Polymerization Of Actin Filaments Research Articles

Oxidation of the actin cytoskeleton regulator cofilin-1 as a mediator of endothelial stiffening and dysfunction in type 2 diabetes

Vascular disease is a feature of type 2 diabetes (T2D) that is caused in part by oxidative stress and endothelial dysfunction. An emerging instigator of endothelial dysfunction is stiffening of the cell, which is mediated by enhanced polymerization of filamentous actin (F-actin). Actin polymerization is regulated by cofilin-1, an actin severing protein that can be phosphorylated or oxidized, leading to its inactivation and loss of severing capability. Yet, the mechanisms contributing to endothelial stiffening in T2D remain largely unknown. Herein, we tested the hypothesis that endothelial stiffening in T2D is mediated by inactivation of cofilin-1, leading to actin polymerization. In support of this hypothesis, human umbilical vein endothelial cells (HUVECs) treated with plasma from subjects with T2D, and the aortic endothelium of diabetic mice (db/db), demonstrate increased cellular stiffness and F-actin presence. Of note, these findings were not associated with enhanced phosphorylation of cofilin-1, suggesting that its inactivation may instead occur through oxidation. Indeed, we show that cofilin-1 can be oxidized by H2O2, a strong oxidizing agent, and that exposure of HUVECs to H2O2 induces F-actin formation. These findings implicate oxidation and inactivation of cofilin-1 as a putative driver of endothelial F-actin formation and cellular stiffening. In further support of the notion that inactivation of cofilin-1 in endothelial cells promotes cytoskeletal remodeling, we show that genetic silencing or pharmacological inhibition of LIM kinase-1 (LIMK-1), a key enzyme responsible for phosphorylating and thus inactivating cofilin-1, reduces F-actin content and cellular stiffness. Congruently, we show that LIMK inhibition reduces endothelial stiffness in aortic explants from db/db mice. It is conceivable that increased endothelial actin polymerization and stiffening impact vasomotor function. As proof-of-concept, we show that intraluminal exposure of isolated arteries to jasplakinolide, a pharmacological inducer of actin polymerization, impairs endothelial-dependent vasodilation, as assessed via flow-mediated dilation (FMD), while endothelium-independent vasodilation remains unaffected. Conversely, cytochalasin D, an inhibitor of actin polymerization, improves FMD, without influencing endothelium-independent vasodilation. Taken together, these findings suggest the existence of a potential new paradigm of T2D-associated endothelial dysfunction whereby oxidative stress inactivates cofilin-1, contributing to F-actin-dependent endothelial stiffening. Moreover, this work supports the idea that LIMK-1 may be considered a potential target for reducing cofilin-1 inactivation and reversing endothelial stiffening in T2D. Funding: R01HL151384, R01HL153264, R01HL137769, and AHA23PRE1020897. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Drebrin Protects Assembled Actin from INF2-FFC-mediated Severing and Stabilizes Cell Protrusions

Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation – contributing to cell shape maintenance – is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin-binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin – DrbA1-300 – is sufficient for this protection, though not as effective as the full-length protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy.

Probing the ligand binding specificity of FNBP4 WW domains and interaction with FH1 domain of FMN1

Formins are a group of actin-binding proteins that mediate nascent actin filament polymerization, filament elongation, and barbed end-capping function, thereby regulating different cellular and developmental processes. Developmental processes like vertebrate gastrulation, neural growth cone dynamics, and limb development require formins functioning in a regulated manner. Formin-binding proteins like Rho GTPase regulate the activation of auto-inhibited conformation of diaphanous formins. Unlike other diaphanous formins, Formin1 (FMN1) a non-diaphanous formin is not regulated by Rho GTPase. FMN1 acts as an antagonist of the Bone Morphogenetic Protein (BMP) signaling pathway during limb development. Several previous reports demonstrated that WW domain-containing proteins can interact with poly-proline-rich amino acid stretches of formins and play a crucial role in developmental processes. In contrast, WW domain-containing Formin-binding Protein 4 (FNBP4) protein plays an essential role in limb development. It has been hypothesized that the interaction between FNBP4 and FMN1 can further attribute to the role in limb development through the BMP signaling pathway. In this study, we have elucidated the binding kinetics of FNBP4 and FMN1 using surface plasmon resonance (SPR) and enzyme-linked immunosorbent assays (ELISA). Our findings confirm that the FNBP4 exhibits interaction with the poly-proline-rich formin homology 1 (FH1) domain of FMN1. Furthermore, only the first WW1 domains are involved in the interaction between the two domains. Thus, this study sheds light on the binding potentialities of WW domains of FNBP4 that might contribute to the regulation of FMN1 function.

