Polymerization Of G-actin Research Articles

Mild hypothermia therapy attenuates early BBB leakage in acute ischaemic stroke.

Reperfusion therapy inevitably leads to brain-blood barrier (BBB) disruption and promotes damage despite its benefits for acute ischaemic stroke (AIS). An effective brain cytoprotective treatment is still needed as an adjunct to reperfusion therapy. Here, we explore the potential benefits of therapeutic hypothermia (HT) in attenuating early BBB leakage and improving neurological outcomes. Mild HT was induced during the early and peri-recanalization stages in a mouse model of transient middle cerebral artery occlusion and reperfusion (tMCAO/R). The results showed that mild HT attenuated early BBB leakage in AIS, decreased the infarction volume, and improved functional outcomes. RNA sequencing data of the microvessels indicated that HT decreased the transcription of the actin polymerization-related pathway. We further discovered that HT attenuated the ROCK1/MLC pathway, leading to a decrease in the polymerization of G-actin to F-actin. Arachidonic acid (AA), a known structural ROCK agonist, partially counteracted the protective effects of HT in the tMCAO/R model. Our study highlights the importance of early vascular protection during reperfusion and provides a new strategy for attenuating early BBB leakage by HT treatment for ischaemic stroke.

Actin polymerization and depolymerization in developing vertebrates.

Development is a complex process that occurs throughout the life cycle. F-actin, a major component of the cytoskeleton, is essential for the morphogenesis of tissues and organs during development. F-actin is formed by the polymerization of G-actin, and the dynamic balance of polymerization and depolymerization ensures proper cellular function. Disruption of this balance results in various abnormalities and defects or even embryonic lethality. Here, we reviewed recent findings on the structure of G-actin and F-actin and the polymerization of G-actin to F-actin. We also focused on the functions of actin isoforms and the underlying mechanisms of actin polymerization/depolymerization in cellular and organic morphogenesis during development. This information will extend our understanding of the role of actin polymerization in the physiologic or pathologic processes during development and may open new avenues for developing therapeutics for embryonic developmental abnormalities or tissue regeneration.

Abstract P2015: A Human Flii Gene Variant Shortens Sarcomeric Actin Thin Filament Length And Predisposes To Cardiomyopathy

Objective: We identified a human variant in the Flightless-I homolog ( FLII ) gene that generates a R1243H missense change associated with cardiomyopathy in human genome-wide association studies. However, the causality and its molecular mechanism remain unclear. Methods and Results: To examine how the human variant resulting in R1243H amino acid substitution might be pathogenic in vivo, we generated a mouse model harboring the R1245H mutation in Flii locus using CRISPR/Cas9 technology. The Flii R1245H/R1245H homozygotes showed reduced cardiac function by 16 weeks of age compared with control ( P =0.01). Eight-week-old Flii R1245H/R1245H mice subjected to 8 weeks of TAC showed significantly worse cardiac function with greater cardiac hypertrophy and premature lethality compared with control ( P <0.01). Here we show that Flii functions in the heart to regulate sarcomeric actin thin filament length. Indeed, hearts from Flii R1245H/R1245H mice at 16 weeks of age showed reduced thin filament length compared with controls ( P =0.009). To further investigate the mechanistic effects of the R1245H mutation in mouse Flii , we examined actin polymerization dynamics in vitro between wild-type (WT) Flii and the R1245H mutant proteins. The data showed that WT Flii augmented G-actin polymerization into F-actin in a concentration dependent manner, while Flii-R1245H mutant protein antagonized polymerization ( P <0.001). Since LMOD2 is reported to elongate sarcomeric actin thin filaments, we overexpressed LMOD2 using AAV9 system in Flii R1245H/R1245H mice and conducted TAC surgeries at 8 weeks of age. AAV9-LMOD2 rescued the shortening of thin filament length in Flii R1245H/R1245H mice and rescued the exacerbated TAC induced pathology associated with this homozygous mutation (P<0.05). Conclusions: A human FLII variant, R1243H, shortens the sarcomeric actin thin filament, which predisposes to cardiomyopathy. Elongation of thin filament length by LMOD2 overexpression is a possible therapeutic strategy in humans harboring this R1243H mutation.

