Actin Filament Remodeling Research Articles

Arp2/3 complex activity enables nuclear YAP for naïve pluripotency of human embryonic stem cells.

Our understanding of the transitions of human embryonic stem cells (hESCs) between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of hESCs as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for formation of the actin ring, to establish uniform cell mechanics within naïve colonies, to promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and for effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

Differential interference with actin-binding protein function by acute Cytochalasin B.

Dynamic actin filament remodeling is crucial for a plethora of fundamental cell biological processes, ranging from cell division and migration to cell communication, intracellular trafficking or tissue development. Cytochalasin B and -D are fungal secondary metabolites frequently used for interference with such processes. Although generally assumed to block actin filament polymerization at their rapidly growing barbed ends and compete with regulators at these sites, our molecular understanding of their precise effects in dynamic actin structures is scarce. Here we combine live cell imaging and analysis of fluorescent actin-binding protein dynamics with acute treatment of lamellipodia in migrating cells with cytochalasin B. Our results show that in spite of an abrupt halt of lamellipodium protrusion, cytochalasin B affects various actin filament barbed end-binding proteins in a differential fashion. Cytochalasin B enhances instead of diminishes the accumulation of prominent barbed end-binding factors such as Ena/VASP family proteins and heterodimeric capping protein (CP) in the lamellipodium. Similar results were obtained with cytochalasin D. All these effects are highly specific, as cytochalasin-induced VASP accumulation requires the presence of CP, but not vice versa , and coincides with abrogation of both actin and VASP turnover. Cytochalasin B can also increase apparent barbed end interactions with the actin-binding β-tentacle of CP and partially mimic its Arp2/3 complex-promoting activity in the lamellipodium. In conclusion, our results reveal a new spectrum of cytochalasin activities on barbed end-binding factors, with important implications for the interpretation of their effects on dynamic actin structures.

Conditional nmy-1 and nmy-2 alleles establish that nonmuscle myosins are required for late Caenorhabditis elegans embryonic elongation.

The elongation of Caenorhabditis elegans embryos allows examination of mechanical interactions between adjacent tissues. Muscle contractions during late elongation induce the remodeling of epidermal circumferential actin filaments through mechanotransduction. Force inputs from the muscles deform circumferential epidermal actin filament, which causes them to be severed, eventually reformed, and shortened. This squeezing force drives embryonic elongation. We investigated the possible role of the nonmuscle myosins NMY-1 and NMY-2 in this process using nmy-1 and nmy-2 thermosensitive alleles. Our findings show these myosins act redundantly in late elongation, since double nmy-2(ts); nmy-1(ts) mutants immediately stop elongation when raised to 25°C. Their inactivation does not reduce muscle activity, as measured from epidermis deformation, suggesting that they are directly involved in the multistep process of epidermal remodeling. Furthermore, NMY-1 and NMY-2 inactivation is reversible when embryos are kept at the nonpermissive temperature for a few hours. However, after longer exposure to 25°C double mutant embryos fail to resume elongation, presumably because NMY-1 was seen to form protein aggregates. We propose that the two C. elegans nonmuscle myosin II act during actin remodeling either to bring severed ends or hold them.

Spearmint targets microtubules by (-)-carvone.

Allelopathy can provide sustainable alternatives to herbicides because it is based on specific signals rather than generic toxicity. We show that the allelopathic activity of Spearmint and Watermint is linked with their main compounds, (-)-carvone and (+)-menthofuran, both deriving from (-)-limonene. Germination of Poppy and Cress, and root growth of Arabidopsis thaliana are inhibited by very low concentrations of (-)-carvone, acting even through the gas phase. (+)-Menthofuran is active as well, but at lower efficacy. Using fluorescently tagged marker lines in tobacco BY-2 cells and Arabidopsis roots, we demonstrate a rapid degradation of microtubules and a remodeling of actin filaments in response to (-)-carvone and, to a milder extent, to (+)-menthofuran. This cytoskeletal response is followed by cell death. By means of a Root Chip system, we can follow the tissue dependent response of the cytoskeleton and show a cell-type dependent gradient of sensitivity between meristem and distal elongation zone, accompanied by programmed cell death.

The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability.

Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser665 and Tyr685. It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr416 and Ser144, respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor.

