Inhibition Of Actin Polymerization Research Articles

A WICB 50th Favorite: A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis.

Molecular Biology of the CellVol. 32, No. 19 ASCB WICB 50th Anniversary FavoriteFree AccessA WICB 50th Favorite: A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosisKristen SkruberKristen Skruber*Address correspondence to: Kristen Skruber (E-mail Address: [email protected]).Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143Search for more papers by this authorAvital Rodal, Monitoring EditorPublished Online:31 Aug 2021https://doi.org/10.1091/mbc.E21-04-0216AboutSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail To celebrate the 50th anniversary of the American Society for Cell Biology's Women in Cell Biology Committee (WICB), members of WICB and the MBoC Editorial Board invited a diverse group of scientists to highlight MBoC papers by women that have had a scientific or personal impact on the authors of the highlight.While a primary role of actin polymerization is in cellular organization and motility, actin also provides the force and scaffold for trafficking processes such as endocytosis. Actin's role in endocytosis has been challenging to elucidate due to the small scale and transient nature of endocytic structures. Inhibiting actin polymerization in yeast completely halts endocytosis. However, until this landmark paper, the corollary process in mammalian cells was less clear, because ablating actin polymerization only partially stalled trafficking. Yarar et al. (2005) used a mixture of electron and fluorescence microscopy and biochemical methods to establish a clear role for actin polymerization during late stages of clathrin-mediated endocytosis. A clever combination of TIRF and wide-field fluorescence imaging allowed precise vesicle tracking. Importantly, understanding the differences in actin's contributions to endocytosis in yeast and metazoa opened up exciting questions about actin's role in plasma membrane tension and shape.FOOTNOTESDOI: 10.1091/mbc.E21-04-0216REFERENCEYarar D, Waterman-Storer CM, Schmid SL (2005). A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis. Mol Biol Cell 16, 964–975. Link, Google ScholarFiguresReferencesRelatedDetails Vol. 32, No. 19 September 01, 20211793-1866 Metrics Downloads & Citations Downloads: 184 History Information© 2021 Skruber. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).PDF download

Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans.

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant-pathogen interactions.

Acetylation of Abelson interactor 1 at K416 regulates actin cytoskeleton and smooth muscle contraction.

Actin cytoskeletal reorganization plays an important role in regulating smooth muscle contraction, which is essential for the modulation of various physiological functions including airway tone. The adapter protein Abi1 (Abelson interactor 1) participates in the control of smooth muscle contraction. The mechanisms by which Abi1 coordinates smooth muscle function are not fully understood. Here, we found that contractile stimulation elicited Abi1 acetylation in human airway smooth muscle (HASM) cells. Mutagenesis analysis identified lysine‐416 (K416) as a major acetylation site. Replacement of K416 with Q (glutamine) enhanced the interaction of Abi1 with neuronal Wiskott‐Aldrich syndrome protein (N‐WASP), an important actin‐regulatory protein. Moreover, the expression of K416Q Abi1 promoted actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser‐19 and vimentin phosphorylation at Ser‐56. Furthermore, p300 is a lysine acetyltransferase that catalyzes acetylation of histone and non‐histone proteins in various cell types. Here, we discovered that a portion of p300 was localized in the cytoplasm of HASM cells. Knockdown of p300 reduced the agonist‐induced Abi1 acetylation in HASM cells and in mouse airway smooth muscle tissues. Smooth muscle conditional knockout of p300 inhibited actin polymerization and the contraction of airway smooth muscle tissues without affecting myosin light chain phosphorylation and vimentin phosphorylation. Together, our results suggest that contractile stimulation induces Abi1 acetylation via p300 in smooth muscle. Acetylation at K416 promotes the coupling of Abi1 with N‐WASP, which facilitates actin polymerization and smooth muscle contraction. This is a novel acetylation‐dependent regulation of the actin cytoskeleton in smooth muscle.

Using kICS to Reveal Changed Membrane Diffusion of AQP-9 Treated with Drugs.