Low disease activity of microscopic polyangiitis in patients with anti-myosin light chain 6 antibody that disrupts actin rearrangement necessary for neutrophil extracellular trap formation

BackgroundNeutrophil extracellular traps (NETs) are critically involved in microscopic polyangiitis (MPA) pathogenesis, and some patients with MPA possess anti-NET antibody (ANETA). Anti-myosin light chain 6 (MYL6) antibody is an ANETA that affects NETs. This study aimed to determine the significance of anti-MYL6 antibody in MPA.MethodsThe influence of anti-MYL6 antibody on NET formation and actin rearrangement necessary for NET formation was assessed by fluorescent staining. An enzyme-linked immunosorbent assay was established to detect serum anti-MYL6 antibody, and the prevalence of this antibody in MPA was determined. Furthermore, the disease activity and response to remission-induction therapy of MPA were compared between anti-MYL6 antibody-positive and anti-MYL6 antibody-negative MPA patients.ResultsAnti-MYL6 antibody disrupted G-actin polymerization into F-actin, suppressing phorbol 12-myristate 13-acetate-induced NET formation. Serum anti-MYL6 antibody was detected in 7 of 59 patients with MPA. The Birmingham vasculitis activity score (BVAS) of anti-MYL6 antibody-positive MPA patients was significantly lower than anti-MYL6 antibody-negative MPA patients. Among the nine BVAS evaluation items, the cutaneous, cardiovascular, and nervous system scores of anti-MYL6 antibody-positive MPA patients were significantly lower than anti-MYL6 antibody-negative MPA patients, although other items, including the renal and chest scores, were equivalent between the two groups. The proportion of patients with remission 6 months after initiation of remission-induction therapy in anti-MYL6 antibody-positive MPA patients was significantly higher than in anti-MYL6 antibody-negative MPA patients.ConclusionsCollective findings suggested that anti-MYL6 antibody disrupted actin rearrangement necessary for NET formation and could reduce the disease activity of MPA.

Molecular Mechanism of Mouse Uterine Smooth Muscle Regulation on Embryo Implantation.

Myometrium plays critical roles in multiple processes such as embryo spacing through peristalsis during mouse implantation, indicating vital roles of smooth muscle in the successful establishment and quality of implantation. Actin, a key element of cytoskeleton structure, plays an important role in the movement and contraction of smooth muscle cells (SMCs). However, the function of peri-implantation uterine smooth muscle and the regulation mechanism of muscle tension are still unclear. This study focused on the molecular mechanism of actin assembly regulation on implantation in smooth muscle. Phalloidin is a highly selective bicyclic peptide used for staining actin filaments (also known as F-actin). Phalloidin staining showed that F-actin gradually weakened in the CD-1 mouse myometrium from day 1 to day 4 of early pregnancy. More than 3 mice were studied for each group. Jasplakinolide (Jasp) used to inhibit F-actin depolymerization promotes F-actin polymerization in SMCs during implantation window and consequently compromises embryo implantation quality. Transcriptome analysis following Jasp treatment in mouse uterine SMCs reveals significant molecular changes associated with actin assembly. Tagln is involved in the regulation of the cell cytoskeleton and promotes the polymerization of G-actin to F-actin. Our results show that Tagln expression is gradually reduced in mouse uterine myometrium from day 1 to 4 of pregnancy. Furthermore, progesterone inhibits the expression of Tagln through the progesterone receptor. Using siRNA to knock down Tagln in day 3 SMCs, we found that phalloidin staining is decreased, which confirms the critical role of Tagln in F-actin polymerization. In conclusion, our data suggested that decreases in actin assembly in uterine smooth muscle during early pregnancy is critical to optimal embryo implantation. Tagln, a key molecule involved in actin assembly, regulates embryo implantation by controlling F-actin aggregation before implantation, suggesting moderate uterine contractility is conducive to embryo implantation. This study provides new insights into how the mouse uterus increases its flexibility to accommodate implanting embryos in the early stage of pregnancy.

Cholesterol-Dependent Dynamics of the Serotonin1A Receptor Utilizing Single Particle Tracking: Analysis of Diffusion Modes.