Magneto-Mechanical Actuation Induces Endothelial Permeability.

Cancer treatment is one of the major health problems that burden our society. According to the American Cancer Society, over 1.9 million new cancer cases and ∼0.6 million deaths from cancer are expected in the US in 2023. Therapeutic targeting is considered to be the gold standard in cancer treatment. However, when a tumor grows beyond a critical size, its vascular system differentiates abnormally and erratically, creating a heterogeneous endothelial barrier that further restricts drug delivery into tumors. While several methods exist, these prompt tumor migration and the appearance of new metastatic sites. Herein, we propose an innovative method based on magneto-mechanical actuation (MMA) to induce endothelial permeability. This method employs FDA-approved PEGylated superparamagnetic iron oxide nanoparticles (PEG-SPIONs) and alternating nonheating magnetic fields. MMA lies in the translation of magnetic forces into mechanical agitation. As a proof of concept, we developed a 2D cell culture model based on human umbilical vein endothelial cells (HUVEC), which were incubated with PEG-SPIONs and then exposed to different magnetic doses. After adjusting the particle concentration, incubation times, and parameters (amplitude, frequency, and exposure time) of the magnetic field generator, we induced actin filament remodeling and subsequent vascular endothelial-cadherin junction disruption. This led to transient gaps in cell monolayers, through which fluorescein isothiocyanate-dextran was translocated. We observed no cell viability reduction for 3 h of particle incubation up to a concentration of 100 μg/mL in the presence and absence of magnetic fields. For optimal permeability studies, the magnetic field parameters were adjusted to 100 mT, 65 Hz, and 30 min in a pulse mode with 5 min OFF intervals. We found that the endothelial permeability reached the highest value (33%) when 2 h postmagnetic field treatment was used. To explain these findings, a magneto-mechanical transduced stress mechanism mediated by intracellular forces was proposed. This method can open new avenues for targeted drug delivery into anatomic regions within the body for a broad range of disease interventions.

Dynamic remodeling of actin networks by cyclase-associated protein and CAP-Abp1 complexes

How actin filaments are spatially organized and remodeled into diverse higher-order networks invivo is still not well understood. Here, we report an unexpected F-actin "coalescence" activity driven by cyclase-associated protein (CAP) and enhanced by its interactions with actin-binding protein 1 (Abp1). We directly observe S.cerevisiae CAP and Abp1 rapidly transforming branched or linear actin networks by bundling and sliding filaments past each other, maximizing filament overlap, and promoting compaction into bundles. This activity does not require ATP and is conserved, as similar behaviors are observed for the mammalian homologs of CAP and Abp1. Coalescence depends on the CAP oligomerization domain but not the helical folded domain (HFD) that mediates its functions in F-actin severing and depolymerization. Coalescence by CAP-Abp1 further depends on interactions between CAP and Abp1 and interactions between Abp1 and F-actin. Our results are consistent with a mechanism in which the formation of energetically favorable sliding CAP and CAP-Abp1 crosslinks drives F-actin bundle compaction. Roles for CAP and CAP-Abp1 in actin remodeling invivo are supported by strong phenotypes arising from deletion of the CAP oligomerization domain and by genetic interactions between sac6Δ and an srv2-301 mutant that does not bind Abp1. Together, these observations identify a new actin filament remodeling function for CAP, which is further enhanced by its direct interactions with Abp1.

Cytochalasans and Their Impact on Actin Filament Remodeling.

The eukaryotic actin cytoskeleton comprises the protein itself in its monomeric and filamentous forms, G- and F-actin, as well as multiple interaction partners (actin-binding proteins, ABPs). This gives rise to a temporally and spatially controlled, dynamic network, eliciting a plethora of motility-associated processes. To interfere with the complex inter- and intracellular interactions the actin cytoskeleton confers, small molecular inhibitors have been used, foremost of all to study the relevance of actin filaments and their turnover for various cellular processes. The most prominent inhibitors act by, e.g., sequestering monomers or by interfering with the polymerization of new filaments and the elongation of existing filaments. Among these inhibitors used as tool compounds are the cytochalasans, fungal secondary metabolites known for decades and exploited for their F-actin polymerization inhibitory capabilities. In spite of their application as tool compounds for decades, comprehensive data are lacking that explain (i) how the structural deviances of the more than 400 cytochalasans described to date influence their bioactivity mechanistically and (ii) how the intricate network of ABPs reacts (or adapts) to cytochalasan binding. This review thus aims to summarize the information available concerning the structural features of cytochalasans and their influence on the described activities on cell morphology and actin cytoskeleton organization in eukaryotic cells.