The formation of nanodomains in the plasma membrane are thought to be part of membrane proteins regulation and signaling. Plasma membrane proteins are often investigated by analyzing the lateral mobility. k-space ICS (kICS) is a powerful image correlation spectroscopy (ICS) technique and a valuable supplement to fluorescence correlation spectroscopy (FCS). Here, we study the diffusion of aquaporin-9 (AQP9) in the plasma membrane, and the effect of different membrane and cytoskeleton affecting drugs, and therefore nanodomain perturbing, using kICS. We measured the diffusion coefficient of AQP9 after addition of these drugs using live cell Total Internal Reflection Fluorescence imaging on HEK-293 cells. The actin polymerization inhibitors Cytochalasin D and Latrunculin A do not affect the diffusion coefficient of AQP9. Methyl-β-Cyclodextrin decreases GFP-AQP9 diffusion coefficient in the plasma membrane. Human epidermal growth factor led to an increase in the diffusion coefficient of AQP9. These findings led to the conclusion that kICS can be used to measure diffusion AQP9, and suggests that the AQP9 is not part of nanodomains.

Cell-fate decision of mesenchymal stem cells toward osteocyte differentiation is committed by spheroid culture

Osteocytes are mechanosensory commander cells to regulate bone remodeling throughout the lifespan. While the osteocytes are known as terminally differentiated cells derived from mesenchymal stem cells, the detailed mechanisms of osteocyte differentiation remain unclear. In this study, we fabricated 3D self-organized spheroids using human mesenchymal stem cells (MSCs). Under the osteogenesis induction medium, the spheroid culture model exerted the osteocyte-likeness within 2 days compared to a conventional 2D monolayer model. Moreover, we showed that an inhibition of actin polymerization in the spheroid further up-regulated the osteocyte gene expressions. Notably, we represented that the cell condensed condition acquired in the 3D spheroid culture model determined a differentiation fate of MSCs to osteocytes. Taken together, we suggest that our self-organized spheroid model can be utilized as a new in vitro model to represent the osteocyte and to recapitulate an in vitro ossification process.

CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation.

The Scar/WAVE complex drives actin nucleation during cell migration. Interestingly, the same complex is important in forming membrane ruffles during macropinocytosis, a process mediating nutrient uptake and membrane receptor trafficking. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A, has not been characterized. Here, we implicate CYRI-A as a key regulator of macropinosome formation and integrin internalization. We find that CYRI-A is transiently recruited to nascent macropinosomes, dependent on PI3K and RAC1 activity. CYRI-A recruitment precedes RAB5A recruitment but follows sharply after RAC1 and actin signaling, consistent with it being a local inhibitor of actin polymerization. Depletion of both CYRI-A and -B results in enhanced surface expression of the α5β1 integrin via reduced internalization. CYRI depletion enhanced migration, invasion, and anchorage-independent growth in 3D. Thus, CYRI-A is a dynamic regulator of macropinocytosis, functioning together with CYRI-B to regulate integrin trafficking.

Synaptosomal Actin Dynamics in the Developmental Visual Cortex Regulate Behavioral Visual Acuity in Rats.

PurposeSynaptosomal actin dynamics are essential for synaptic structural stability. Whether actin dynamics are involved in structural and functional synaptic plasticity within the primary visual cortex (V1) or behavioral visual acuity in rats has still not been thoroughly investigated.MethodsSynaptosome preparation and western blot analysis were used to analyze synaptosomal actin dynamics. Transmission electron microscopy was used to detect synaptic density and mitochondrial area alterations. A visual water maze task was applied to assess behavioral visual acuity. Microinjection of the actin polymerization inhibitor or stabilizer detected the effect of actin dynamics on visual function.ResultsActin dynamics, the mitochondrial area, and synaptic density within the area of V1 are increased during the critical period for the development of binocularity. Microinjection of the actin polymerization inhibitor cytochalasin D into the V1 decreased the mitochondrial area, synaptic density, and behavioral visual acuity. Long-term monocular deprivation reduced actin dynamics, the mitochondrial area, and synaptic density within the V1 contralateral to the deprived eye compared with those ipsilateral to the deprived eye and impaired visual acuity in the amblyopic eye. In addition, the mitochondrial area, synaptic density, and behavioral visual acuity were improved by stabilization of actin polymerization by jasplakinolide microinjection.ConclusionsDuring the critical period of visual development of binocularity, synaptosomal actin dynamics regulate synaptic structure and function and play roles in behavioral visual acuity in rats.

MYO1F Regulates IgE and MRGPRX2-Dependent Mast Cell Exocytosis.

