Stabilization Of F-actin Research Articles

Epithelial Gpr116 regulates pulmonary alveolar homeostasis via Gq/11 signaling.

Pulmonary function is dependent upon the precise regulation of alveolar surfactant. Alterations in pulmonary surfactant concentrations or function impair ventilation and cause tissue injury. Identification of the molecular pathways that sense and regulate endogenous alveolar surfactant concentrations, coupled with the ability to pharmacologically modulate them both positively and negatively, would be a major therapeutic advance for patients with acute and chronic lung diseases caused by disruption of surfactant homeostasis. The orphan adhesion GPCR GPR116 (also known as Adgrf5) is a critical regulator of alveolar surfactant concentrations. Here, we show that human and mouse GPR116 control surfactant secretion and reuptake in alveolar type II (AT2) cells by regulating guanine nucleotide-binding domain α q and 11 (Gq/11) signaling. Synthetic peptides derived from the ectodomain of GPR116 activated Gq/11-dependent inositol phosphate conversion, calcium mobilization, and cortical F-actin stabilization to inhibit surfactant secretion. AT2 cell-specific deletion of Gnaq and Gna11 phenocopied the accumulation of surfactant observed in Gpr116-/- mice. These data provide proof of concept that GPR116 is a plausible therapeutic target to modulate endogenous alveolar surfactant pools to treat pulmonary diseases associated with surfactant dysfunction.

Optimisation and asymptotic stability

ABSTRACTThe problems of unconstrained optimisation and establishing asymptotic stability have much in common. Understanding the analogy between these two sheds light on their interconnection and may lead to a number of new results. For instance, in this paper, we provide estimates of the rate of convergence when analysing asymptotic stability of differential equations, rather than just ascertain the very fact of stability. Also, standard methods for the design of Lyapunov functions (e.g. those having the meaning of the full energy of the system) turn out to be unsatisfactory from this point of view and have to be modified. These claims are exemplified in the paper by considering the heavy-ball method for function minimisation ‘in parallel’ with the problem of asymptotic stability for the synchronous motor equation.

Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons from Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization.

Chorea-acanthocytosis (ChAc) is a fatal neurodegenerative disease without a known cure. To gain pathophysiological insight, we newly established a human in vitro model using skin biopsies from ChAc patients to generate disease-specific induced pluripotent stem cells (iPSCs) and developed an efficient iPSC differentiation protocol providing striatal medium spiny neurons. Using patch-clamp electrophysiology, we detected a pathologically enhanced synaptic activity in ChAc neurons. Healthy control levels of synaptic activity could be restored by treatment of ChAc neurons with the F-actin stabilizer phallacidin and the Src kinase inhibitor PP2. Because Src kinases are involved in bridging the membrane to the actin cytoskeleton by membrane protein phosphorylation, our data suggest an actin-dependent mechanism of this dysfunctional phenotype and potential treatment targets in ChAc.

