RESUMEN
Activation of the Arp2/3 complex by VCA-motif-bearing actin nucleation-promoting factors results in the formation of "daughter" actin filaments branching off the sides of pre-existing "mother" filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates "linear" actin filaments. Uncovering the similarities and differences between these two mechanisms is fundamental to understanding how actin cytoskeleton dynamics are regulated. Here, analysis of individual filaments reveals that, unexpectedly, the VCA motifs of WASP, N-WASP, and WASH destabilize existing branches, as well as SPIN90-Arp2/3 at linear filament ends. Furthermore, branch stabilizer cortactin and destabilizer GMF each have a similar impact on SPIN90-activated Arp2/3. However, unlike branch junctions, SPIN90-Arp2/3 at the ends of linear filaments is not destabilized by piconewton forces and does not become less stable with time. It thus appears that linear and branched Arp2/3-generated filaments respond similarly to the regulatory proteins we have tested, albeit with some differences, but significantly differ in their responses to aging and mechanical stress. These kinetic differences likely reflect the small conformational differences recently reported between Arp2/3 in branch junctions and linear filaments and suggest that their turnover in cells may be differently regulated.
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Citoesqueleto de Actina , Complejo 2-3 Proteico Relacionado con la Actina , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Citoesqueleto/metabolismo , Actinas/metabolismoRESUMEN
Lissencephaly is a neurodevelopmental disorder characterized by a loss of brain surface convolutions caused by genetic variants that disrupt neuronal migration. However, the genetic origins of the disorder remain unidentified in nearly one-fifth of people with lissencephaly. Using whole-exome sequencing, we identified a de novo BAIAP2 variant, p.Arg29Trp, in an individual with lissencephaly with a posterior more severe than anterior (P>A) gradient, implicating BAIAP2 as a potential lissencephaly gene. Spatial transcriptome analysis in the developing mouse cortex revealed that Baiap2 is expressed in the cortical plate and intermediate zone in an anterior low to posterior high gradient. We next used in utero electroporation to explore the effects of the Baiap2 variant in the developing mouse cortex. We found that Baiap2 knockdown caused abnormalities in neuronal migration, morphogenesis and differentiation. Expression of the p.Arg29Trp variant failed to rescue the migration defect, suggesting a loss-of-function effect. Mechanistically, the variant interfered with the ability of BAIAP2 to localize to the cell membrane. These results suggest that the functions of BAIAP2 in the cytoskeleton, cell morphogenesis and migration are important for cortical development and for the pathogenesis of lissencephaly in humans.
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Lisencefalia , Animales , Humanos , Ratones , Encéfalo/metabolismo , Movimiento Celular/genética , Citoesqueleto/metabolismo , Lisencefalia/genética , Lisencefalia/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
After ATP-actin monomers assemble filaments, the ATP's [Formula: see text]-phosphate is hydrolyzedwithin seconds and dissociates over minutes. We used all-atom molecular dynamics simulations to sample the release of phosphate from filaments and study residues that gate release. Dissociation of phosphate from Mg2+ is rate limiting and associated with an energy barrier of 20 kcal/mol, consistent with experimental rates of phosphate release. Phosphate then diffuses within an internal cavity toward a gate formed by R177, as suggested in prior computational studies and cryo-EM structures. The gate is closed when R177 hydrogen bonds with N111 and is open when R177 forms a salt bridge with D179. Most of the time, interactions of R177 with other residues occlude the phosphate release pathway. Machine learning analysis reveals that the occluding interactions fluctuate rapidly, underscoring the secondary role of backdoor gate opening in Pi release, in contrast with the previous hypothesis that gate opening is the primary event.
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Citoesqueleto de Actina , Adenosina Trifosfato , Simulación de Dinámica Molecular , Fosfatos , Fosfatos/metabolismo , Fosfatos/química , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Adenosina Trifosfato/metabolismo , Actinas/metabolismo , Actinas/química , Enlace de Hidrógeno , Magnesio/metabolismo , Magnesio/química , Microscopía por CrioelectrónRESUMEN
Molecular motors employ chemical energy to generate unidirectional mechanical output against a track while navigating a chaotic cellular environment, potential disorder on the track, and against Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.