The activation and degranulation of mast cells is critical in the pathogenesis of allergic inflammation and modulation of inflammation. Recently, we demonstrated that the unconventional long-tailed myosin, MYO1F, localizes with cortical F-actin and mediates adhesion and migration of mast cells. In this study, we show that knockdown of MYO1F by short hairpin RNA reduces human mast cell degranulation induced by both IgE crosslinking and by stimulation of the Mas-related G protein-coupled receptor X2 (MRGPRX2), which has been associated with allergic and pseudoallergic drug reactions, respectively. Defective degranulation was accompanied by a reduced reassembly of the cortical actin ring after activation but reversed by inhibition of actin polymerization. Our data show that MYO1F is required for full Cdc42 GTPase activation, a critical step in exocytosis. Furthermore, MYO1F knockdown resulted in less granule localization in the cell membrane and fewer fissioned mitochondria along with deficient mitochondria translocation to exocytic sites. Consistent with that, AKT and DRP1 phosphorylation are diminished in MYO1F knockdown cells. Altogether, our data point to MYO1F as an important regulator of mast cell degranulation by contributing to the dynamics of the cortical actin ring and the distribution of both the secretory granules and mitochondria.

Protein synthesis and actin polymerization in the rapid effects of 17β-estradiol on short-term social memory and dendritic spine dynamics in female mice

Estrogens rapidly facilitate learning and memory, including social recognition – the ability of an animal to recognize another. In ovariectomized female mice, systemic or dorsal hippocampal administration of 17β-estradiol (E2) facilitates short-term social recognition memory within 40 min. Within the same timeframe, E2 increases dendritic spine density in CA1 dorsal hippocampal neurons of behavioural task-naïve mice and in hippocampal sections. Mechanisms underlying these effects remain unclear. Estrogens rapidly modulate actin cytoskeletal dynamics through actin polymerization and the translation of key synaptic proteins. We first determined doses of actin polymerization inhibitor latrunculin A (LAT) and protein synthesis inhibitor anisomycin (ANI) that would block short-term social recognition memory when infused into the dorsal hippocampus of ovariectomized female mice 15 min prior to testing. The highest doses that did not block social recognition prevented the facilitating effects of E2, whereas DNA transcription inhibitor, actinomycin D, could not block social recognition. As task performance may interfere with E2-facilitated increases in dendritic spine density, dendritic spine density and length were examined in task-performing and task-naïve mice. E2 increased dendritic spine density 15 but not 40 min following treatment, regardless of whether the animal had performed the social recognition task. This effect was blocked by LAT, but not ANI. Thus, both actin polymerization and protein synthesis are necessary for E2 to rapidly facilitate social recognition, whereas actin polymerization, but not protein synthesis, is required for the rapid increase in dendritic spine density brought on by E2.

Epigenetic modulation of immune synaptic-cytoskeletal networks potentiates \u03b3\u03b4 T cell-mediated cytotoxicity in lung cancer

γδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune system and can attack cancer cells in an MHC-unrestricted manner. Trials of adoptive γδ T cell transfer in solid tumors have had limited success. Here, we show that DNA methyltransferase inhibitors (DNMTis) upregulate surface molecules on cancer cells related to γδ T cell activation using quantitative surface proteomics. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded Vδ1-enriched γδ T cells. Mechanistically, DNMTi enhances immune synapse formation and mediates cytoskeletal reorganization via coordinated alterations of DNA methylation and chromatin accessibility. Genetic depletion of adhesion molecules or pharmacological inhibition of actin polymerization abolishes the potentiating effect of DNMTi. Clinically, the DNMTi-associated cytoskeleton signature stratifies lung cancer patients prognostically. These results support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management.

Phenotypic Models of CAR T-Cell Activation Elucidate the Pivotal Regulatory Role of CAR Downmodulation.