Abstract 5064: CORO1B amplification and expression status identifies an aggressive subset of chromosome 11q13 amplified HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) is highly invasive cancer type that occurs with greater incidence in West Virginia and the rest of Appalachia. The chromosome 11q13 region is amplified in approximately 30% of late stage HNSCC cases and is associated with a poor patient prognosis. The core 11q13 region encodes the well-studied tumor genes CCND1 (cyclin D1) and CTTN (cortactin). The CORO1B (coronin 1B) gene flanks the 11q13 core and is amplified in 9-14% of all HNSCC, but how CORO1B amplification contributes to HNSCC is unknown. Coronin 1B governs cell motility by negatively regulating F-actin stability through displacement of cortactin at actin related protein 2/3 (Arp 2/3) branch points, generating dynamic turnover of Arp2/3-F-actin networks required for productive cell movement. Kaplan-Meier analysis of two independent patient cohorts indicates that HNSCC cases with CORO1B amplification have significantly reduced overall survival. Cox hazard ratios also indicate the CORO1B amplification is strongly associated with increased mortality in both cohorts. Automated quantitative analysis (AQUA) of coronin 1B expression in HNSCC tissue microarrays indicates that coronin 1B overexpression decreases median survival of HNSCC patients from 81 to 29 months. Reduced expression of coronin 1B by RNAi-mediated knockdown in established HNSCC cell lines decreases several actin-mediated invasive processes, including invadopodia formation and extracelluar matrix degradation. Collectively these results suggest that elevated coronin 1B expression levels in HNSCC function to enhance tumor invasion, potentially through accelerated downregulation of cortactin stabilizing activity at Arp2/3-F-actin junctions. Importantly, CORO1B amplification and coronin 1B expression status may serve a role as a precision biomarker for identification of a subset of late-stage HNSCC patients that would benefit from more aggressive forms of initial clinical treatment. Citation Format: Jessica L. Allen, Elyse L. Walk, Kristen L. Rhodes, Colleen J. Beatty, Steven M. Markwell, Brenen W. Papenberg, Erik T. Interval, Hong Wu, Marileila Varella Garcia, James E. Bear, Scott A. Weed. CORO1B amplification and expression status identifies an aggressive subset of chromosome 11q13 amplified HNSCC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5064.

High-mobility group box 1 inhibits HCO3- absorption in the medullary thick ascending limb through RAGE-Rho-ROCK-mediated inhibition of basolateral Na+/H+ exchange.

High-mobility group box 1 (HMGB1) is a nuclear protein released extracellularly in response to infection or injury, where it activates immune responses and contributes to the pathogenesis of kidney dysfunction in sepsis and sterile inflammatory disorders. Recently, we demonstrated that HMGB1 inhibits HCO3 (-) absorption in perfused rat medullary thick ascending limbs (MTAL) through a basolateral receptor for advanced glycation end products (RAGE)-dependent pathway that is additive to Toll-like receptor 4 (TLR4)-ERK-mediated inhibition by LPS (Good DW, George T, Watts BA III. Am J Physiol Renal Physiol 309: F720-F730, 2015). Here, we examined signaling and transport mechanisms that mediate inhibition by HMGB1. Inhibition of HCO3 (-) absorption by HMGB1 was eliminated by the Rho-associated kinase (ROCK) inhibitor Y27632 and by a specific inhibitor of Rho, the major upstream activator of ROCK. HMGB1 increased RhoA and ROCK1 activity. HMGB1-induced ROCK1 activation was eliminated by the RAGE antagonist FPS-ZM1 and by inhibition of Rho. The Rho and ROCK inhibitors had no effect on inhibition of HCO3 (-) absorption by bath LPS. Inhibition of HCO3 (-) absorption by HMGB1 was eliminated by bath amiloride, 0 Na(+) bath, and the F-actin stabilizer jasplakinolide, three conditions that selectively prevent inhibition of MTAL HCO3 (-) absorption mediated through NHE1. HMGB1 decreased basolateral Na(+)/H(+) exchange activity through activation of ROCK. We conclude that HMGB1 inhibits HCO3 (-) absorption in the MTAL through a RAGE-RhoA-ROCK1 signaling pathway coupled to inhibition of NHE1. The HMGB1-RAGE-RhoA-ROCK1 pathway thus represents a potential target to attenuate MTAL dysfunction during sepsis and other inflammatory disorders. HMGB1 and LPS inhibit HCO3 (-) absorption through different receptor signaling and transport mechanisms, which enables these pathogenic mediators to act directly and independently to impair MTAL function.

Syndapin promotes pseudocleavage furrow formation by actin organization in the syncytial Drosophila embryo.