Actin Polymerization Defects Induce Mitochondrial Dysfunction in Cellular Models of Nemaline Myopathies.

Nemaline myopathy (NM) is one of the most common forms of congenital myopathy and it is identified by the presence of "nemaline bodies" (rods) in muscle fibers by histopathological examination. The most common forms of NM are caused by mutations in the Actin Alpha 1 (ACTA1) and Nebulin (NEB) genes. Clinical features include hypotonia and muscle weakness. Unfortunately, there is no curative treatment and the pathogenetic mechanisms remain unclear. In this manuscript, we examined the pathophysiological alterations in NM using dermal fibroblasts derived from patients with mutations in ACTA1 and NEB genes. Patients' fibroblasts were stained with rhodamine-phalloidin to analyze the polymerization of actin filaments by fluorescence microscopy. We found that patients' fibroblasts showed incorrect actin filament polymerization compared to control fibroblasts. Actin filament polymerization defects were associated with mitochondrial dysfunction. Furthermore, we identified two mitochondrial-boosting compounds, linoleic acid (LA) and L-carnitine (LCAR), that improved the formation of actin filaments in mutant fibroblasts and corrected mitochondrial bioenergetics. Our results indicate that cellular models can be useful to study the pathophysiological mechanisms involved in NM and to find new potential therapies. Furthermore, targeting mitochondrial dysfunction with LA and LCAR can revert the pathological alterations in NM cellular models.

Identification of small molecules that suppress cell invasion and metastasis promoted by WASF3 activation

The WASF3 gene promotes cancer cell invasion and metastasis, and genetic inactivation leads to suppression of metastasis. To identify small molecules that might interfere with WASF3 function, we performed an in silico docking study to the regulatory pocket of WASF3 using the National Cancer Institute (NCI) diversity set VI small molecule library. Compounds that showed the maximum likelihood of interaction with WASF3 were screened for their effect on cell movement in breast and prostate cancer cells, a well-established predictor of invasion and metastasis. Three hit compounds were identified that affected cell movement, and the same compounds also suppressed cell migration and invasion in vitro in both MDA-MB-231 breast cancer cells and Du145 prostate cancer cells. Using a zebrafish metastasis assay, one of these compounds, NSC670283, showed significant suppression of metastasis in vivo while not affecting cell proliferation. NSC670283 showed a consistent effect on suppression of invasion and metastasis, and cellular temperature shift assays provided support for physical interaction with WASF3. In addition, suppression of cell movement and invasion was accompanied by a decrease in actin filament polymerization. The data in this study suggest that these small molecules inhibit cancer cell invasion and metastasis, and to our knowledge, it is the first identification of a small molecule that can potentially inhibit WASF3-directed metastasis, laying the foundation for medicinal chemistry approaches to enhance the potency of the identified compounds.

Computational modeling of the variation of the transmembrane potential of the endothelial cells of the blood-brain-barrier subject to an external electric field.

The electromechanical properties of the membrane of endothelial cells forming the blood-brain barrier play a vital role in the function of this barrier. The mechanical effect exerted by external electric fields on the membrane could change its electrical properties. In this study the effect of extremely low frequency (ELF) external electric fields on the electrical activity of these cells has been studied by considering the mechanical effect of these fields on the capacitance of the membrane. The effect of time-dependent capacitance of the membrane is incorporated in the current components of the parallel conductance model for the electrical activity of the cells. The results show that the application of ELF electric fields induces hyperpolarization, having an indirect effect on the release of nitric oxide from the endothelial cell and the polymerization of actin filaments. Accordingly, this could play an important role in the permeability of the barrier. Our finding can have possible consequences in the field of drug delivery into the central nervous system.