G protein-coupled receptors (GPCRs) are signaling hubs in cell membranes that regulate a wide range of physiological processes and are popular drug targets. Serotonin1A receptors are important members of the GPCR family and are implicated in neuropsychiatric disorders. Cholesterol is a key constituent of higher eukaryotic membranes and is believed to contribute to the segregated distribution of membrane constituents into domains. To explore the role of cholesterol in lateral dynamics of GPCRs, we utilized single particle tracking (SPT) to monitor diffusion of serotonin1A receptors under acute and chronic cholesterol-depleted conditions. Our results show that the short-term diffusion coefficient of the receptor decreases upon cholesterol depletion, irrespective of the method of cholesterol depletion. Analysis of SPT trajectories revealed that relative populations of receptors undergoing various modes of diffusion change upon cholesterol depletion. Notably, in cholesterol-depleted cells, we observed an increase in the confined population of the receptor accompanied by a reduction in diffusion coefficient for chronic cholesterol depletion. These results are supported by our recent work and present observations that show polymerization of G-actin in response to chronic cholesterol depletion. Taken together, our results bring out the interdependence of cholesterol and actin cytoskeleton in regulating diffusion of GPCRs in membranes.

Structure and activity of a thermally stable mutant of Acanthamoeba actophorin.

Actophorin, which was recently tested for crystallization under microgravity on the International Space Station, was subjected to mutagenesis to identify a construct with improved biophysical properties that were expected to improve the extent of diffraction. First, 20 mutations, including one C-terminal deletion of three residues, were introduced individually into actophorin, resulting in modest increases in thermal stability of between +0.5°C and +2.2°C. All but two of the stabilizing mutants increased both the rates of severing F-actin filaments and of spontaneous polymerization of pyrenyl G-actin in vitro. When the individual mutations were combined into a single actophorin variant, Acto-2, the overall thermal stability was 22°C higher than that of wild-type actophorin. When an inactivating S2P mutation in Acto-2 was restored, Acto-2/P2S was more stable by 20°C but was notably more active than the wild-type protein. The inactivating S2P mutation reaffirms the importance that Ser2 plays in the F-actin-severing reaction. The crystal structure of Acto-2 was solved to 1.7 Å resolution in a monoclinic space group, a first for actophorin. Surprisingly, despite the increase in thermal stability, the extended β-turn region, which is intimately involved in interactions with F-actin, is disordered in one copy of Acto-2 in the asymmetric unit. These observations emphasize the complex interplay among protein thermal stability, function and dynamics.

Cyclic mechanical strain with high-tensile triggers autophagy in growth plate chondrocytes

BackgroundMechanical loading has been widely considered to be essential for growth plate to maintain metabolism and development. Cyclic mechanical strain has been demonstrated to induce autophagy, whereas the relationship between cyclic tensile strain (CTS) and autophagy in growth plate chondrocytes (GPCs) is not clear. The objective of this study was to investigate whether CTS can regulate autophagy in GPCs in vitro and explore the potential mechanisms of this regulation.MethodsThe 2-week-old Sprague–Dawley rat GPCs were subjected to CTS of varying magnitude and duration at a frequency of 2.0 Hz. The mRNA levels of autophagy-related genes were measured by RT-qPCR. The autophagy in GPCs was verified by transmission electron microscopy (TME), immunofluorescence and Western blotting. The fluorescence-activated cell sorting (FACS) was employed to detect the percentage of apoptotic and necrotic cells.ResultsIn GPCs, CTS significantly increased the mRNA and protein levels of autophagy-related genes, such as LC3, ULK1, ATG5 and BECN1 in a magnitude- and time-dependent manner. There was no significant difference in the proportion of apoptotic and necrotic cells between control group and CTS group. The autophagy inhibitors, 3-methyladenine (3MA) and chloroquine (CQ) reversed the CTS-induced autophagy via promoting the formation of autophagosomes. Cytochalasin D (cytoD), an inhibitor of G-actin polymerization into F-actin, could effectively block the CTS-induced autophagy in GPCs.ConclusionCyclic mechanical strain with high-tensile triggers autophagy in GPCs, which can be suppressed by 3MA and CQ, and cytoskeletal F-actin microfilaments organization plays a key role in chondrocytes’ response to mechanical loading.