Adoptive cell immunotherapy with chimeric antigen receptor (CAR) showed limited potency in solid tumors, despite durable remissions for hematopoietic malignancies. Therefore, an investigation of ways to enhance the efficacy of CARs' antitumor response has been engaged upon. We previously examined the interplay between the biophysical parameters of CAR binding (i.e., affinity, avidity, and antigen density), as regulators of CAR T-cell activity and detected nonmonotonic behaviors of affinity and antigen density and an interrelation between avidity and antigen density. Here, we built an evolving phenotypic model of CAR T-cell regulation, which suggested that receptor downmodulation is a key determinant of CAR T-cell function. We verified this assumption by measuring and manipulating receptor downmodulation and intracellular signaling processes. CAR downmodulation inhibition, via actin polymerization inhibition, but not inhibition of regulatory inhibitory phosphatases, was able to increase CAR T-cell responses. In addition, we documented trogocytosis in CAR T cells that depends on actin polymerization. In summary, our study modeled the parameters that govern CAR T-cell engagement and revealed an underappreciated mechanism of T-cell regulation. These results have a potential to predict and therefore advance the rational design of CAR T cells for adoptive cell treatments.See related article on p. 872.

Stage-dependent sequential organization of nascent smooth muscle cells and its implications for the gut coiling morphogenesis in Xenopus larva

In vertebrates, gut coiling proceeds left-right asymmetrically during expansion of the gastrointestinal tract with highly organized muscular structures facilitating peristalsis. In this report, we explored the mechanisms of larval gut coiling morphogenesis relevant to its nascent smooth muscle cells using highly transparent Xenopus early larvae. First, to visualize the dynamics of intestinal smooth muscle cells, whole-mount specimens were immunostained with anti-smooth muscle-specific actin (SM-actin) antibody. We found that the nascent gut of Xenopus early larvae gradually expands the SM-actin-positive region in a stage-dependent manner. Transverse orientation of smooth muscle cells was first established, and next, the cellular longitudinal orientation along the gut axis was followed to make a meshwork of the contractile cells. Finally, anisotropic torsion by the smooth muscle cells was generated in the center of gut coiling, suggesting that twisting force might be involved in the late phase of coiling morphogenesis of the gut. Administration of S-(–)-Blebbistatin to attenuate the actomyosin contraction in vivo resulted in cancellation of coiling of the gut. Development of decapitation embryos, trunk ‘torso’ explants, and gut-only explants revealed that initial coiling of the gut proceeds without interactions with the other parts of the body including the central nervous system. We newly developed an in vitro model to assess the gut coiling morphogenesis, indicating that coiling pattern of the nascent Xenopus gut is partially gut-autonomous. Using this gut explant culture technique, inhibition of actomyosin contraction was performed by administrating either actin polymerization inhibitor, myosin light chain kinase inhibitor, or calmodulin antagonist. All of these reagents decreased the extent of gut coiling morphogenesis in vitro. Taken together, these results suggest that the contraction force generated by actomyosin-rich intestinal smooth muscle cells during larval stages is essential for the normal coiling morphogenesis of this muscular tubular organ.

Cyclophosphamide alters the tumor cell secretome to potentiate the anti-myeloma activity of daratumumab through augmentation of macrophage-mediated antibody dependent cellular phagocytosis

ABSTRACT Multiple Myeloma (MM) is a malignant disorder of plasma cells which, despite significant advances in treatment, remains incurable. Daratumumab, the first CD38 directed monoclonal antibody, has shown promising activity alone and in combination with other agents for MM treatment. Daratumumab is thought to have pleiotropic mechanisms of activity including natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). With the knowledge that CD38-expressing NK cells are depleted by daratumumab, we sought to investigate a potential mechanism of enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) by combining daratumumab with cyclophosphamide (CTX). Cyclophosphamide’s immunomodulatory function was investigated by conditioning macrophages with tumor cell secretome collected from cyclophosphamide treated MM cell lines (CTX-TCS). Flow cytometry analysis revealed that CTX-TCS conditioning augmented the migratory capacity of macrophages and increased CD32 and CD64 Fcγ receptor expression on their cell surface. Daratumumab-specific tumor clearance was increased by conditioning macrophages with CTX-TCS in a dose-dependent manner. This effect was impeded by pre-incubating macrophages with Cytochalasin D (CytoD), an inhibitor of actin polymerization, indicating macrophage-mediated ADCP as the mechanism of clearance. CD64 expression on macrophages directly correlated with MM cell clearance and was essential to the observed synergy between cyclophosphamide and daratumumab, as tumor clearance was attenuated in the presence of a FcγRI/CD64 blocking agent. Cyclophosphamide independently enhances daratumumab-mediated killing of MM cells by altering the tumor microenvironment to promote macrophage recruitment, polarization to a pro-inflammatory phenotype, and directing ADCP. These findings support the addition of cyclophosphamide to existing or novel monoclonal antibody-containing MM regimens.