Investigating the Role of MK2 in Head and Neck Squamous Cell Carcinoma Growth, Metastasis and STING Pathway Activation

Our prior work demonstrated that inhibition of MAPKAPK2 (MK2) can enhance radiation (RT)-mediated in vivo head and neck squamous cell carcinoma (HNSCC) tumor control and survival in preclinical immune incompetent models. The cytosolic DNA sensor cyclic GMP-AMP synthetase (cGAS) and its downstream adaptor protein, stimulator of interferon genes (STING), are conserved proteins within the innate immune signaling pathways and are important for mediating host defense against microbial infection and can play a role in anti-cancer immunity. We hypothesized that loss of MK2 enhances radiation-induced cGAS-STING pathway activation leading to improved tumor control and survival. MK2 shRNA knockdown human (Tu167, CAL27) and MK2 Cas9/CRISPR knockout (KO) syngeneic murine (Ly2, MLM3) HNSCC cell lines were treated with 10 Gy irradiation. Micronuclei were quantitated by DAPI-immunofluorescence (IF). Protein changes in cGAS-STING were evaluated by immunoblot. Inflammatory cytokine production including Type I IFNβ1 were evaluated by RT-qPCR. Ly2 and MLM3 cells were orthotopically or flank engrafted into immune competent mice (Balb/c, C57Bl/6, respectively) for animal tumor control-survival studies. Tumor immune cell infiltrate was examined using FACS and immunohistochemistry. Selected drug studies using the MK2 inhibitor, ATI-450, were performed with RT. Loss of MK2 in HNSCC (Tu167, CAL27, Ly2, MLM3) treated with RT led to a significant increase in micronuclei formation compared to control cells. MK2-enhanced micronuclei generation following RT could be inhibited with the actin filament polymerization inhibitor, cytochalasin B. RT treatment of MK2 shRNA cells led to increased cGAS and phospho-STING levels compared to either treatment alone. IFNβ1 levels were significantly higher in HNSCC cell lines treated with RT and with MK2 inhibited by an MK2 inhibitor (ATI-450) or genetic reduction compared to either treatment alone. In-vivo implantation of MLM3 cells into C57Bl/6 comparing control vs MK2 KO tumors treated with ±8 Gy RT demonstrated improved mouse survival favoring RT+MK2 KO over RT, MK2 KO or parental (63, 58, 58.5, 35 days, respectively). FACS analysis of MLM3 WT v KO tumors 3 days post RT showed an overall increase in the number of CD3/CD8 T-cells infiltrating into the tumor in all groups except for parental tumors. Further analysis demonstrated that loss of MK2 reversed CD8 T-cell exhaustion and when combined with radiotherapy led to increased CD8 T-cell activation. Furthermore, activated CD4 and CD8 T cells were reduced in WT+RT cells compared to WT tumors whereas no reduction was seen in the KO or KO+RT. HNSCC tumor MK2 inhibition enhances RT-mediated micronuclei formation and subsequent cGAS-STING-IFNβ1 levels. Loss of HNSCC MK2 leads to increased CD4-CD8 T-cell infiltration into the tumor and this effect is enhanced following RT. Targeting tumor MK2 may facilitate improved tumor control.

Nascent adhesions shorten the period of lamellipodium protrusion through the Brownian ratchet mechanism.

Directional cell migration is driven by the conversion of oscillating edge motion into lasting periods of leading edge protrusion. Actin polymerization against the membrane and adhesions control edge motion, but the exact mechanisms that determine protrusion period remain elusive. We addressed this by developing a computational model in which polymerization of actin filaments against a deformable membrane and variable adhesion dynamics support edge motion. Consistent with previous reports, our model showed that actin polymerization and adhesion lifetime power protrusion velocity. However, increasing adhesion lifetime decreased the protrusion period. Measurements of adhesion lifetime and edge motion in migrating cells confirmed that adhesion lifetime is associated with and promotes protrusion velocity, but decreased duration. Our model showed that adhesions' control of protrusion persistence originates from the Brownian ratchet mechanism for actin filament polymerization. With longer adhesion lifetime or increased-adhesion density, the proportion of actin filaments tethered to the substrate increased, maintaining filaments against the cell membrane. The reduced filament-membrane distance generated pushing force for high edge velocity, but limited further polymerization needed for protrusion duration. We propose a mechanism for cell edge protrusion in which adhesion strength regulates actin filament polymerization to control the periods of leading edge protrusion.