Fluoride disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated RhoA/ROCK signaling and myosin light chain kinase

Fluoride is a common contaminant of groundwater and agricultural commodity, which poses challenges to animal and human health. A wealth of research has demonstrated its detrimental effects on intestinal mucosal integrity; however, the underlying mechanisms remain obscure. This study aimed to investigate the role of the cytoskeleton in fluoride-induced barrier dysfunction. After sodium fluoride (NaF) treatment of the cultured Caco-2 cells, both cytotoxicity and cytomorphological changes (internal vacuoles or massive ablation) were observed. NaF lowered transepithelial electrical resistance (TEER) and enhanced paracellular permeation of fluorescein isothiocyanate dextran 4 (FD-4), indicating Caco-2 monolayers hyperpermeability. In the meantime, NaF treatment altered both the expression and distribution of the tight junction protein ZO-1. Fluoride exposure increased myosin light chain II (MLC2) phosphorylation and triggered actin filament (F-actin) remodeling. While inhibition of myosin II by Blebbistatin blocked NaF-induced barrier failure and ZO-1 discontinuity, the corresponding agonist Ionomycin had effects comparable to those of fluoride, suggesting that MLC2 serves as an effector. Given the mechanisms upstream of p-MLC2 regulation, further studies demonstrated that NaF activated RhoA/ROCK signaling pathway and myosin light chain kinase (MLCK), strikingly increasing the expression of both. Pharmacological inhibitors (Rhosin, Y-27632 and ML-7) reversed NaF-induced barrier breakdown and stress fiber formation. The role of intracellular calcium ions ([Ca2+]i) in NaF effects on Rho/ROCK pathway and MLCK was investigated. We found that NaF elevated [Ca2+]i, whereas chelator BAPTA-AM attenuated increased RhoA and MLCK expression as well as ZO-1 rupture, thus, restoring barrier function. Collectively, abovementioned results suggest that NaF induces barrier impairment via Ca2+-dependent RhoA/ROCK pathway and MLCK, which in turn triggers MLC2 phosphorylation and rearrangement of ZO-1 and F-actin. These results provide potential therapeutic targets for fluoride-induced intestinal injury.

Convolutional neural network segmentation of bare and decorated actin filaments in cellular cryo-tomography.

Developing neurons have a highly dynamic structure at the tip of growing axons, the growth cone, that guides the neuron to its synaptic target. Rapid remodeling of actin filaments and bundles is vital to the movement of these structures. Recently, an actin binding protein that canonically plays a severing role in filaments, cofilin, has been shown to play a role in filament stabilization and flexibility within growth cones when fully decorated at a 1:1 ratio. Decorated filaments, called cofilactin, are visibly distinct from f-actin within cryo-electron tomograms due to the helical pitch tightening that cofilin induces along the length of the decorated portion. To better characterize the spatial arrangement and role of cofilactin within growth cones, we are training convolutional neural networks (CNNs) to identify and segment f-actin, cofilactin, and the transition zones between decorated and undecorated regions within cryo-tomographic data. Due to the low signal-to-noise ratio and native image aberrations of cryo-electron tomography (cryo-ET), distinguishing filaments accurately by hand within very noisy data is both tedious and time consuming. Actin networks in growth cones can be densely packed and annotating a single tomogram can take upwards of a week for an expert hand segmenter. Convolutional neural networks excel at distinguishing true signal from noise. With proper training, it is feasible that a CNN, in this case a U-Net, will be able to segment filaments rapidly and accurately within growth cones. We have used simulated tomographic data to create ‘perfect’ ground truth training sets and generated trained U-Nets that can accurately identify cofilin and actin in both in vitro and cellular tomograms.