Coordinated membrane and cytoskeletal remodeling activities are required for membrane extension in processes such as cytokinesis and syncytial nuclear division cycles in Drosophila Pseudocleavage furrow membranes in the syncytial Drosophila blastoderm embryo show rapid extension and retraction regulated by actin-remodeling proteins. The F-BAR domain protein Syndapin (Synd) is involved in membrane tubulation, endocytosis, and, uniquely, in F-actin stability. Here we report a role for Synd in actin-regulated pseudocleavage furrow formation. Synd localized to these furrows, and its loss resulted in short, disorganized furrows. Synd presence was important for the recruitment of the septin Peanut and distribution of Diaphanous and F-actin at furrows. Synd and Peanut were both absent in furrow-initiation mutants of RhoGEF2 and Diaphanous and in furrow-progression mutants of Anillin. Synd overexpression in rhogef2 mutants reversed its furrow-extension phenotypes, Peanut and Diaphanous recruitment, and F-actin organization. We conclude that Synd plays an important role in pseudocleavage furrow extension, and this role is also likely to be crucial in cleavage furrow formation during cell division.

Nogo‐A controls structural plasticity at dendritic spines by rapidly modulating actin dynamics

Nogo-A and its receptors have been shown to control synaptic plasticity, including negatively regulating long-term potentiation (LTP) in the cortex and hippocampus at a fast time scale and restraining experience-dependent turnover of dendritic spines over days. However, the molecular mechanisms and the precise time course mediating these actions of Nogo-A are largely unexplored. Here we show that Nogo-A signaling in the adult nervous system rapidly modulates the spine actin cytoskeleton within minutes to control structural plasticity at dendritic spines of CA3 pyramidal neurons. Indeed, acute Nogo-A loss-of-function transiently increases F-actin stability and results in an increase in dendritic spine density and length. In addition, Nogo-A acutely restricts AMPAR insertion and mEPSC amplitude at hippocampal synaptic sites. These data indicate a crucial function of Nogo-A in modulating the very tight balance between plasticity and stability of the neuronal circuitry underlying learning processes and the ability to store long-term information in the mature CNS. © 2016 Wiley Periodicals, Inc.

A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics.

The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development.

Tropomyosin-1 protects transformed alveolar epithelial cells against cigaret smoke extract through the stabilization of F-actin-dependent cell–cell junctions

The aim of the study was to estimate the effect of tropomyosin-1-based structural stabilization of F-actin in transformed human alveolar epithelial line H1299 cells subjected to high oxidative stress induced by cigaret smoke extract. We demonstrated here that cigaret smoke extract induces cell shrinking and detachment as a consequence of F-actin cytoskeleton degradation in H1299 cells not overexpressing tropomyosin-1. Furthermore, the treatment of these cells with cigaret smoke extract resulted in the loss of peripheral localization of ZO-1 and initiated apoptosis. In contrast, structural stabilization of F-actin, by overexpression of tropomyosin-1, preserved cell to cell interactions through the attenuation of cortical actin organization into thin fibers and thus protected these cells against oxidative stress-induced degradation of actin cytoskeleton and cell death. In conclusion, we suggest that structural stabilization of thin cortical F-actin fibers increases link between tight junctions proteins and actin cytoskeleton and thus protects H1299 cells against cigaret smoke extract.

Aura (mid1ip1l) regulates the cytoskeleton at the zebrafish egg-to-embryo transition.

Embryos from females homozygous for a recessive maternal-effect mutation in the gene aura exhibit defects including reduced cortical integrity, defective cortical granule (CG) release upon egg activation, failure to complete cytokinesis, and abnormal cell wound healing. We show that the cytokinesis defects are associated with aberrant cytoskeletal reorganization during furrow maturation, including abnormal F-actin enrichment and microtubule reorganization. Cortical F-actin prior to furrow formation fails to exhibit a normal transition into F-actin-rich arcs, and drug inhibition is consistent with aura function promoting F-actin polymerization and/or stabilization. In mutants, components of exocytic and endocytic vesicles, such as Vamp2, Clathrin and Dynamin, are sequestered in unreleased CGs, indicating a need for CG recycling in the normal redistribution of these factors. However, the exocytic targeting factor Rab11 is recruited to the furrow plane normally at the tip of bundling microtubules, suggesting an alternative anchoring mechanism independent of membrane recycling. A positional cloning approach indicates that the mutation in aura is associated with a truncation of Mid1 interacting protein 1 like (Mid1ip1l), previously identified as an interactor of the X-linked Opitz G/BBB syndrome gene product Mid1. A Cas9/CRISPR-induced mutant allele in mid1ip1l fails to complement the originally isolated aura maternal-effect mutation, confirming gene assignment. Mid1ip1l protein localizes to cortical F-actin aggregates, consistent with a direct role in cytoskeletal regulation. Our studies indicate that maternally provided aura (mid1ip1l) acts during the reorganization of the cytoskeleton at the egg-to-embryo transition and highlight the importance of cytoskeletal dynamics and membrane recycling during this developmental period.

