MYB52 negatively regulates ADF9-meditated actin filament bundling in Arabidopsis pavement cell morphogenesis.

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.

A kinesin-like protein, KAC, is required for light-induced and actin-based chloroplast movement in Marchantia polymorpha.

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.

Mechanism of phosphate release from actin filaments.

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.

Chloroplast-actin filaments decide the direction of chloroplast avoidance movement under strong light in Arabidopsis thaliana.

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.

Lipid droplets in Arabidopsis thaliana leaves contain myosin-binding proteins and enzymes associated with furan-containing fatty acid biosynthesis.

Lipid droplets (LDs) are lipid storage organelles in plant leaves and seeds. Seed LD proteins are well known, and their functions in lipid metabolism have been characterized; however, many leaf LD proteins remain to be identified. We therefore isolated LDs from leaves of the leaf LD-overaccumulating mutant high sterol ester 1 (hise1) of Arabidopsis thaliana by centrifugation or co-immunoprecipitation. We then performed LD proteomics by mass spectrometry and identified 3,206 candidate leaf LD proteins. In this study, we selected 31 candidate proteins for transient expression assays using a construct encoding the candidate protein fused with green fluorescent protein (GFP). Fluorescence microscopy showed that MYOSIN BINDING PROTEIN14 (MYOB14) and two uncharacterized proteins localized to LDs labeled with the LD marker. Subcellular localization analysis of MYOB family members revealed that MYOB1, MYOB2, MYOB3, and MYOB5 localized to LDs. LDs moved along actin filaments together with the endoplasmic reticulum. Co-immunoprecipitation of myosin XIK with MYOB2-GFP or MYOB14-GFP suggested that LD-localized MYOBs are involved in association with the myosin XIK-LDs. The two uncharacterized proteins were highly similar to enzymes for furan fatty acid biosynthesis in the photosynthetic bacterium Cereibacter sphaeroides, suggesting a relationship between LDs and furan fatty acid biosynthesis. Our findings thus reveal potential molecular functions of LDs and provide a valuable resource for further studies of the leaf LD proteome.

Myosin-5 varies its step length to carry cargo straight along the irregular F-actin track.

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.

Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin.

Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the groove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved by a combination of fast association via the core and very slow dissociation dictated by the arm. Similar to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by actin depolymerizing factor (ADF)/cofilin, which correlated with increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

Effect of temperature on actin filament corkscrewing driven by nonprocessive myosin IC.

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.

Methionine redox regulation of actin-interacting proteins primarily governs antioxidative signaling and response to the salvianolic acid B treatment in EA.hy926 cells.

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.

Mechanism of Phosphate Release from Actin Filaments.

After ATP-actin monomers assemble filaments, the γ-phosphate is hydrolyzed from ATP within seconds and dissociates from the filament over several minutes. We used all-atom well-tempered metadynamics molecular dynamics simulations to sample the release of phosphate from filaments along with unbiased molecular dynamics simulations to 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 in an internal cavity toward a gate formed by R177 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 occludes the phosphate release pathway. Machine learning analysis reveals that the occluding interactions fluctuate rapidly. These occluded states have not been documented in cryo-EM reconstructions.

Rare loss-of-function variants in FLNB cause non-syndromic orofacial clefts.

Orofacial clefts (OFCs) are the most common congenital craniofacial disorders, of which the etiology is closely related to rare coding variants. Filamin B (FLNB) is an actin-binding protein implicated in bone formation. FLNB mutations have been identified in several types of syndromic OFCs and previous studies suggest a role of FLNB in the onset of non-syndromic OFCs (NSOFCs). Here, we report two rare heterozygous variants (p.P441T and p.G565R) in FLNB in two unrelated hereditary families with NSOFCs. Bioinformatics analysis suggests that both variants may disrupt the function of FLNB. In mammalian cells, p.P441T and p.G565R variants are less potent to induce cell stretches than wild type FLNB, suggesting that they are loss-of-function mutations. Immunohistochemistry analysis demonstrates that FLNB is abundantly expressed during palatal development. Importantly, Flnb-/- embryos display cleft palates and previously defined skeletal defects. Taken together, our findings reveal that FLNB is required for development of palates in mice and FLNB is a bona fide causal gene for NSOFCs in humans.