Changes in nanomechanical properties of single neuroblastoma cells as a model for oxygen and glucose deprivation (OGD)

Although complex, the biological processes underlying ischemic stroke are better known than those related to biomechanical alterations of single cells. Mechanisms of biomechanical changes and their relations to the molecular processes are crucial for understanding the function and dysfunction of the brain. In our study, we applied atomic force microscopy (AFM) to quantify the alterations in biomechanical properties in neuroblastoma SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) and reoxygenation (RO). Obtained results reveal several characteristics. Cell viability remained at the same level, regardless of the OGD and RO conditions, but, in parallel, the metabolic activity of cells decreased with OGD duration. 24 h RO did not recover the metabolic activity fully. Cells subjected to OGD appeared softer than control cells. Cell softening was strongly present in cells after 1 h of OGD and with longer OGD duration, and in RO conditions, cells recovered their mechanical properties. Changes in the nanomechanical properties of cells were attributed to the remodelling of actin filaments, which was related to cofilin-based regulation and impaired metabolic activity of cells. The presented study shows the importance of nanomechanics in research on ischemic-related pathological processes such as stroke.

Vasoactive intestinal peptide-VIPR2 signaling regulates tumor cell migration.

Phosphoinositide metabolism is critically involved in human cancer cell migration and metastatic growth. The formation of lamellipodia at the leading edge of migrating cells is regulated by metabolism of the inositol phospholipid PI(4,5)P2 into PI(3,4,5)P3. The synthesized PI(3,4,5)P3 promotes the translocation of WASP family verprolin homologous protein 2 (WAVE2) to the plasma membrane and regulates guanine nucleotide exchange factor Rac-mediated actin filament remodeling. Here, we investigated if VIPR2, a receptor for vasoactive intestinal peptide (VIP), has a potential role in regulating cell migration via this pathway. We found that silencing of VIPR2 in MDA-MB-231 and MCF-7 human breast cancer cells inhibited VIP-induced cell migration. In contrast, stable expression of exogenous VIPR2 promoted VIP-induced tumor cell migration, an effect that was inhibited by the addition of a PI3-kinase (PI3K)γ inhibitor or a VIPR2-selective antagonist. VIPR2 stably-expressing cells exhibited increased PI3K activity. Membrane localization of PI(3,4,5)P3 was significantly attenuated by VIPR2-silencing. VIPR2-silencing in MDA-MB-231 cells suppressed lamellipodium extension; in VIPR2-overexpressing cells, VIPR2 accumulated in the cell membrane on lamellipodia and co-localized with WAVE2. Conversely, VIPR2-silencing reduced WAVE2 level on the cell membrane and inhibited the interaction between WAVE2, actin-related protein 3, and actin. These findings suggest that VIP–VIPR2 signaling controls cancer migration by regulating WAVE2-mediated actin nucleation and elongation for lamellipodium formation through the synthesis of PI(3,4,5)P3.

Hypergravity Attenuates Reactivity in Primary Murine Astrocytes.

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2g and 10g). We revealed spreading rates, migration velocities, and stellation to be diminished under 2g hypergravity. In contrast, proliferation and apoptosis rates were not affected. In particular, hypergravity attenuated reactivity induction. We observed cytoskeletal remodeling of actin filaments and microtubules under hypergravity. Hence, the reorganization of these key elements of cell structure demonstrates that fundamental mechanisms on shape and mobility of astrocytes are affected due to altered gravity conditions. In future experiments, potential target molecules for pharmacological interventions that attenuate astrocytic reactivity will be investigated. The ultimate goal is to enhance neuronal regeneration for novel therapeutic approaches.

The Chemerin/CMKLR1 Axis Is Involved in the Recruitment of Microglia to Aβ Deposition through p38 MAPK Pathway.