Sinoporphyrin sodium mediated photodynamic therapy inhibits the migration associated with collapse of F-actin filaments cytoskeleton in MDA-MB-231 cells

ObjectiveWe previously demonstrated that the photosensitizer sinoporphyrin sodium (DVDMS) mediated photodynamic therapy (PDT) had potential advantages in inhibiting tumor growth and metastasis. However, details regarding the mechanism of cell migration inhibition remain unclear. Therefore, in this study, we aimed to investigate the effects of DVDMS-PDT on F-actin filaments, cell migration, apoptotic response and the possible interactions between them in human breast cancer MDA-MB-231 cells. Materials and methodsThe cell viability was evaluated by MTT and Guava ViaCount assays. The subcellular localization of DVDMS and reactive oxygen species (ROS) generation were analyzed by fluorescence microscope and flow cytometry. FITC-phalloidin was used to evaluate the changes of F-actin filaments. Cell migration was analyzed by scratch assay and Transwell assay. Cell apoptosis was determined by nuclear TUNEL staining and Annexin V-PE/7-AAD assay. Jasplakinolide, an F-actin stabilizer, was applied to dissect the influences of F-actin filaments disruption on cell migration and apoptosis. ResultsDVDMS-PDT significantly suppressed cell proliferation, promoted early apoptotic response, triggered collapse of F-actin filaments and inhibited cell migration in MDA-MB-231 cells. Cell migration significantly increased when cells were pretreated with F-actin stabilizer jasplakinolide after PDT, while cell apoptosis was not obviously affected. Moreover, ROS was a key factor in causing collapse of F-actin filaments. ConclusionWe demonstrated that DVDMS-PDT triggered cell apoptosis and collapse of F-actin filaments through the induction of ROS in MDA-MB-231 cells. F-actin filaments contributed to cell migration but produced no obvious effect on cell apoptosis.

Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation

Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.

14-3-3 λ protein interacts with ADF1 to regulate actin cytoskeleton dynamics in Arabidopsis.

Actin cytoskeleton dynamics is critical for variety of cellular events including cell elongation, division and morphogenesis, and is tightly regulated by numerous groups of actin binding proteins. However it is not well understood how these actin binding proteins are modulated in a physiological condition by their interaction proteins. In this study, we describe that Arabidopsis 14-3-3 λ protein interacted with actin depolymerizing factor 1 (ADF1) in plant to regulate F-actin stability and dynamics. Loss of 14-3-3 λ in Arabidopsis resulted in longer etiolated hypocotyls in dark and changed actin cytoskeleton architecture in hypocotyl cells. Overexpression of ADF1 repressed 14-3-3 λ mutant hypocotyl elongation and actin dynamic phenotype. In addition, the phosphorylation level of ADF1 was increased and the subcellular localization of ADF1 was altered in 14-3-3 λ mutant. Consistent with these observations, the actin filaments were more stable in 14-3-3 λ mutant. Our results indicate that 14-3-3 λ protein mediates F-actin dynamics possibly through inhibiting ADF1 phosphorylation in vivo.

Overexpressed Calponin3 by Subsonic Vibration Induces Neural Differentiation of hUC-MSCs by Regulating the Ionotropic Glutamate Receptor.