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Actinas , Miosina Tipo V , Actinas/química , Miosinas/química , Citoesqueleto de Actina/química , Movimiento (Física) , Miosina Tipo V/químicaRESUMEN
Chloroplasts accumulate on the cell surface under weak light conditions to efficiently capture light but avoid strong light to minimize photodamage. The blue light receptor phototropin regulates the chloroplast movement in various plant species. In Arabidopsis thaliana, phototropin mediates the light-induced chloroplast movement and positioning via specialized actin filaments on the chloroplasts, chloroplast-actin filaments. KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT (KAC) and CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) are pivotal for chloroplast-actin-based chloroplast movement and positioning in land plants. However, the mechanisms by which KAC and CHUP1 regulate chloroplast movement and positioning remain unclear. In this study, we characterized KAC and CHUP1 orthologs in the liverwort Marchantia polymorpha, MpKAC and MpCHUP1, respectively. Their knockout mutants, Mpkack° and Mpchup1k°, impaired the light-induced chloroplast movement. Although Mpchup1k° showed mild chloroplast aggregation, Mpkack° displayed severe chloroplast aggregation, suggesting the greater contribution of MpKAC to the chloroplast anchorage to the plasma membrane. Analysis of the subcellular localization of the functional MpKAC-Citrine indicated that MpKAC-Citrine formed a punctate structure on the plasma membrane. Structure-function analysis of MpKAC revealed that a deletion of the conserved C-terminal domain abrogates the targeting to the plasma membrane and its function. A deletion of the N-terminal motor domain retained the plasma membrane targeting but abrogates the formation of punctate structure and showed severe defect in the light-induced chloroplast movement. Our findings suggest that the formation of the punctate structure on the plasma membrane of MpKAC is essential for chloroplast movement.
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Myosin family proteins are ATP-driven, actin filament-based motor proteins that generate force along actin filaments. In in vitro actin filament gliding assays, certain myosins generate rotation of gliding actin filaments around their long axes. In this study, we assessed the effects of temperature on the corkscrewing motion of actin filaments, including factors like gliding and rotational velocities and corkscrewing pitch. The corkscrewing motion was driven by a nonprocessive, full-length single-headed Drosophila myosin IC attached to an antibody adsorbed onto a cover glass. We performed an in vitro actin filament corkscrewing assay at temperatures ranging from 25 °C to 35 °C. We found that the gliding and rotational velocities and the pitch of corkscrewing actin filaments generated by myosin IC molecules increased with increasing temperature. Since the pitch is determined by dividing the gliding velocity by the rotational velocity, an increase in the pitch indicates that the gliding velocity increased faster than the rotational velocity with increasing temperature. These results suggest that temperature has distinct effects on the gliding and rotational forces produced by myosin IC, with implications for interpreting the temperature effect on torque-generation mechanisms driven by myosins on actin filaments at physiological temperatures.