E-cigarette vapor renders neutrophils dysfunctional due to filamentous actin accumulation

Electronic cigarette (e-cigarette) use continues to rise despite concerns of long-term effects, especially the risk of developing lung diseases such as chronic obstructive pulmonary disease. Neutrophils are central to the pathogenesis of chronic obstructive pulmonary disease, with changes in phenotype and function implicated in tissue damage. We sought to measure the impact of direct exposure to nicotine-containing and nicotine-free e-cigarette vapor on human neutrophil function and phenotype. Neutrophils were isolated from the whole blood of self-reported nonsmoking, nonvaping healthy volunteers. Neutrophils were exposed to 40 puffs of e-cigarette vapor generated from e-cigarette devices using flavorless e-cigarette liquids with and without nicotine before functions, deformability, and phenotype were assessed. Neutrophil surface marker expression was altered, with CD62L and CXCR2 expression significantly reduced in neutrophils treated with e-cigarette vapor containing nicotine. Neutrophil migration to IL-8, phagocytosis of Escherichia coli and Staphylococcus aureus pHrodo bioparticles, oxidative burst response, and phorbol 12-myristate 13-acetate-stimulated neutrophil extracellular trap formation were all significantly reduced by e-cigarette vapor treatments, independent of nicotine content. E-cigarette vapor induced increased levels of baseline polymerized filamentous actin levels in the cytoplasm, compared with untreated controls. The significant reduction in effector neutrophil functions after exposure to high-power e-cigarette devices, even in the absence of nicotine, is associated with excessive filamentous actin polymerization. This highlights the potentially damaging impact of vaping on respiratory health and reinforces the urgency of research to uncover the long-term health implications of e-cigarettes.

Polymerization force–regulated actin filament–Arp2/3 complex interaction dominates self-adaptive cell migrations

Cells migrate by adapting their leading-edge behaviors to heterogeneous extracellular microenvironments (ECMs) during cancer invasions and immune responses. Yet it remains poorly understood how such complicated dynamic behaviors emerge from millisecond-scale assembling activities of protein molecules, which are hard to probe experimentally. To address this gap, we establish a spatiotemporal "resistance-adaptive propulsion" theory based on the interactions between Arp2/3 complexes and polymerizing actin filaments and a multiscale dynamic modeling system spanning from molecular proteins to the cell. We quantitatively find that cells can accurately self-adapt propulsive forces to overcome heterogeneous ECMs via a resistance-triggered positive feedback mechanism, dominated by polymerization-induced actin filament bending and the bending-regulated actin-Arp2/3 binding. However, for high resistance regions, resistance triggers a negative feedback, hindering branched filament assembly, which adapts cellular morphologies to circumnavigate the obstacles. Strikingly, the synergy of the two opposite feedbacks not only empowers the cell with both powerful and flexible migratory capabilities to deal with complex ECMs but also enables efficient utilization of intracellular proteins by the cell. In addition, we identify that the nature of cell migration velocity depending on ECM history stems from the inherent temporal hysteresis of cytoskeleton remodeling. We also show that directional cell migration is dictated by the competition between the local stiffness of ECMs and the local polymerizing rate of actin network caused by chemotactic cues. Our results reveal that it is the polymerization force-regulated actin filament-Arp2/3 complex binding interaction that dominates self-adaptive cell migrations in complex ECMs, and we provide a predictive theory and a spatiotemporal multiscale modeling system at the protein level.

Basally distributed actin array drives embryonic hypocotyl elongation during the seed-to-seedling transition in Arabidopsis.