A lissencephaly-associated BAIAP2 variant causes defects in neuronal migration during brain development.

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.

Trifunctional Linkers Enable Improved Visualization of Actin by Expansion Microscopy.

Expansion microscopy (ExM) revolutionized the field of super-resolution microscopy by allowing for subdiffraction resolution fluorescence imaging on standard fluorescence microscopes. However, it has been found that it is hard to visualize actin filaments efficiently using ExM. To improve actin imaging, multifunctional molecules have been designed with moderate success. Here, we present optimized methods for phalloidin conjugate grafting that have a high efficiency for both cellular and tissue samples. Our optimized strategy improves anchoring and signal retention by ∼10 times. We demonstrate the potential of optimized trifunctional linkers (TRITON) for actin imaging in combination with immunolabeling using different ExM protocols. 10X ExM of actin labeled with optimized TRITON enabled us to visualize the periodicity of actin rings in cultured hippocampal neurons and brain slices by Airyscan confocal microscopy. Thus, TRITON linkers provide an efficient grafting method, especially in cases in which the concentration of target-bound monomers is insufficient for high-quality ExM.

Bisphenol A is more potent than bisphenol S in influencing the physiological and pathological functions of lungs via inducing lung fibrosis and stimulating metastasis.

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.

Lipocalin 10 is essential for protection against inflammation-triggered vascular leakage by activating LDL receptor-related protein 2-slingshot homologue 1 signalling pathway.

AIMS: Systemic inflammation occurs commonly during many human disease settings and increases vascular permeability, leading to organ failure, and lethal outcomes. Lipocalin 10 (Lcn10), a poorly characterized member of the lipocalin family, is remarkably altered in the cardiovascular system of human patients with inflammatory conditions. Nonetheless, whether Lcn10 regulates inflammation-induced endothelial permeability remains unknown. METHODS AND RESULTS: Systemic inflammation models were induced using mice by injection of endotoxin lipopolysaccharide (LPS) or caecal ligation and puncture (CLP) surgery. We observed that the expression of Lcn10 was dynamically altered only in endothelial cells (ECs), but not in either fibroblasts or cardiomyocytes isolated from mouse hearts following the LPS challenge or CLP surgery. Using in vitro gain- and loss-of-function approaches and an in vivo global knockout mouse model, we discovered that Lcn10 negatively regulated endothelial permeability upon inflammatory stimuli. Loss of Lcn10 augmented vascular leakage, leading to severe organ damage and higher mortality following LPS challenge, compared to wild-type controls. By contrast, overexpression of Lcn10 in ECs displayed opposite effects. A mechanistic analysis revealed that both endogenous and exogenous elevation of Lcn10 in ECs could activate slingshot homologue 1 (Ssh1)-Cofilin signalling cascade, a key axis known to control actin filament dynamics. Accordingly, a reduced formation of stress fibre and increased generation of cortical actin band were exhibited in Lcn10-ECs, when compared to controls upon endotoxin insults. Furthermore, we identified that Lcn10 interacted with LDL receptor-related protein 2 (LRP2) in ECs, which acted as an upstream factor of the Ssh1-Confilin signalling. Finally, injection of recombinant Lcn10 protein into endotoxic mice showed therapeutic effects against inflammation-induced vascular leakage. CONCLUSION: This study identifies Lcn10 as a novel regulator of EC function and illustrates a new link in the Lcn10-LRP2-Ssh1 axis to controlling endothelial barrier integrity. Our findings may provide novel strategies for the treatment of inflammation-related diseases.

Effects of Simvastatin on RBL-2H3 Cell Degranulation.

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.

Myosin-5 varies its steps along the irregular F-actin track.

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast to the majority of macroscopic machines, they need to navigate a chaotic cellular environment, potential disorder in the track and 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 (iSCAT) 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.

mRNA sequencing reveals the distinct gene expression and biological functions in cardiac fibroblasts regulated by recombinant fibroblast growth factor 2.

