanomedicine.

Invasion and trafficking of hypervirulent group B streptococci in polarized enterocytes.

Streptococcus agalactiae (group B streptococcus or GBS) is a commensal bacterium that can frequently behave as a pathogen, particularly in the neonatal period and in the elderly. The gut is a primary site of GBS colonization and a potential port of entry during neonatal infections caused by hypervirulent clonal complex 17 (CC17) strains. Here we studied the interactions between the prototypical CC17 BM110 strain and polarized enterocytes using the Caco-2 cell line. GBS could adhere to and invade these cells through their apical or basolateral surfaces. Basolateral invasion was considerably more efficient than apical invasion and predominated under conditions resulting in weakening of cell-to-cell junctions. Bacterial internalization occurred by a mechanism involving caveolae- and lipid raft-dependent endocytosis and actin re-organization, but not clathrin-dependent endocytosis. In the first steps of Caco-2 invasion, GBS colocalized with the early endocytic marker EEA-1, to later reside in acidic vacuoles. Taken together, these data suggest that CC17 GBS selectively adheres to the lateral surface of enterocytes from which it enters through caveolar lipid rafts using a classical, actin-dependent endocytic pathway. These data may be useful to develop alternative preventive strategies aimed at blocking GBS invasion of the intestinal barrier.

Role of Intermediate Filaments in Blood-Brain Barrier in Health and Disease.

The blood–brain barrier (BBB) is a highly selective cellular monolayer unique to the microvasculature of the central nervous system (CNS), and it mediates the communication of the CNS with the rest of the body by regulating the passage of molecules into the CNS microenvironment. Limitation of passage of substances through the BBB is mainly due to tight junctions (TJ) and adherens junctions (AJ) between brain microvascular endothelial cells. The importance of actin filaments and microtubules in establishing and maintaining TJs and AJs has been indicated; however, recent studies have shown that intermediate filaments are also important in the formation and function of cell–cell junctions. The most common intermediate filament protein in endothelial cells is vimentin. Vimentin plays a role in blood–brain barrier permeability in both cell–cell and cell–matrix interactions by affecting the actin and microtubule reorganization and by binding directly to VE-cadherin or integrin proteins. The BBB permeability increases due to the formation of stress fibers and the disruption of VE–cadherin interactions between two neighboring cells in various diseases, disrupting the fiber network of intermediate filament vimentin in different ways. Intermediate filaments may be long ignored key targets in regulation of BBB permeability in health and disease.

Treponema denticola-Induced RASA4 Upregulation Mediates Cytoskeletal Dysfunction and MMP-2 Activity in Periodontal Fibroblasts.

The periodontal complex consists of the periodontal ligament (PDL), alveolar bone, and cementum, which work together to turn mechanical load into biological responses that are responsible for maintaining a homeostatic environment. However oral microbes, under conditions of dysbiosis, may challenge the actin dynamic properties of the PDL in the context of periodontal disease. To study this process, we examined host-microbial interactions in the context of the periodontium via molecular and functional cell assays and showed that human PDL cell interactions with Treponema denticola induce actin depolymerization through a novel actin reorganization signaling mechanism. This actin reorganization mechanism and loss of cell adhesion is a pathological response characterized by an initial upregulation of RASA4 mRNA expression resulting in an increase in matrix metalloproteinase-2 activity. This mechanism is specific to the T. denticola effector protein, dentilisin, thereby uncovering a novel effect for Treponema denticola-mediated RASA4 transcriptional activation and actin depolymerization in primary human PDL cells.