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Citoesqueleto de Actina , Miosinas , Temperatura , Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Rotación , Actinas/metabolismoRESUMEN
Actin-interacting proteins are important molecules for filament assembly and cytoskeletal signaling within vascular endothelium. Disruption in their interactions causes endothelial pathogenesis through redox imbalance. Actin filament redox regulation remains largely unexplored, in the context of pharmacological treatment. This work focused on the peptidyl methionine (M) redox regulation of actin-interacting proteins, aiming at elucidating its role on governing antioxidative signaling and response. Endothelial EA.hy926 cells were subjected to treatment with salvianolic acid B (Sal B) and tert-butyl-hydroperoxide (tBHP) stimulation. Mass spectrometry was employed to characterize redox status of proteins, including actin, myosin-9, kelch-like erythroid-derived cap-n-collar homology-associated protein 1 (Keap1), plastin-3, prelamin-A/C and vimentin. The protein redox landscape revealed distinct stoichiometric ratios or reaction site transitions mediated by M sulfoxide reductase and reactive oxygen species. In comparison with effects of tBHP stimulation, Sal B treatment prevented oxidation at actin M325, myosin-9 M1489/1565, Keap1 M120, plastin-3 M592, prelamin-A/C M187/371/540 and vimentin M344. For Keap1, reaction site was transitioned within its scaffolding region to the actin ring. These protein M oxidation regulations contributed to the Sal B cytoprotective effects on actin filament. Additionally, regarding the Keap1 homo-dimerization region, Sal B preventive roles against M120 oxidation acted as a primary signal driver to activate nuclear factor erythroid 2-related factor 2 (Nrf2). Transcriptional splicing of non-POU domain-containing octamer-binding protein was validated during the Sal B-mediated overexpression of NAD(P)H dehydrogenase [quinone] 1. This molecular redox regulation of actin-interacting proteins provided valuable insights into the phenolic structures of Sal B analogs, showing potential antioxidative effects on vascular endothelium.
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Actinas , Antioxidantes , Benzofuranos , Depsidos , Antioxidantes/farmacología , Antioxidantes/metabolismo , Actinas/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Vimentina/metabolismo , Estrés Oxidativo , Metionina , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Proteínas del Citoesqueleto/metabolismo , Miosinas/metabolismo , Miosinas/farmacologíaRESUMEN
Chloroplast-actin (cp-actin) filaments are crucial for light-induced chloroplast movement, and appear in the front region of moving chloroplasts when visualized using GFP-mouse Talin. They are short and thick, exist between a chloroplast and the plasma membrane, and move actively and rapidly compared to cytoplasmic long actin filaments that run through a cell. The average period during which a cp-actin filament was observed at the same position was less than 0.5 s. The average lengths of the cp-actin filaments calculated from those at the front region of the moving chloroplast and those around the chloroplast periphery after stopping the movement were almost the same, approximately 0.8 µm. Each cp-actin filament is shown as a dotted line consisting of 4-5 dots. The vector sum of cp-actin filaments in a moving chloroplast is parallel to the moving direction of the chloroplast, suggesting that the direction of chloroplast movement is regulated by the vector sum of cp-actin filaments. However, once the chloroplasts stopped moving, the vector sum of the cp-actin filaments around the chloroplast periphery was close to zero, indicating that the direction of movement was undecided. To determine the precise structure of cp-actin filaments under electron microscopy, Arabidopsis leaves and fern Adiantum capillus-veneris gametophytes were frozen using a high-pressure freezer, and observed under electron microscopy. However, no bundled microfilaments were found, suggesting that the cp-actin filaments were unstable even under high-pressure freezing.
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Citoesqueleto de Actina , Arabidopsis , Cloroplastos , Luz , Cloroplastos/fisiología , Cloroplastos/metabolismo , Cloroplastos/efectos de la radiación , Cloroplastos/ultraestructura , Arabidopsis/fisiología , Arabidopsis/efectos de la radiación , Adiantum/fisiología , Adiantum/efectos de la radiación , Hojas de la Planta/fisiología , Hojas de la Planta/efectos de la radiación , Actinas/metabolismo , MovimientoRESUMEN
It has been proposed that cortical fine actin filaments are needed for the morphogenesis of pavement cells (PCs). However, the precise role and regulation mechanisms of actin filaments in PC morphogenesis are not well understood. Here, we found that Arabidopsis thaliana ACTIN DEPOLYMERIZING FACTOR9 (ADF9) is required for the morphogenesis of PC, which is negatively regulated by the R2R3 MYELOBLASTOSIS (MYB) transcription factor MYB52. In adf9 mutants, the lobe number of cotyledon PCs was significantly reduced, while the average lobe length did not differ significantly compared to that of wild type (Col-0), except for the variations in cell area and circularity, whereas the PC shapes in ADF9 overexpression seedlings showed different results. ADF9 decorated actin filaments, and colocalized with plasma membrane. The extent of filament bundling and actin filament bundling activity in adf9 mutant decreased. In addition, MYB52 directly targeted the promoter of ADF9 and negatively regulated its expression. The myb52-2 mutant showed increased lobe number and cell area, reduced cell circularity of PCs, and the PC phenotypes were suppressed when ADF9 was knocked out. Taken together, our data demonstrate that actin filaments play an important role in the morphogenesis of PC and reveal a transcriptional mechanism underlying MYB52 regulation of ADF9-mediated actin filament bundling in PC morphogenesis.