Preparation of Clostridium perfringens binary iota-toxin pore complex for structural analysis using cryo-EM.

Iota toxin, a type of A-B toxin produced by Clostridium perfringens, comprises an enzymatic component (Ia) and a membrane-binding component (Ib). The translocation of Ia to the target cell via the pore formed by Ib allows it to function as an ADP-ribosyltransferase that inhibits actin polymerization in the host cell. The structure of Ia-bound Ib-pore has been determined using cryo-electron microscopy (cryo-EM), thereby elucidating the mechanism of the initial Ia translocation; however, open questions regarding Ia translocation still exist. In this chapter, we describe a new method of preparing Ia-bound Ib-pore complex samples for structural analysis at high resolution using cryo-EM. This method is different from previously reported methods for other A-B toxins. Consequently, it produces Ib-pore with two different states with short and long membrane-spanning β-barrel stem. We expect that this method will be useful in functional and structural studies of iota toxin and other binary toxins.

Cytoskeleton-dependent clustering of membrane-bound prion protein on the cell surface

Prion diseases are a group of neurodegenerative disorders that infect animals and humans with proteinaceous particles called prions. Prions consist of scrapie prion protein (PrPSc), a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc. Attachment of PrPC to cellular membranes via a glycosylphosphatidylinositol (GPI) anchor is critical for the conversion of PrPC into PrPSc. However, the mechanisms governing PrPC conversion and replication on the membrane remain largely unclear. Here, a site-selectively modified PrP variant equipped with a fluorescent GPI anchor mimic (PrP-GPI) was employed to directly observe PrP at the cellular membrane in neuronal SH-SY5Y cells. PrP-GPI exhibits a cholesterol-dependent membrane accumulation and a cytoskeleton-dependent mobility. More specifically, inhibition of actin polymerization reduced the diffusion of PrP-GPI indicating protein clustering, which resembles the initial step of PrP aggregation and conversion into its pathogenic isoform. An intact actin cytoskeleton might therefore prevent conversion of PrPC into PrPSc and offer new therapeutic angles.

Primary Cilia, Ciliogenesis and the Actin Cytoskeleton: A Little Less Resorption, A Little More Actin Please.

Primary cilia are microtubule-based organelles that extend from the apical surface of most mammalian cells, forming when the basal body (derived from the mother centriole) docks at the apical cell membrane. They act as universal cellular “antennae” in vertebrates that receive and integrate mechanical and chemical signals from the extracellular environment, serving diverse roles in chemo-, mechano- and photo-sensation that control developmental signaling, cell polarity and cell proliferation. Mutations in ciliary genes cause a major group of inherited developmental disorders called ciliopathies. There are very few preventative treatments or new therapeutic interventions that modify disease progression or the long-term outlook of patients with these conditions. Recent work has identified at least four distinct but interrelated cellular processes that regulate cilia formation and maintenance, comprising the cell cycle, cellular proteostasis, signaling pathways and structural influences of the actin cytoskeleton. The actin cytoskeleton is composed of microfilaments that are formed from filamentous (F) polymers of globular G-actin subunits. Actin filaments are organized into bundles and networks, and are attached to the cell membrane, by diverse cross-linking proteins. During cell migration, actin filament bundles form either radially at the leading edge or as axial stress fibers. Early studies demonstrated that loss-of-function mutations in ciliopathy genes increased stress fiber formation and impaired ciliogenesis whereas pharmacological inhibition of actin polymerization promoted ciliogenesis. These studies suggest that polymerization of the actin cytoskeleton, F-actin branching and the formation of stress fibers all inhibit primary cilium formation, whereas depolymerization or depletion of actin enhance ciliogenesis. Here, we review the mechanistic basis for these effects on ciliogenesis, which comprise several cellular processes acting in concert at different timescales. Actin polymerization is both a physical barrier to both cilia-targeted vesicle transport and to the membrane remodeling required for ciliogenesis. In contrast, actin may cause cilia loss by localizing disassembly factors at the ciliary base, and F-actin branching may itself activate the YAP/TAZ pathway to promote cilia disassembly. The fundamental role of actin polymerization in the control of ciliogenesis may present potential new targets for disease-modifying therapeutic approaches in treating ciliopathies.