Mechanisms of the modulation of actin-myosin interactions by A1-type myosin light chains

BackgroundThe interaction of N-terminal extension of the myosin A1 essential light chain (A1 ELC) with actin is receiving increasing attention as a target in utilizing synthetic A1 ELC N-terminal-derived peptides in cardiac dysfunction therapy. MethodsTo elucidate the mechanism by which these peptides regulate actin-myosin interaction, here we have investigated their effects on the myosin subfragment 1 (S1)-induced polymerization of G-actin. ResultsThe MLCFpep and MLCSpep peptides spanning the 3–12 of A1 ELC sequences from fast and slow skeletal muscle, respectively, increased the rate of actin polymerization not only by S1(A2) but also the rate of S1(A1)-induced actin polymerization, suggesting that they did not interfere with the direct binding of A1 ELC with actin. The efficiency of actin polymerization in the presence of the N-terminal ELC peptides depended on their sequence. Substitution of aspartic acid for neutral asparagine at position 5 of MLCFpep dramatically enhanced its ability to stimulate S1-induced polymerization and enabled it to initiate polymerization of G-actin in the absence of S1. ConclusionsThese and other results presented in this work suggest that the modulation of myosin motor activity by N-terminal ELC peptides is exerted through a change in actin filament conformation rather than through blocking the A1 ELC-actin interaction. General significanceThe results imply the possibility of enhancing therapeutic effects of these peptides by modifications of their sequence.

F-Actin Dysplasia Involved in Organ of Corti Deformity in Gjb2 Knockdown Mouse Model.

Mutations in the GJB2 gene encoding connexin26 (Cx26) protein are one of the most common causes of hereditary deafness. Previous studies have found that different Cx26-null mouse models have severe hearing loss and deformity of the organ of Corti (OC) as well as a reduction in microtubules in pillar cells (PCs). To explore the underlying mechanism of OC deformity caused by Cx26 downregulation further, we established Cx26 knockdown (KD) mouse models at postnatal days (P)0 and P8. The actin filaments contained in the pillar cells of mice in the P0 KD group were reduced by 54.85% and vinculin was increased by 22%, while the outer hair cells (OHCs) showed normal F-actin content. In the P8 KD group, PCs and OHCs of mice also showed almost normal F-actin content. The G-actin/F-actin ratio increased by 38% in the P0 KD group. No significant change was found in the mRNA or protein expression level of G-actin or the cadherin–catenin core complex in the P0 KD group at P6. Moreover, immunofluorescence showed that the intensity of LRRK2 was reduced by 97% in the P0 KD group at P6. Our results indicate that Cx26 is involved in the maturation of the cytoskeleton during the development of the OC at the early postnatal stage. The polymerization of G-actin into F-actin is prevented in Cx26 KD mice.

Ca2+-dependent binding of S100A6 to cofilin-1 regulates actin filament polymerization-depolymerization dynamics

S100A6 is a Ca2+-binding protein belonging to the S100 family. Many reports indicate that S100A6 is involved in actin filament organization, however the mechanism of S100A6 action in this process is not fully understood. By screening S100A6 binding partners in NIH3T3 mouse fibroblasts, we have found that S100A6 binds cofilin-1, a protein required for the dynamics of actin polymerization and depolymerization. By applying various biochemical and cell biology assays, we have shown that S100A6 bound to cofilin-1 in a Ca2+-dependent manner and increased cofilin-1 affinity for F-actin. Microscopic analysis indicated that S100A6 significantly decreased severing of the actin filaments induced by cofilin-1. Moreover, in the presence of cofilin-1, S100A6 stabilized the filaments by inhibiting their depolymerization. When S100A6 was present at sub-stoichiometric concentrations in relation to actin, polymerization of G-actin accelerated by cofilin-1 was increased. At higher S100A6:actin ratios the polymerization rate was decreased. Altogether, these results show that S100A6 regulates actin filament dynamics by controlling activity of cofilin-1 and suggest that this regulation is Ca2+ -dependent.

Truncated Actin-Targeting Macrolide Derivative Blocks Cancer Cell Motility and Invasion of Extracellular Matrix.