The accumulation of microglia around senile plaques is one of the pathological features of Alzheimer’s disease (AD). Chemerin is an adipokine with immune-modulating properties. Our previous study showed that chemokine-like receptor 1 (CMKLR1), the receptor for chemerin, is also a functional receptor of Aβ. However, it remains unclear whether and how the chemerin/CMKLR1 axis affects the migration of microglia. The impact of CMKLR1 on microglial activation and recruitment toward Aβ deposits was examined in APP/PS1 mice mated with CMKLR1 knockout (CMKLR1−/−) mice. CMKLR1 deficiency reduced the number of microglia around Aβ deposits in aged APP/PS1-CMKLR1−/− mice compared with APP/PS1 mice. Chemerin expression was significantly decreased in the hippocampus and cortex of aged APP/PS1 mice compared with WT mice. In vitro assays demonstrated that activation of the chemerin/CMKLR1 axis promoted the migration of primary cultures of microglia and murine microglial N9 cells. Mechanistic studies found that chemerin/CMKLR1 induced polarization and protrusion formation of microglia by promoting the remodeling of actin filaments and microtubules, and Golgi apparatus reorientation. The inhibition of p38 MAPK attenuated the promotion of the chemerin/CMKLR1 axis on microglial migration and polarization. In addition, chemerin inhibited Aβ-induced microglial clustering. The inhibition of p38 MAPK alleviated the suppressive effect of chemerin on Aβ-induced microglial aggregation. Our data indicate that the chemerin/CMKLR1 axis is involved in the migration and recruitment of microglia to senile plaques via the p38 MAPK pathway. Modulation of the chemerin/CMKLR1 axis is a potential new strategy for AD therapy.

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments.

Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell contractile apparatus. Cell exposure to different types of stress is accompanied by the translocation of sHsps onto actin filaments; therefore, it is commonly believed that the sHsps are true actin-binding proteins. Investigations of last years have shown that this assumption is incorrect. Stress-induced translocation of sHsp to actin filaments is not the result of direct interaction of these proteins with intact actin, but results from the chaperone-like activity of sHsps and their interaction with various actin-binding proteins. HspB1 and HspB5 interact with giant elastic proteins titin and filamin thus providing an integrity of the contractile apparatus and its proper localization in the cell. HspB6 binds to the universal adapter protein 14-3-3 and only indirectly affects the structure of actin filament. HspB7 interacts with filamin C and controls actin filament assembly. HspB8 forms tight complex with the universal regulatory and adapter protein Bag3 and participates in the chaperone-assisted selective autophagy (CASA) of actin-binding proteins (e.g., filamin), as well as in the actin-depending processes taking place in mitoses. Hence, the mechanisms of sHsp participation in the maintenance of the contractile apparatus and cytoskeleton are much more complicated and diverse than it has been postulated earlier and are not limited to direct interactions of sHsps with actin. The old hypothesis on the direct binding of sHsps to intact actin should be revised and further detailed investigation on the sHsp interaction with minor proteins participating in the formation and remodeling of actin filaments is required.

LIM Kinases, Promising but Reluctant Therapeutic Targets: Chemistry and Preclinical Validation In Vivo.

LIM Kinases are important actors in the regulation of cytoskeleton dynamics by controlling microtubule and actin filament turnover. The signaling pathways involving LIM kinases for actin filament remodeling are well established. They are downstream effectors of small G proteins of the Rho-GTPases family and have become promising targets for the treatment of several major diseases because of their position at the lower end of these signaling cascades. Cofilin, which depolymerizes actin filaments, is the best-known substrate of these enzymes. The phosphorylation of cofilin to its inactive form by LIM kinases avoids actin filament depolymerization. The balance between phosphorylated and non-phosphorylated cofilin is thought to play an important role in tumor cell invasion and metastasis. Since 2006, many small molecules have been developed for LIMK inhibition, and in this review article, we will discuss the structure–activity relationships of the few inhibitor families that have been tested in vivo on different pathological models.

Novel roles of mTORC2 in regulation of insulin secretion by actin filament remodeling.

Mammalian target of rapamycin (mTOR) kinase is an essential hub where nutrients and growth factors converge to control cellular metabolism. mTOR interacts with different accessory proteins to form complexes 1 and 2 (mTORC), and each complex has different intracellular targets. Although mTORC1's role in β-cells has been extensively studied, less is known about mTORC2's function in β-cells. Here, we show that mice with constitutive and inducible β-cell-specific deletion of RICTOR (βRicKO and iβRicKO mice, respectively) are glucose intolerant due to impaired insulin secretion when glucose is injected intraperitoneally. Decreased insulin secretion in βRicKO islets was caused by abnormal actin polymerization. Interestingly, when glucose was administered orally, no difference in glucose homeostasis and insulin secretion were observed, suggesting that incretins are counteracting the mTORC2 deficiency. Mechanistically, glucagon-like peptide-1 (GLP-1), but not gastric inhibitory polypeptide (GIP), rescued insulin secretion in vivo and in vitro by improving actin polymerization in βRicKO islets. In conclusion, mTORC2 regulates glucose-stimulated insulin secretion by promoting actin filament remodeling.NEW & NOTEWORTHY The current studies uncover a novel mechanism linking mTORC2 signaling to glucose-stimulated insulin secretion by modulation of the actin filaments. This work also underscores the important role of GLP-1 in rescuing defects in insulin secretion by modulating actin polymerization and suggests that this effect is independent of mTORC2 signaling.