In this study, we used proteomics to investigate the effects of sonic vibration (SV) on mesenchymal stem cells derived from human umbilical cords (hUC-MSCs) during neural differentiation to understand how SV enhances neural differentiation of hUC-MSCs. We investigated the levels of gene and protein related to neural differentiation after 3 or 5 days in a group treated with 40-Hz SV. In addition, protein expression patterns were compared between the control and the 40-Hz SV-treated hUC-MSC groups via a proteomic approach. Among these proteins, calponin3 (CNN3) was confirmed to have 299 % higher expression in the 40-Hz SV stimulated hUC-MSCs group than that in the control by Western blotting. Notably, overexpression of CNN3-GFP in Chinese hamster ovary (CHO)-K1 cells had positive effects on the stability and reorganization of F-actin compared with that in GFP-transfected cells. Moreover, CNN3 changed the morphology of the cells by making a neurite-like form. After being subjected to SV, messenger RNA (mRNA) levels of glutamate receptors such as PSD95, GluR1, and NR1 as well as intracellular calcium levels were upregulated. These results suggest that the activity of glutamate receptors increased because of CNN3 characteristics. Taken together, these results demonstrate that overexpressed CNN3 during SV increases expression of glutamate receptors and promotes functional neural differentiation of hUC-MSCs.

CXCL16/CXCR6 chemokine signaling mediates breast cancer progression by pERK1/2-dependent mechanisms.

Our previous studies demonstrate that CXCL6/CXCR6 chemokine axis induces prostate cancer progression by the AKT/mTOR signaling pathway; however, its role and mechanisms underlying invasiveness and metastasis of breast cancer are yet to be elucidated. In this investigation, CXCR6 protein expression was examined using high-density tissue microarrays and immunohistochemistry. Expression of CXCR6 shows a higher epithelial staining in breast cancer nest site and metastatic lymph node than the normal breast tissue, suggesting that CXCR6 may be involved in breast cancer (BC) development. In vitro and in vivo experiments indicate that overexpression of CXCR6 in BC cells has a marked effect on increasing cell migration, invasion and metastasis. In contrast, reduction of CXCR6 expression by shRNAs in these cells greatly reduce its invasion and metastasis ability. Mechanistic analyses show that CXCL16/CXCR6 chemokine axis is capable of modulating activation of RhoA through activating ERK1/2 signaling pathway, which then inhibits the activity of cofilin, thereby enhancing the stability of F-actin, responsible for invasiveness and metastasis of BC. Taken together, our data shows for the first time that the CXCR6 / ERK1/2/ RhoA / cofilin /F-actin pathway plays a central role in the development of BC. Targeting the signaling pathway may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for BC.

Blockade of intracellular Zn2+ signaling in the dentate gyrus erases recognition memory via impairment of maintained LTP.

There is no evidence on the precise role of synaptic Zn2+ signaling on the retention and recall of recognition memory. On the basis of the findings that intracellular Zn2+ signaling in the dentate gyrus is required for object recognition, short-term memory, the present study deals with the effect of spatiotemporally blocking Zn2+ signaling in the dentate gyrus after LTP induction and learning. Three-day-maintained LTP was impaired 1 day after injection of clioquinol into the dentate gyrus, which transiently reduced intracellular Zn2+ signaling in the dentate gyrus. The irreversible impairment was rescued not only by co-injection of ZnCl2 , which ameliorated the loss of Zn2+ signaling, but also by pre-injection of Jasplakinolide, a stabilizer of F-actin, prior to clioquinol injection. Simultaneously, 3-day-old space recognition memory was impaired 1 day after injection of clioquinol into the dentate gyrus, but not by pre-injection of Jasplakinolide. Jasplakinolide also rescued both impairments of 3-day-maintained LTP and 3-day-old memory after injection of ZnAF-2DA into the dentate gyrus, which blocked intracellular Zn2+ signaling in the dentate gyrus. The present paper indicates that the blockade and/or loss of intracellular Zn2+ signaling in the dentate gyrus coincidently impair maintained LTP and recognition memory. The mechanism maintaining LTP via intracellular Zn2+ signaling in dentate granule cells, which may be involved in the formation of F-actin, may retain space recognition memory.