Cancer metastasis is a complex process involving highly motile tumor cells that breach tissue barriers, enter the bloodstream and lymphatic system, and disseminate throughout the body as circulating tumor cells. The primary cellular mechanism contributing to these critical events is the reorganization of the actin cytoskeleton. Mycalolide B (MycB) is an actin-targeting marine macrolide that can suppress proliferation, migration, and invasion of breast and ovarian cancer cells at low nanomolar doses. Through structure-activity relationship studies focused on the actin-binding tail region (C24-C35) of MycB, we identified a potent truncated derivative that inhibits polymerization of G-actin and severs F-actin by binding to actin's barbed end cleft. Biological analyses of this miniature MycB derivative demonstrate that it causes a rapid collapse of the actin cytoskeleton in ovarian cancer cells and impairs cancer cell motility and invasion of the extracellular matrix (ECM) by inhibiting invadopodia-mediated ECM degradation. These studies provide essential proof-of-principle for developing actin-targeting therapeutic agents to block cancer metastasis and establish a synthetically tractable barbed end-binding pharmacophore that can be further improved by adding targeting groups for precision drug design.

The BAR domain of the Arf GTPase-activating protein ASAP1 directly binds actin filaments

The Arf GTPase-activating protein (Arf GAP) with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) establishes a connection between the cell membrane and the cortical actin cytoskeleton. The formation, maintenance, and turnover of actin filaments and bundles in the actin cortex are important for cell adhesion, invasion, and migration. Here, using actin cosedimentation, polymerization, and depolymerization assays, along with total internal reflection fluorescence (TIRF), confocal, and EM analyses, we show that the N-terminal N-BAR domain of ASAP1 directly binds to F-actin. We found that ASAP1 homodimerization aligns F-actin in predominantly unipolar bundles and stabilizes them against depolymerization. Furthermore, the ASAP1 N-BAR domain moderately reduced the spontaneous polymerization of G-actin. The overexpression of the ASAP1 BAR-PH tandem domain in fibroblasts induced the formation of actin-filled projections more effectively than did full-length ASAP1. An ASAP1 construct that lacked the N-BAR domain failed to induce cellular projections. Our results suggest that ASAP1 regulates the dynamics and the formation of higher-order actin structures, possibly through direct binding to F-actin via its N-BAR domain. We propose that ASAP1 is a hub protein for dynamic protein-protein interactions in mechanosensitive structures, such as focal adhesions, invadopodia, and podosomes, that are directly implicated in oncogenic events. The effect of ASAP1 on actin dynamics puts a spotlight on its function as a central signaling molecule that regulates the dynamics of the actin cytoskeleton by transmitting signals from the plasma membrane.

Role and dynamics of an agmatinase-like protein (AGM-1) in Neurospora crassa

Agmatinase is known as a metalloenzyme which hydrolyzes agmatine to produce urea and putrescine, being crucial in the alternative pathway to produce polyamines. In this study, an agmatinase-like protein (AGM-1) (NCU 01348) in the filamentous fungus Neurospora crassa is reported. Purified AGM-1 from N. crassa displays enzymatic activity hydrolyzing agmatine; therefore, it can be considered as an agmatinase-like protein. However, its role in the alternative pathway to produce polyamines apparently is not its main function since only a slight reduction of polyamines concentration was detected in the Δagm-1 het strain. Moreover, the null mutant Δagm-1 (homokaryon strain) was unable to grow and the deficiency of agm-1 in the heterokaryon strain provoked a decrease in elongation rate, conidia and biomass production, despite of having de constitutive pathway via the ornithine decarboxylase (ODC). Additionally, mature hyphae of the Δagm-1 het strain presented unusual apical branching and a disorganized Spitzenkörper (Spk). Trying to reveal the role of AGM-1in N. crassa, the protein was tagged with GFP and interestingly the dynamics and intracellular localization of AGM-1 closely resembles the F-actin population. This finding was further examined in order to elucidate if AGM-1is in a close association with F-actin. Since polyamines, among them agmatine, have been reported to act as stabilizers of actin filaments, we evaluated in vitro G-actin polymerization in the presence of agmatine and the effect of purified AGM-1 from N. crassa on these polymerized actin filaments. It was found that polymerization of actin filaments increases in the presence of agmatine and the addition of purified AGM-1 from N. crassa depolymerizes these actin filaments. Also, it was determined that an intact substrate binding site of the enzyme is necessary for the localization pattern of the native AGM-1. These results suggest that in N. crassa AGM-1 has a close association with the F-actin population via its substrate agmatine, playing an essential role during cell development.