Seed germination is a vital developmental transition for the production of progeny by sexual reproduction in spermatophytes. The seed-to-seedling transition is predominately driven by hypocotyl cell elongation. However, the mechanism that underlies hypocotyl growth remains largely unknown. In this study, we characterized the actin array reorganization in embryonic hypocotyl epidermal cells. Live-cell imaging revealed a basally organized actin array formed during hypocotyl cell elongation. This polarized actin assembly is a barrel-shaped network, which comprises a backbone of longitudinally aligned actin cables and a fine actin cap linking these cables. We provide genetic evidence that the basal actin array formation requires formin-mediated actin polymerization and directional movement of actin filaments powered by myosin XIs. In fh1-1 and xi3ko mutants, actin filaments failed to reorganize into the basal actin array, and the hypocotyl cell elongation was inhibited compared with wild-type plants. Collectively, our work uncovers the molecular mechanisms for basal actin array assembly and demonstrates the connection between actin polarization and hypocotyl elongation during seed-to-seedling transition.

Chiral growth of adherent filopodia

Adherent filopodia are elongated finger-like membrane protrusions, extending from the edges of diverse cell types and participating in cell adhesion, spreading, migration, and environmental sensing. The formation and elongation of filopodia are driven by the polymerization of parallel actin filaments, comprising the filopodia cytoskeletal core. Here, we report that adherent filopodia, formed during the spreading of cultured cells on galectin-8-coated substrates, tend to change the direction of their extension in a chiral fashion, acquiring a left-bent shape. Cryoelectron tomography examination indicated that turning of the filopodia tip to the left is accompanied by the displacement of the actin core bundle to the right of the filopodia midline. Reduction of the adhesion to galectin-8 by treatment with thiodigalactoside abolished this filopodia chirality. By modulating the expression of a variety of actin-associated filopodia proteins, we identified myosin-X and formin DAAM1 as major filopodia chirality promoting factors. Formin mDia1, actin filament elongation factor VASP, and actin filament cross-linker fascin were also shown to be involved. Thus, the simple actin cytoskeleton of filopodia, together with a small number of associated proteins are sufficient to drive a complex navigation process, manifested by the development of left-right asymmetry in these cellular protrusions.

Oral exposure to polystyrene nanoplastics reduced male fertility and even caused male infertility by inducing testicular and sperm toxicities in mice

Nanoplastics (NPs) are the novel hazardous materials and ubiquitous in environment with different sizes. Although recent studies showed testicular toxicity of PS-NPs, whether and how NPs affect male fertility and whether they have the size-dependent effect remain ambiguous in mammals. In this study, the male mice were orally exposed to 25-, 50-, and 100-nm polystyrene NPs (PS-NPs) for 56 days. All three sized PS-NPs reduced male fertility and even caused male infertility. They accumulated in the testes, induced oxidative stress, affected the expression of apoptosis- and inflammation-related genes, and compromised energy metabolism, resulting in damaged testicular microstructure and functions. PS-NPs caused more severe testicular toxicity in infertile mice than in fertile mice. In addition, PS-NPs inhibited sperm capacitation and capacitation-dependent processes in infertile mice but not in fertile mice. In infertile mice, PS-NPs reduced the sperm levels of two Rho GTPases (RAC1 and CDC42) via increasing their ubiquitination levels and diminished sperm filamentous actin polymerization, thus inhibiting sperm capacitation. However, these testicular and sperm toxicities showed no size-dependent effect among three sized PS-NPs. In conclusion, PS-NPs inhibit male fertility by their multifaceted toxicity on testes and sperm in mice, providing novel insights into reproductive risks of NPs to mammals.

Actin based motility unveiled: How chemical energy is converted into motion

Actin-based motility is a complex process in which the actin-polymerization is the primary force-generating motor machinery. It can produce protrusive forces through actin filaments polymerization and cross-link during lamellipodia protrusion in migrating cells and it is responsible for the intracellular motion of certain pathogens in infected host cells. We propose a multi-physics model for actin-based motility, stemming from continuity equations that account for the actin chemical kinetics. Thermodynamic restrictions are identified, moving from the multiplicative decomposition of the deformation gradient into chemical and elastic parts. Constitutive theory and chemical kinetics are prescribed to finally write governing equations for the multi-physics problem. The field equations are solved numerically with the finite element method. As a proof of concept, a one-dimensional model for actin-based motility of bacteria pathogens is studied.