A mechanical refractory period of chondrocytes after dynamic hydrostatic pressure

Purpose: Mechanical stimulation, a crucial factor for maintaining the cartilaginous phenotype and promoting the chondrogenesis, has been widely used in autologous chondrocyte transplantation. This study was designed to investigate a novel concept of mechanical refractory period of chondrocytes after dynamic hydrostatic pressure (dHP).Materials and methods: dHP protocols (0.1 Hz, 2 MPa) were applied. The variation in type II collagen (Col II) expression induced by each dHP unit was measured. The dynamic remodeling of F-actin during the mechanical protocols was observed morphologically and mechanically by laser confocal microscopy and optical magnetic twisting cytometry (OMTC), respectively. About 20 ng/ml VEGF was used to stabilize the F-actin and restrain the mechanical refractory period.Results: Compared with the remarkable increase of Col II (16-fold) induced by the initial dHP unit, the chondrocytes entered a mechanical refractory period and the second unit hardly elevated Col II expression (only 2.9-fold). This refractory period recovered partially within 2 h. The uniform, parallel, and coarse fibers of F-actin before dHP became thin, sparse, and disordered, and the cell stiffness decreased concomitantly. The variations in both the morphology and the mechanical property of F-actin were highly synchronous to the mechanical refractory period and recovered in a time-dependent manner. VEGF postponed the appearance of this refractory period and maintained the high expression of Col II by VEGF/p38/MAPKAPK-2/LIMK/cofilin pathway.Conclusion: A mechanical refractory period of chondrocytes has been discovered and defined in this study. The F-actin depolymerization is the putative mechanism, and this refractory period can be postponed by VEGF-induced F-actin stabilization.

Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy.

Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells. Importance: HIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.

F-actin cytoskeleton reorganization is associated with hepatic stellate cell activation

The activation of hepatic stellate cells (HSCs) is involved in the development of hepatic fibrosis. Previous studies have indicated that the acquisition of certain properties by activated HSCs is highly dependent on the reorganization of the actin cytoskeleton. However, direct evidence showing that the reorganization of the actin cytoskeleton is responsible for HSC activation is lacking. The aim of the present study was to investigate the role of cytoskeletal reorganization during HSC activation and to clarify the underlying mechanism. HSC-T6 cells were treated either with the F-actin stabilizer jasplakinolide (Jas) or the depolymerizer cytochalasin D (Cyto D). The actin cytoskeleton was evaluated via assessment of stress fiber formation. Furthermore, the activation properties of HSCs, including proliferation, adhesion, migration and the expression of α-smooth muscle actin (α-SMA) and collagen 1, were investigated in vitro. The results showed that Jas and Cyto D affected the actin distribution in HSC-T6 cells. Treatment with Jas resulted in thick actin bundles and a patchy appearance in the cytoplasm in HSC-T6 cells. In parallel, polymerization of actin microfilaments induced by Jas upregulated the expression of α-SMA and collagen 1, and also enhanced the migration and adhesion properties of HSC-T6 cells. Furthermore, the activation of HSC-T6 cells induced by the reorganization of the actin cytoskeleton was associated with the p38 mitogen-activated protein kinase (p38 MAPK) pathway. In conclusion, the present study suggests that the reorganization of the F-actin cytoskeleton is associated with HSC activation and that the p38 MAPK pathway is involved in this process. The inhibition of F-actin reorganization may thus be a potential key factor or molecular target for the control of liver fibrosis or cirrhosis.

Lateral junction dynamics lead the way out

Epithelial cell layers need to be tightly regulated to maintain their integrity and correct function. Cell integration into epithelial sheets is now shown to depend on the N-WASP-regulated stabilization of cortical F-actin, which generates distinct patterns of apical-lateral contractility at E-cadherin-based cell-cell junctions.