Abstract 16815: GJA1-20k, a Truncated Isoform of Connexin 43, Nucleates De Novo Actin Clusters

Introduction: Connexin 43 (Cx43) gap junctions allow rapid communication of signals between cardiomyocytes. Loss of Cx43 gap junction coupling contributes to arrhythmogenesis. GJA1-20k, a truncated internally translated isoform of the Cx43 gene GJA1 , is necessary for full length Cx43 trafficking to intercalated discs for gap junction formation. GJA1-20k stabilizes actin filaments, necessary for the Cx43 delivery highway. The mechanism of GJA1-20k interaction with actin, while essential for gap junction formation, is still not known. Methods: Cell free individual actin dynamics were observed by TIRF microscopy. For real time imaging of actin nucleation and polymerization, glass bottom chamber slides were pre-incubated with 100nM N-ethylmaleimide myosin and loaded with Alexa Fluor 488-conjugated actin (Thermo Fisher) with GJA1-20k in ATP containing buffer. Actin binding assays were performed using a spin-down assay kit (Cytoskeleton, Inc.). Actin polymerization was studied by using pyrene labeled actin (Cytoskeleton, Inc.). Results: Cell free biochemical binding assays show that GJA1-20k binds to G-actin molecules, nucleating de novo actin clusters yet also surprisingly preventing G-actin polymerization. Pyrene actin assays further confirm that GJA1-20k alone does not promote polymerization. Cell free TIRF imaging also reveals that GJA1-20k promotes the formation of de novo G-actin clusters (Figure 1). Once a critical degree of nucleation is present however, polymerization occurs at a rate that promotes the formation of thicker actin fibers. Because GJA1-20k preferentially supports nucleation over polymerization, GJA1-20k introduces novel foci of new actin growth that, when a critical mass is present, rapidly overflows to polymerization. Conclusions: GJA1-20k nucleates de novo actin clusters, and thus is able to seed novel cytoskeleton growth patterns, dictating the direction of gap junction delivery.

TAGLN2 polymerizes G-actin in a low ionic state but blocks Arp2/3-nucleated actin branching in physiological conditions

TAGLN is an actin-binding protein family that comprises three isoforms with theorized roles in smooth muscle differentiation, tumour development, lymphocyte activation, and brain chemistry. However, their fundamental characteristics in regulation of the actin-based cytoskeleton are not fully understood. Here we show that TAGLN2 (including TAGLN1 and TAGLN3) extensively nucleates G-actin polymerization under low-salt conditions, where polymerization would be completely suppressed. The calponin homology domain and actin-binding loop are essential to mechanically connect two adjacent G-actins, thereby mediating multimeric interactions. However, TAGLN2 blocked the Arp2/3 complex binding to actin filaments under physiological salt conditions, thereby inhibiting branched actin nucleation. In HeLa and T cells, TAGLN2 enhanced filopodium-like membrane protrusion. Collectively, the dual functional nature of TAGLN2—G-actin polymerization and Arp2/3 complex inhibition—may account for the mechanisms of filopodia development at the edge of Arp2/3-rich lamellipodia in various cell types.