Fibroblast fate determination during cardiac reprogramming by remodeling of actin filaments.

SummaryFibroblasts can be reprogrammed into induced cardiomyocyte-like cells (iCMs) by forced expression of cardiogenic transcription factors. However, it remains unknown how fibroblasts adopt a cardiomyocyte (CM) fate during their spontaneous ongoing transdifferentiation toward myofibroblasts (MFs). By tracing fibroblast lineages following cardiac reprogramming in vitro, we found that most mature iCMs are derived directly from fibroblasts without transition through the MF state. This direct conversion is attributable to mutually exclusive induction of cardiac sarcomeres and MF cytoskeletal structures in the cytoplasm of fibroblasts during reprogramming. For direct fate switch from fibroblasts to iCMs, significant remodeling of actin isoforms occurs in fibroblasts, including induction of α-cardiac actin and decrease of the actin isoforms predominant in MFs. Accordingly, genetic or pharmacological ablation of MF-enriched actin isoforms significantly enhances cardiac reprogramming. Our results demonstrate that remodeling of actin isoforms is required for fibroblast to CM fate conversion by cardiac reprogramming.

Ena/VASP proteins in cell edge protrusion, migration and adhesion.

The tightly coordinated, spatiotemporal control of actin filament remodeling provides the basis of fundamental cellular processes, such as cell migration and adhesion. Specific protein assemblies, composed of various actin-binding proteins, are thought to operate in these processes to nucleate and elongate new filaments, arrange them into complex three-dimensional (3D) arrays and recycle them to replenish the actin monomer pool. Actin filament assembly is not only necessary to generate pushing forces against the leading edge membrane or to propel pathogens through the cytoplasm, but also coincides with the generation of stress fibers (SFs) and focal adhesions (FAs) that generate, transmit and sense mechanical tension. The only protein families known to date that directly enhance the elongation of actin filaments are formins and the family of Ena/VASP proteins. Their mechanisms of action, however, in enhancing processive filament elongation are distinct. The aim of this Review is to summarize our current knowledge on the molecular mechanisms of Ena/VASP-mediated actin filament assembly, and to discuss recent insights into the cell biological functions of Ena/VASP proteins in cell edge protrusion, migration and adhesion.

PCARE requires coiled coil, RP62 kinase-binding and EVH1 domain-binding motifs for ciliary expansion.

Retinitis pigmentosa (RP) is a genetically heterogeneous form of inherited retinal disease that leads to progressive visual impairment. One genetic subtype of RP, RP54, has been linked to mutations in PCARE (photoreceptor cilium actin regulator). We have recently shown that PCARE recruits WASF3 to the tip of a primary cilium, and thereby activates an Arp2/3 complex which results in the remodeling of actin filaments that drives the expansion of the ciliary tip membrane. On the basis of these findings, and the lack of proper photoreceptor development in mice lacking Pcare, we postulated that PCARE plays an important role in photoreceptor outer segment disk formation. In this study, we aimed to decipher the relationship between predicted structural and function amino acid motifs within PCARE and its function. Our results show that PCARE contains a predicted helical coiled coil domain together with evolutionary conserved binding sites for photoreceptor kinase MAK (type RP62), as well as EVH1 domain-binding linear motifs. Upon deletion of the helical domain, PCARE failed to localize to the cilia. Furthermore, upon deletion of the EVH1 domain-binding motifs separately or together, co-expression of mutant protein with WASF3 resulted in smaller ciliary tip membrane expansions. Finally, inactivation of the lipid modification on the cysteine residue at amino acid position 3 also caused a moderate decrease in the sizes of ciliary tip expansions. Taken together, our data illustrate the importance of amino acid motifs and domains within PCARE in fulfilling its physiological function.