RESUMEN
The cell motility of smooth muscle cells (SMCs) is essential for vascular and internal organ development and tissue regeneration in response to damage. Cell migration requires dynamic changes in the actin-cytoskeleton via the p-21 activated kinase (Pak)-Cofilin signaling cascade, which is the central axis of the actin filaments. We previously identified that the Inka2 gene was preferentially expressed in the central nervous system (CNS) and revealed that Inka2 directly binds Pak4 to suppress its kinase activity, thereby regulating actin de-polymerization in dendritic spine formation of the forebrain neurons. However, its physiological significance outside the CNS remains unclear. Here we determined the Inka2 expression profile in various organs using in situ hybridization analysis and lacZ staining on Inka2flox/+ mice. Robust Inka2 expression was consistently detected in the SMCs of many peripheral organs, including the arteries, esophagus, stomach, intestine, and bladder. The scratch assay was used on primary cultured SMCs and revealed that Inka2-/- SMC exhibits accelerated cell migration ability without a change in the cell proliferation rate. Inka2-/- SMCs displayed Cofilin activation/phosphorylation, a downstream molecule of Pak4 signal cascade. These results suggest that Inka2 regulates SMC motility through modulating actin reorganization as the endogenous inhibitor of Pak4.
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Actinas , Miocitos del Músculo Liso , Animales , Ratones , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Movimiento Celular/fisiología , Células Cultivadas , Miocitos del Músculo Liso/metabolismoRESUMEN
BACKGROUND: The actin filament-associated protein (AFAP) family genes include AFAP1/AFAP-110, AFAP1L1 and AFAP1L2/XB130. Increasing evidence indicates these three AFAP family members participate in tumor progression, but their clinical significance and molecular mechanisms in gastric cancer (GC) remain unclear. METHODS: We first analyzed expression of AFAP family genes using public datasets and verified the results. The clinical significance of AFAP family genes in GC patients was also analyzed. In vitro and in vivo experiments were applied to explore the function of AFAP1L1. Enrichment analysis was used to explore potential molecular mechanisms. We then performed additional experiments, such as cell adhesion assay, co-immunoprecipitation and so on to confirm the downstream molecular mechanisms of AFAP1L1. RESULTS: Public data analyses and our verification both showed AFAP1L1 was the only AFAP family members that was significantly upregulated in GC compared with normal gastric tissues. Besides, only AFAP1L1 could predict poor prognosis and act as an independent risk factor for GC patients. In addition, AFAP1L1 promotes GC cells proliferation, migration, invasion in vitro and tumor growth, metastasis in vivo by inducing epithelial-to-mesenchymal transition (EMT). In terms of mechanism, AFAP1L1 interacts with VAV guanine nucleotide exchange factor 2 (VAV2) to activate Rho family GTPases CDC42, which finally promotes expression of integrin subunit alpha 5 (ITGA5) and activation of integrin signaling pathway. CONCLUSION: AFAP1L1 promotes GC progression by inducing EMT through VAV2-mediated activation of CDC42 and ITGA5 signaling pathway, indicating AFAP1L1 may be a promising prognostic biomarker and therapeutic target for GC patients.