The Anti-Cancer Effect of Linusorb B3 from Flaxseed Oil through the Promotion of Apoptosis, Inhibition of Actin Polymerization, and Suppression of Src Activity in Glioblastoma Cells.

Linusorbs (LOs) are natural peptides found in flaxseed oil that exert various biological activities. Of LOs, LOB3 ([1–9-NαC]-linusorb B3) was reported to have antioxidative and anti-inflammatory activities; however, its anti-cancer activity has been poorly understood. Therefore, this study investigated the anti-cancer effect of LOB3 and its underlying mechanism in glioblastoma cells. LOB3 induced apoptosis and suppressed the proliferation of C6 cells by inhibiting the expression of anti-apoptotic genes, B cell lymphoma 2 (Bcl-2) and p53, as well as promoting the activation of pro-apoptotic caspases, caspase-3 and -9. LOB3 also retarded the migration of C6 cells, which was achieved by suppressing the formation of the actin cytoskeleton critical for the progression, invasion, and metastasis of cancer. Moreover, LOB3 inhibited the activation of the proto-oncogene, Src, and the downstream effector, signal transducer and activator of transcription 3 (STAT3), in C6 cells. Taken together, these results suggest that LOB3 plays an anti-cancer role by inducing apoptosis and inhibiting the migration of C6 cells through the regulation of apoptosis-related molecules, actin polymerization, and proto-oncogenes.

YAP Circular RNA, circYap, Attenuates Cardiac Fibrosis via Binding with Tropomyosin-4 and Gamma-Actin Decreasing Actin Polymerization

Cardiac fibrosis is a common pathological feature of cardiac hypertrophy. This study was designed to investigate a novel function of Yes-associated protein (YAP) circular RNA, circYap, in modulating cardiac fibrosis and the underlying mechanisms. By circular RNA sequencing, we found that three out of fifteen reported circYap isoforms were expressed in nine human heart tissues, with the isoform hsa_circ_0002320 being the highest. The levels of this isoform in the hearts of patients with cardiac hypertrophy were found to be significantly decreased. In the pressure overload mouse model, the levels of circYap were reduced in mouse hearts with transverse aortic constriction (TAC). Upon circYap plasmid injection, the cardiac fibrosis was attenuated, and the heart function was improved along with the elevation of cardiac circYap levels in TAC mice. Tropomyosin-4 (TMP4) and gamma-actin (ACTG) were identified to bind with circYap in cardiac cells and mouse heart tissues. Such bindings led to an increased TPM4 interaction with ACTG, resulting in the inhibition of actin polymerization and the following fibrosis. Collectively, our study uncovered a novel molecule that could regulate cardiac remodeling during cardiac fibrosis and implicated a new function of circular RNA. This process may be targeted for future cardio-therapy.

Pluripotency of embryonic stem cells lacking clathrin-mediated endocytosis cannot be rescued by restoring cellular stiffness

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells, which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young's modulus, indicative of greater cellular stiffness, compared with WT mESCs. The increase in stiffness was accompanied by the presence of actin stress fibers and accumulation of the inactive, phosphorylated, actin-binding protein cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young's modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, whereas WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, whereas physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate.

Crucial role of framework with cytoskeletal actin filaments for shaping microstructure of footpad setae in the ladybird beetle, Harmonia axyridis

Insects that can walk on smooth surfaces have specialized structures, footpads, on their legs. Footpads play an important role in adhesion to the substrate surface. Although the morphology and function of footpads have been studied, the mechanism of their formation is still elusive. In the ladybird beetle (Harmonia axyridis), hairy footpads are present on the first and second tarsal segments of the legs. The footpads are covered with hundreds of hairs, i.e. setae, whose tips consist of four types: pointed, lanceolate, spatular, and discoidal. We examined the formation of the footpad during the pupal stage using immuno-staining and fluorescent-conjugated phalloidin staining. We found that a seta was composed of a shaft and a socket and some setae were accompanied by a neuron. By the mid-pupal stages, the shaft cells elongated to form a setal structure. Cytoskeletal actin bundles ramified to create a framework for the setal tip structure of the cells. We examined the effects of the application of cytochalasin D, which inhibits actin polymerization, on the formation of footpad setal structures. The results showed that the setal tips were deformed by the inhibition of actin polymerization. Our observations reveal that cytoskeletal actin filaments are involved in shaping the setae.