After myocardial injury, cardiac fibroblasts (CFs) differentiate into myofibroblasts, which express and secrete extracellular matrix (ECM) components for myocardial repair, but also promote myocardial fibrosis. Recombinant fibroblast growth factor 2 (FGF2) protein drug with low molecular weight can promote cell survival and angiogenesis, and it was found that FGF2 could inhibit the activation of CFs, suggesting FGF2 has great potential in myocardial repair. However, the regulatory role of FGF2 on CFs has not been fully elucidated. Here, we found that recombinant FGF2 significantly suppressed the expression of alpha smooth muscle actin (α-SMA) in CFs. Through RNA sequencing, we analyzed mRNA expression in CFs and the differently expressed genes regulated by FGF2, including 430 up-regulated genes and 391 down-regulated genes. Gene ontology analysis revealed that the differentially expressed genes were strongly enriched in multiple biological functions, including ECM organization, cell adhesion, actin filament organization and axon guidance. The results of gene set enrichment analysis (GSEA) show that ECM organization and actin filament organization are down-regulated, while axon guidance is up-regulated. Further cellular experiments indicate that the regulatory functions of FGF2 are consistent with the findings of the gene enrichment analysis. This study provides valuable insights into the potential therapeutic role of FGF2 in treating cardiac fibrosis and establishes a foundation for further research to uncover the underlying mechanisms of CFs gene expression regulated by FGF2.

Characterization of Mechanical and Cellular Effects of Rhythmic Vertical Vibrations on Adherent Cell Cultures.

This paper presents an innovative experimental setup that employs the principles of audio technology to subject adherent cells to rhythmic vertical vibrations. We employ a novel approach that combines three-axis acceleration measurements and particle tracking velocimetry to evaluate the setup's performance. This allows us to estimate crucial parameters such as root mean square acceleration, fluid flow patterns, and shear stress generated within the cell culture wells when subjected to various vibration types. The experimental conditions consisted of four vibrational modes: No Vibration, Continuous Vibration, Regular Pulse, and Variable Pulse. To evaluate the effects on cells, we utilized fluorescence microscopy and a customized feature extraction algorithm to analyze the F-actin filament structures. Our findings indicate a consistent trend across all vibrated cell cultures, revealing a reduction in size and altered orientation (2D angle) of the filaments. Furthermore, we observed cell accumulations in the G1 cell cycle phase in cells treated with Continuous Vibration and Regular Pulse. Our results demonstrate a negative correlation between the magnitude of mechanical stimuli and the size of F-actin filaments, as well as a positive correlation with the accumulations of cells in the G1 phase of the cell cycle. By unraveling these analyses, this study paves the way for future investigations and provides a compelling framework for comprehending the intricate cellular responses to rhythmic mechanical stimulation.

3,5,3'-Triiodo-L-Thyronine Regulates Actin Cytoskeleton Dynamic in The Differentiated PC-12 Cells during Hypoxia through An αvß3 Integrin.

OBJECTIVE: Thyroid hormones are involved in the pathogenesis of various neurological disorders. Ischemia/hypoxia that induces rigidity of the actin filaments, which initiates neurodegeneration and reduces synaptic plasticity. We hypothesized that thyroid hormones via alpha-v-beta-3 (αvß3) integrin could regulate the actin filament rearrangement during hypoxia and increase neuronal cell viability. MATERIALS AND METHODS: In this experimental study, we analysed the dynamics of actin cytoskeleton according to the G/F actin ratio, cofilin-1/p-cofilin-1 ratio, and p-Fyn/Fyn ratio in differentiated PC-12 cells with/without T3 hormone (3,5,3'-triiodo-L-thyronine) treatment and blocking αvß3-integrin-antibody under hypoxic conditions using electrophoresis and western blotting methods. We assessed NADPH oxidase activity under the hypoxic condition by the luminometric method and Rac1 activity using the ELISA-based (G-LISA) activation assay kit. RESULTS: The T3 hormone induces the αvß3 integrin-dependent dephosphorylation of the Fyn kinase (P=0.0010), modulates the G/F actin ratio (P=0.0010) and activates the Rac1/NADPH oxidase/cofilin-1 (P=0.0069, P=0.0010, P=0.0045) pathway. T3 increases PC-12 cell viability (P=0.0050) during hypoxia via αvß3 integrin-dependent downstream regulation systems. CONCLUSION: The T3 thyroid hormone may modulate the G/F actin ratio via the Rac1 GTPase/NADPH oxidase/ cofilin1signaling pathway and αvß3-integrin-dependent suppression of Fyn kinase phosphorylation.