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Neoplasias Gástricas , Humanos , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Integrinas/metabolismo , Proteínas de Microfilamentos/genética , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , Transducción de Señal , Neoplasias Gástricas/patologíaRESUMEN
Hypercholesterolemia is a major complication of arteriosclerosis. Mast cells in arteriosclerosis plaques induce inflammatory reactions and promote arterial sclerosis. In this study, we evaluated the pharmacological effects of simvastatin (SV)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors on the degranulation of rat basophilic leukemia (RBL)-2H3 cells, which are commonly used as mast cell models. SV significantly decreased the degranulation induced by three types of stimulation: antigen antibody reaction (Ag-Ab), thapsigargin (Tg) serosal endoplasmic reticulum calcium ATPase (SERCA) inhibitor, and A23187 calcium ionophore. SV had a stronger inhibitory effect on degranulation induced by Ag-Ab stimulation than the other two stimulations. However, SV did not inhibit increase of intracellular Ca2+ concentrations. Mevalonate or geranylgeraniol co-treatment with SV completely prevented the inhibitory effect of SV on the degranulation induced by these stimulations. Immunoblotting results showed that SV inhibited protein kinase C (PKC) delta translocation induced by Ag-Ab but not by Tg or A23187. SV induced a reduction in active Rac1, and actin filament rearrangement. In conclusion, SV inhibits RBL-2H3 cell degranulation by inhibiting downstream signaling pathways, including the sequential degranulation pathway. These inhibitory effects were completely reversed by the addition of geranylgeraniol and might be induced by changes in the translocation of the small guanosine 5'-triphosphatase (GTPase) families Rab and Rho, which are related to vesicular transport PKC delta translocation and actin filament formation, respectively. These changes are caused by the inhibition of HMG-CoA reductase by SV following the synthesis of geranylgeranyl pyrophosphates, which play important roles in the activation of small GTPases, Rab.
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Degranulación de la Célula , Simvastatina , Animales , Ratas , Degranulación de la Célula/fisiología , Calcimicina/farmacología , Simvastatina/farmacología , Transducción de Señal , Mastocitos , Calcio/metabolismoRESUMEN
Actin filaments elongate and shorten much faster at their barbed end than their pointed end, but the molecular basis of this difference has not been understood. We use all-atom molecular dynamics simulations to investigate the properties of subunits at both ends of the filament. The terminal subunits tend toward conformations that resemble actin monomers in solution, while contacts with neighboring subunits progressively flatten the conformation of internal subunits. At the barbed end the terminal subunit is loosely tethered by its DNase-1 loop to the third subunit, because its monomer-like conformation precludes stabilizing contacts with the penultimate subunit. The motions of the terminal subunit make the partially flattened penultimate subunit accessible for binding monomers. At the pointed end, unique contacts between the penultimate and terminal subunits are consistent with existing cryogenic electron microscopic (cryo-EM) maps, limit binding to incoming monomers, and flatten the terminal subunit, which likely promotes ATP hydrolysis and rapid phosphate release. These structures explain the distinct polymerization kinetics of the two ends.
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Citoesqueleto de Actina/química , Actinas/química , Modelos Moleculares , Conformación Proteica , Adenosina Difosfato/química , Adenosina Trifosfato/química , Sitios de Unión , Simulación de Dinámica Molecular , Unión Proteica , Multimerización de Proteína , Subunidades de ProteínaRESUMEN
Cell-to-cell transmission of misfolding-prone α-synuclein (α-Syn) has emerged as a key pathological event in Parkinson's disease. This process is initiated when α-Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs. By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing "scars" on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to α-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for α-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments.