Dark-induced chloroplast relocation depends on actin filaments in the liverwort Apopellia endiviifolia along with the light- and cold-induced relocations.

Chloroplasts move to the periclinal walls of cells under weak light to harness light energy for photosynthesis and to anticlinal walls to avoid strong light. These responses involve the cytoskeleton components microtubules and/or actin filaments. In the dark, chloroplasts move to the anticlinal cell walls bordering neighbouring cells (dark-positioning response), but this response in various plants normally requires a prolonged dark incubation period, which has hampered analysis. However, we recently demonstrated the dark-positioning response that can be induced after a short period of dark incubation in the liverwort Apopellia endiviifolia. Here, we investigated whether the cytoskeleton components function in the dark-positioning response of A. endiviifolia cells. Microtubules and actin filaments were fluorescentlyvisualised in A. endiviifolia cells and were disrupted following treatment with the microtubule and actin filament polymerisation inhibitors. The dark-positioning response was unaffected in the cells with disrupted microtubules. By contrast, the dark-positioning response was inhibited by the disruption of actin filaments. The disruption of actin filaments also restricted chloroplast mobility during light- and cold-dependent chloroplast movements in A. endiviifolia. Therefore, the dark-positioning response of A. endiviifolia depends solely on an actin filament-associated motility mechanism, as do the light- and cold-dependent chloroplast responses.

Metallothionein-3 attenuates the effect of Cu2+ ions on actin filaments

Metallothionein 3 (MT-3) is a cysteine-rich metal-binding protein that is expressed in the mammalian central nervous system and kidney. Various reports have posited a role for MT-3 in regulating the actin cytoskeleton by promoting the assembly of actin filaments. We generated purified, recombinant mouse MT-3 of known metal compositions, either with zinc (Zn), lead (Pb), or copper/zinc (Cu/Zn) bound. None of these forms of MT-3 accelerated actin filament polymerization in vitro, either with or without the actin binding protein profilin. Furthermore, using a co-sedimentation assay, we did not observe Zn-bound MT-3 in complex with actin filaments. Cu2+ ions on their own induced rapid actin polymerization, an effect that we attribute to filament fragmentation. This effect of Cu2+ is reversed by adding either EGTA or Zn-bound MT-3, indicating that either molecule can chelate Cu2+ from actin. Altogether, our data indicate that purified recombinant MT-3 does not directly bind actin but it does attenuate the Cu-induced fragmentation of actin filaments.

CLASP2 facilitates dynamic actin filament organization along the microtubule lattice.

Coordination between the microtubule and actin networks is essential for cell motility, neuronal growth cone guidance, and wound healing. Members of the CLASP (cytoplasmic linker-associated protein) family of proteins have been implicated in the cytoskeletal crosstalk between microtubules and actin networks, however, the molecular mechanisms underlying CLASPs role in cytoskeletal coordination are unclear. Here, we investigate CLASP2α's crosslinking function with microtubules and F-actin. Our results demonstrate that CLASP2α crosslinks F-actin to the microtubule lattice in vitro. We find that the crosslinking ability is retained by L-TOG2-S, a minimal construct containing the TOG2 domain and serine-arginine rich region of CLASP2α. Furthermore, CLASP2α promotes the accumulation of multiple actin filaments along the microtubule, supporting up to 11 F-actin landing events on a single microtubule lattice region. CLASP2α also facilitates dynamic organization of polymerizing actin filaments templated by the microtubule network, with F-actin forming bridges between individual microtubules. Finally, we find that depletion of CLASPs in vascular smooth muscle cells results in disorganized actin fibers and reduced co-alignment of actin fibers with microtubules, suggesting that CLASP and microtubules contribute to higher-order actin structures. Taken together, our results indicate that CLASP2α can directly crosslink F-actin to microtubules, and that this microtubule-CLASP-actin interaction may influence overall cytoskeletal organization in cells.