Actin polymerization triggers gastric epithelial repair of damage

BackgroundGastric epithelial repair, mediated by cell migration and dead cell exfoliation, is a rapid process. Actin dynamics, in collaboration with myosin, are essential for the cell migration during epithelial repair of damage. We previously demonstrated that actin dynamics play an important role in the repair of damage in vivo mouse stomach and in vitro gastric organoids (gastroids). To extend our current knowledge, we examined the repair of damage in gastroids generated from human actin‐GFP (HuGE) transgenic mouse stomach and investigated the involvement of actin polymerization and myosin in this process.MethodsGastroids were generated from gastric corpus of HuGE mice. Hoechst33342 (10 μg/ml) was added to image the nucleus. Selective high intensity illumination of single gastric epithelial cell nucleus (two‐photon; 730 nm light) caused a microscopic lesion via photodamage. Dynamic changes in actin‐GFP and damage size were quantified over time. In some cases, Lucifer yellow (20 μM) or Alexa647‐10K dextran (10 μM) was added to medium to determine cellular and/or epithelial leakage.ResultsIn response to photodamage of gastroid epithelium, actin‐GFP expression increased within ~1 min in the lateral membranes neighboring the damaged cell, and stayed elevated in this position and in lamellipodia during the cellular migration inward that closes the gap at the basal pole of the dead cell. Synchronous dead cell exfoliation was observed. When no intentional damage was imposed, similar events occurred during natural cell shedding into the lumen of the gastroids. Assuming a bistable switch controlling actin polymerization, we constructed a mathematical model whose simulations recaptured the temporal order of the observed repair processes, and the model predicted actin‐mediated force led to the observed dead cell exfoliation. In undamaged gastroids, Cytochalasin D (inhibitor of actin assembly at F‐actin barded ends: 1 μM) did not alter total cellular actin‐GFP intensity, while Latrunculin A (inhibitor of G‐actin polymerization: 2 μM) reduced GFP intensity. Both inhibitors increased paracellular leakage in the gastroid over the time, and completely inhibited the damage‐induced increase of actin, as well as repair of damage. In contrast, in undamaged gastroids, Jasplakinolide (actin filament stabilizer: 1 μM) facilitated actin‐GFP accumulation in the membrane and decreased GFP in the cytosolic compartment in a time‐dependent manner. Short time exposures to Jasplakinolide or Latrunculin A were imposed such that there was not effect on basal actin‐GFP intensity. In this condition, Jasplakinolide did not affect repair of damage, but Latrunculin A completely inhibited repair. Blebbistatin (Myosin II inhibitor, 10 μM) significantly inhibited repair of damage and slowed dead cell ejection, with no effect on GFP‐actin accumulation.ConclusionNew F‐actin polymerization or elongation is necessary to initiate gastric epithelial repair of damage. Furthermore, recruitment of myosin II supports generation of force to exfoliate the dead cell.Support or Funding InformationNIH RO1 DK102551.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of actin dynamics as potential macrophage subtype-targeting anti-tumour strategy

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells.

Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin.

Actin cytoskeleton dynamics are controlled by various actin binding proteins (ABPs) that modulate the polymerization of the monomeric G-actin and the depolymerization of filamentous F-actin. Although revealing the structures of the actin/ABP complexes is crucial to understand how the ABPs regulate actin dynamics, the X-ray crystallography and cryoEM methods are inadequate to apply for the ABPs that interact with G- or F-actin with lower affinity or multiple binding modes. In this study, we aimed to establish the alternative method to build a structural model of G-actin/ABP complexes, utilizing the paramagnetic relaxation enhancement (PRE) experiments. Thymosin β4 (Tβ4) was used as a test case for validation, since its structure in complex with G-actin was reported recently. Recombinantly expressed G-actin, containing a cysteine mutation, was conjugated with a nitroxyl spin label at the specific site. Based on the intensity ratio of the 1H-15N HSQC spectra of Tβ4 in the complex with G-actin in the paramagnetic and diamagnetic states, the distances between the amide groups of Tβ4 and the spin label of G-actin were estimated. Using the PRE-derived distance constraints, we were able to compute a well-converged docking structure of the G-actin/Tβ4 complex that shows great accordance with the reference structure.

Integrins synergise to induce expression of the MRTF-A-SRF target gene ISG15 for promoting cancer cell invasion.

Integrin-mediated activation of small GTPases induces the polymerisation of G-actin into various actin structures and the release of the transcriptional co-activator MRTF from G-actin. Here we report that pan-integrin-null fibroblasts seeded on fibronectin and expressing β1- and/or αV-class integrin contained different G-actin pools, nuclear MRTF-A (also known as MKL1 or MAL) levels and MRTF-A-SRF activities. The nuclear MRTF-A levels and activities were highest in cells expressing both integrin classes, lower in cells expressing β1 integrins and lowest in cells expressing the αV integrins. Quantitative proteomics and transcriptomics analyses linked the differential MRTF-A activities to the expression of the ubiquitin-like modifier interferon-stimulated gene 15 (ISG15), which is known to modify focal adhesion and cytoskeletal proteins. The malignant breast cancer cell line MDA-MB-231 expressed high levels of β1 integrins, ISG15 and ISGylated proteins, which promoted invasive properties, whereas non-invasive MDA-MB-468 and MCF-7 cell lines expressed low levels of β1 integrins, ISG15 and ISGylated proteins. Our findings suggest that integrin-adhesion-induced MRTF-A-SRF activation and ISG15 expression constitute a newly discovered signalling circuit that promotes cell migration and invasion.