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Endocitosis/fisiología , Miosinas/química , Miosinas/metabolismo , Polielectrolitos/metabolismo , alfa-Sinucleína/metabolismo , Línea Celular , Clatrina/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Modelos Moleculares , Enfermedad de Parkinson/metabolismo , Conformación Proteica , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismoRESUMEN
Bisphenol A (BPA) is widely used in the production of plastics, food containers, and receipt ink globally. However, research has identified it as an endocrine disruptor, affecting the hormonal balance in living organisms. Bisphenol S (BPS), one of the alternative substances, was developed, but its effects on human health and the underlying mechanisms remain unclarified. Specifically, research on the effects of oral exposure to bisphenol on the lungs is lacking. We examined the potential differences in toxicity between these compounds in lung cells in vitro and in vivo. Our toxicity mechanism studies on MRC5 and A549 cells exposed to BPA or BPS revealed that BPA induced actin filament abnormalities and activated epithelial-mesenchymal transition (EMT). This finding suggests an increased potential for lung fibrosis and metastasis in lung cancer. However, given that BPS was not detected at the administered dose and under the specific experimental conditions, the probability of these occurrences is considered minimal. Additionally, animal experiments confirmed that oral exposure to BPA activates EMT in the lungs. Our study provides evidence that prolonged oral exposure to BPA can lead to EMT activation in lung tissue, similar to that observed in cell experiments, suggesting the potential to induce lung fibrosis. This research emphasizes the importance of regulating the use of BPA to mitigate its associated pulmonary toxicity. Furthermore, it is significant that within the parameters of our experimental conditions, BPS did not exhibit the toxicological pathways clearly evident in BPA.
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Fibrosis Pulmonar , Animales , Humanos , Fibrosis Pulmonar/inducido químicamente , Fenoles/toxicidad , PulmónRESUMEN
Dendritic morphology underlies the source and processing of neuronal signal inputs. Morphology can be broadly described by two types of geometric characteristics. The first is dendrogram topology, defined by the length and frequency of the arbor branches; the second is spatial embedding, mainly determined by branch angles and straightness. We have previously demonstrated that microtubules and actin filaments are associated with arbor elongation and branching, fully constraining dendrogram topology. Here, we relate the local distribution of these two primary cytoskeletal components with dendritic spatial embedding. We first reconstruct and analyze 167 sensory neurons from the Drosophila larva encompassing multiple cell classes and genotypes. We observe that branches with a higher microtubule concentration tend to deviate less from the direction of their parent branch across all neuron types. Higher microtubule branches are also overall straighter. F-actin displays a similar effect on angular deviation and branch straightness, but not as consistently across all neuron types as microtubule. These observations raise the question as to whether the associations between cytoskeletal distributions and arbor geometry are sufficient constraints to reproduce type-specific dendritic architecture. Therefore, we create a computational model of dendritic morphology purely constrained by the cytoskeletal composition measured from real neurons. The model quantitatively captures both spatial embedding and dendrogram topology across all tested neuron groups. These results suggest a common developmental mechanism regulating diverse morphologies, where the local cytoskeletal distribution can fully specify the overall emergent geometry of dendritic arbors.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Dendritas/metabolismo , Microtúbulos/metabolismo , Células Receptoras Sensoriales/metabolismo , Citoesqueleto de Actina/metabolismoRESUMEN
In food allergies, antigen-induced aggregation of FcεRI on mast cells initiates highly ordered and sequential signaling events. Dok-1(downstream of tyrosine kinase 1), undergoes intense tyrosine phosphorylation upon FcεRI stimulation, which negatively regulates Ras/Erk signaling and the subsequent cytokine release, but it remains unclear whether Dok-1 regulates Fc-mediated degranulation. In this study, we investigated the role of Dok-1 in FcεRI-mediated degranulation. Dok-1 overexpressing RBL-2H3 cells were established. Degranulation, immunoprecipitation, co-immunoprecipitation, immunoblotting and flow cytometry assay were performed to explore the effects of Dok-1 and its underlying mechanisms. We found that, following FcεRI activation, Dok-1 was recruited to the plasma membrane, leading to tyrosine phosphorylation. Phosphorylated Dok-1 inhibits FcεRI-operated calcium influx, and negatively regulated degranulation by inhibiting calcium-dependent disassembly of actin filaments. Our data revealed that Dok-1 is a negative regulator of FcεRI-mediated mast cell degranulation. These findings contribute to the identification of therapeutic targets for food allergies.