CLASP2 facilitates dynamic actin filament organization along the microtubule lattice.

Coordination between the microtubule and actin networks is essential for cell motility, neuronal growth cone guidance, and wound healing. Members of the CLASP (cytoplasmic linker-associated protein) family of proteins have been implicated in the cytoskeletal cross-talk between microtubules and actin networks; however, the molecular mechanisms underlying the role of CLASP in cytoskeletal coordination are unclear. Here, we investigate CLASP2α's cross-linking function with microtubules and F-actin. Our results demonstrate that CLASP2α cross-links F-actin to the microtubule lattice in vitro. We find that the cross-linking ability is retained by L-TOG2-S, a minimal construct containing the TOG2 domain and serine-arginine-rich region of CLASP2α. Furthermore, CLASP2α promotes the accumulation of multiple actin filaments along the microtubule, supporting up to 11 F-actin landing events on a single microtubule lattice region. CLASP2α also facilitates the dynamic organization of polymerizing actin filaments templated by the microtubule network, with F-actin forming bridges between individual microtubules. Finally, we find that depletion of CLASPs in vascular smooth muscle cells results in disorganized actin fibers and reduced coalignment of actin fibers with microtubules, suggesting that CLASP and microtubules contribute to higher-order actin structures. Taken together, our results indicate that CLASP2α can directly cross-link F-actin to microtubules and that this microtubule-CLASP-actin interaction may influence overall cytoskeletal organization in cells.

Mechanism and Prospect of Gastrodin in Osteoporosis, Bone Regeneration, and Osseointegration.

Gastrodin, a traditional Chinese medicine ingredient, is widely used to treat vascular and neurological diseases. However, recently, an increasing number of studies have shown that gastrodin has anti-osteoporosis effects, and its mechanisms of action include its antioxidant effect, anti-inflammatory effect, and anti-apoptotic effect. In addition, gastrodin has many unique advantages in promoting bone healing in tissue engineering, such as inducing high hydrophilicity in the material surface, its anti-inflammatory effect, and pro-vascular regeneration. Therefore, this paper summarized the effects and mechanisms of gastrodin on osteoporosis and bone regeneration in the current research. Here we propose an assumption that the use of gastrodin in the surface loading of oral implants may greatly promote the osseointegration of implants and increase the success rate of implants. In addition, we speculated on the potential mechanisms of gastrodin against osteoporosis, by affecting actin filament polymerization, renin-angiotensin system (RAS) and ferroptosis, and proposed that the potential combination of gastrodin with Mg2+, angiotensin type 2 receptor blockers or artemisinin may greatly inhibit osteoporosis. The purpose of this review is to provide a reference for more in-depth research and application of gastrodin in the treatment of osteoporosis and implant osseointegration in the future.

Cell-Level Analysis Visualizing Photodynamic Therapy with Porphylipoprotein and Talaporphyrin Sodium.

We revealed the difference in the mechanism of photodynamic therapy (PDT) between two photosensitizers: porphylipoprotein (PLP), which has recently attracted attention for its potential to be highly effective in treating cancer, and talaporphyrin sodium (NPe6). (1) NPe6 accumulates in lysosomes, whereas PLP is incorporated into phagosomes formed by PLP injection. (2) PDT causes NPe6 to generate reactive oxygen species, thereby producing actin filaments and stress fibers. In the case of PLP, however, reactive oxygen species generated by PDT remain in the phagosomes until the phagosomal membrane is destroyed, which delays the initiation of RhoA activation and RhoA*/ROCK generation. (4) After the disruption of the phagosomal membrane, however, the outflow of various reactive oxygen species accelerates the production of actin filaments and stress fibers, and blebbing occurs earlier than in the case of NPe6. (5) PLP increases the elastic modulus of cells without RhoA activity in the early stage. This is because phagosomes are involved in polymerizing actin filaments and pseudopodia formation. Considering the high selectivity and uptake of PLP into cancer cells, a larger effect with PDT can be expected by skillfully combining the newly discovered characteristics, such as the appearance of a strong effect at an early stage.