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Calcio , Degranulación de la Célula , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Calcio/metabolismo , Mastocitos , Fosforilación , Receptores de IgE , Tirosina/metabolismo , Tirosina/farmacologíaRESUMEN
Actin dynamics is essential for T-cell development. We show here that cofilin1 is the key molecule for controlling actin filament turnover in this process. Mice with specific depletion of cofilin1 in thymocytes showed increased steady-state levels of actin filaments, and associated alterations in the pattern of thymocyte migration and adhesion. Our data suggest that cofilin1 is controlling oscillatory F-actin changes, a parameter that influences the migration pattern in a 3-D environment. In a collagen matrix, cofilin1 controls the speed and resting intervals of migrating thymocytes. Cofilin1 was not involved in thymocyte proliferation, cell survival, apoptosis or surface receptor trafficking. However, in cofilin1 mutant mice, impaired adhesion and migration resulted in a specific block of thymocyte differentiation from CD4/CD8 double-positive thymocytes towards CD4 and CD8 single-positive cells. Our data suggest that tuning of the dwelling time of thymocytes in the thymic niches is tightly controlled by cofilin1 and essential for positive selection during T-cell differentiation. We describe a novel role of cofilin1 in the physiological context of migration-dependent cell differentiation.
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Actinas , Timocitos , Actinas/genética , Animales , Linfocitos T CD8-positivos , Diferenciación Celular , Movimiento Celular , Cofilina 1 , RatonesRESUMEN
Self-incompatibility (SI) in the poppy Papaver rhoeas triggers dramatic alterations in actin within pollen tubes. However, how these actin alterations are mechanistically achieved remains largely unexplored. Here, we used treatment with the Ca2+ ionophore A23187 to mimic the SI-induced elevation in cytosolic Ca2+ and trigger formation of the distinctive F-actin foci. Live-cell imaging revealed that this remodeling involves F-actin fragmentation and depolymerization, accompanied by the rapid formation of punctate actin foci and subsequent increase in their size. We established that actin foci are generated and enlarged from crosslinking of fragmented actin filament structures. Moreover, we show that villins associate with actin structures and are involved in this actin reorganization process. Notably, we demonstrate that Arabidopsis VILLIN5 promotes actin depolymerization and formation of actin foci by fragmenting actin filaments, and controlling the enlargement of actin foci via bundling of actin filaments. Our study thus uncovers important novel insights about the molecular players and mechanisms involved in forming the distinctive actin foci in pollen tubes.
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Actinas , Proteínas de Microfilamentos , Tubo Polínico , Citoesqueleto de Actina , Actinas/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/fisiología , Tubo Polínico/genéticaRESUMEN
Actin cytoskeletal dynamics play a critical role in the regulation of myogenesis through mechanotransduction, Hippo signaling modulation, cell proliferation, and morphological changes. Although Twinfilin-1 (TWF1), a highly conserved actin-depolymerizing factor, is known to regulate actin filament assembly by sequestering actin monomer and capping barbed ends, the biological significance of TWF1 during the differentiation of myogenic progenitor cells has not been investigated. In this study, we unveiled the roles played by TWF1 in the proliferation and differentiation of C2C12 myoblasts. TWF1 was the predominant isoform in myoblasts, and its expression was induced during the early stage of differentiation. Knockdown of TWF1 by siRNA (siTWF1) induced the accumulation of actin filaments (F-actin) and promoted the nuclear translocation of Yes-associated protein (YAP) in the Hippo signaling pathway. TWF1 depletion activated transcription of YAP target genes and induced cell cycle and proliferation in myoblasts. Furthermore, TWF1 knockdown markedly reduced the expressions of myogenic regulatory factors, such as MyoD and MyoG, and drastically hindered myoblast differentiation, fusion, and myotube formation. Collectively, this study highlights the essential role of TWF1 in the myogenic differentiation of progenitor cells via modulation of F-actin and YAP, and suggests TWF1 as a potential therapeutic target for muscle wasting and myopathies.