Actin Dynamics Research Articles

Rac1 inhibition regenerates wounds in mouse fetuses via altered actin dynamics

Mammalian wounds leave visible scars, and there are no methods for complete regeneration. However, mouse fetuses regenerate their skin, including epidermal and dermal structures, up to embryonic day (E)13. This regeneration pattern requires the formation of actin cables in the wound margin epithelium; however, the molecular mechanisms are not fully understood. Rac1 alters actin in cells and is involved in the formation of filopodia. We investigated whether actin remodeling and skin regeneration patterns can be reproduced through the regulation of Rac1 signaling. Rac1 expression was downregulated in E13 wounds and upregulated after E15 when scars remained. NSC23766, a Rac1-specific inhibitor, altered actin dynamics at the cell margin from filopodia formation to cable formation and inhibited the migration of mouse epidermal keratinocyte, PAM212, by Rac1 signaling suppression. NSC23766 suppressed Rac1 activity and completely regenerated the fetal mouse wounds, even at E14, by changing actin dynamics. Knocked-out Rac1 transgenic mice experienced delayed epithelialization of wounds with suppressed epidermal migration in adults; however, in fetuses, complete wound regeneration via Rac1 signal suppression was observed. Therefore, Rac1 suppression in the wound epidermis can achieve regenerative wound healing in fetuses and may be a potential candidate for healing scars.

Interpenetrating Polymer Network Hydrogels with Tunable Viscoelasticity and Proteolytic Cleavability to Direct Stem Cells In Vitro.

The dynamic nature of cellular microenvironments, regulated by the viscoelasticity and enzymatic cleavage of the extracellular matrix, remains challenging to emulate in engineered synthetic biomaterials. To address this, a novel platform of cell-instructive hydrogels is introduced, composed of two concurrently forming interpenetrating polymer networks (IPNs). These IPNs consist of the same basic building blocks - four-armed poly(ethylene glycol) and the sulfated glycosaminoglycan (sGAG) heparin - are cross-linked through either chemical or physical interactions, allowing for precise and selective tuning of the hydrogel's stiffness, viscoelasticity, and proteolytic cleavability. The studies of the individual and combined effects of these parameters on stem cell behavior revealed that human mesenchymal stem cells exhibited increased spreading and Yes-associated protein transcriptional activity in more viscoelastic and cleavable sGAG-IPN hydrogels. Furthermore, human induced pluripotent stem cell (iPSC) cysts displayed enhanced lumen formation, growth, and pluripotency maintenance when cultured in sGAG-IPN hydrogels with higher viscoelasticity. Inhibition studies emphasized the pivotal roles of actin dynamics and matrix metalloproteinase activity in iPSC cyst morphology, which varied with the viscoelastic properties of the hydrogels. Thus, the introduced sGAG-IPN hydrogel platform offers a powerful methodology for exogenous stem cell fate control.

Implementation of actin polymerization and depolymerization in a two-dimensional cell migration model and its implications on mammalian cell morphology and velocity

Cell migration, a pivotal process in wound healing, immune response, and even cancer metastasis, manifests through intricate interplay between morphology, speed, and cytoskeletal dynamics. Mathematical modeling emerges as a powerful tool to dissect these complex interactions. This work presents a two-dimensional immersed boundary model for mammalian cell migration, incorporating both filamentous actin (F-actin) and monomeric actin (G-actin) to explicitly capture polymerization and depolymerization. This model builds upon our previous one-dimensional efforts, now enabling us to explore the impact of G-actin on not just cell velocity but also morphology. We compare predictions from both models, revealing that while the one-dimensional model captures core dynamics along the cell’s axis, the two-dimensional model excels in portraying cell shape evolution and transverse variations in actin concentration and velocity. Our findings highlight the crucial role of including G-actin in shaping cell morphology. Actin velocity aligned with migration direction elongates the cell, while velocity normal to the membrane promotes spreading. Importantly, the model establishes a link between these microscopic aspects and macroscopic observables like cell shape, offering a deeper understanding of cell migration dynamics. This work not only provides a more comprehensive picture of cell migration but also paves the way for future studies exploring the interplay of actin dynamics, cell morphology, and biophysical parameters in diverse biological contexts.

METTL3/YTHDF1 stabilizes CORO6 expression promoting osteosarcoma progression through glycolysis

This study investigates the role of CORO6 (Coronin 6) in the development of osteosarcoma. Osteosarcoma is a common malignant bone tumor in children and adolescents, characterized by rapid and irregular bone growth and a high risk of distant lung metastasis. CORO6 is a member of the Coronin family, known for its conserved WD40 repeat domain. This structure allows CORO6 to inhibit actin dynamics through interactions with F-actin and Arp2/3, thereby affecting the organization of the cytoskeleton. Our research found that in osteosarcoma patients, the levels of CORO6 are significantly elevated. Experimental observations showed that reducing the expression of CORO6 significantly inhibits the growth, migration, and invasion abilities of osteosarcoma cells. Moreover, in vivo experiments demonstrated that the absence of CORO6 effectively inhibits the growth of osteosarcoma in animal models. We also discovered that CORO6 promotes the proliferation, migration and invasion capabilities of osteosarcoma cells by activating the Wnt/β-catenin signaling pathway. Moreover, CORO6 play a critical important role in glycosis of osteosarcoma cells. Mechanically, we found that METTL3/YTHDF1 induced m6A modification of CORO6 mRNA promoted the expression of CORO6 by enhancing its stability. These findings offer new directions for the treatment of osteosarcoma, suggesting that CORO6 could be a novel prognostic biomarker and an effective therapeutic target for patients.In summary, CORO6, as an oncogene, plays a key role in the development of osteosarcoma, providing a crucial theoretical basis for the development of new osteosarcoma treatment strategies.

Accumulation of F-actin drives brain aging and limits healthspan in Drosophila

The actin cytoskeleton is a key determinant of cell structure and homeostasis. However, possible tissue-specific changes to actin dynamics during aging, notably brain aging, are not understood. Here, we show that there is an age-related increase in filamentous actin (F-actin) in Drosophila brains, which is counteracted by prolongevity interventions. Critically, decreasing F-actin levels in aging neurons prevents age-onset cognitive decline and extends organismal healthspan. Mechanistically, we show that autophagy, a recycling process required for neuronal homeostasis, is disabled upon actin dysregulation in the aged brain. Remarkably, disrupting actin polymerization in aged animals with cytoskeletal drugs restores brain autophagy to youthful levels and reverses cellular hallmarks of brain aging. Finally, reducing F-actin levels in aging neurons slows brain aging and promotes healthspan in an autophagy-dependent manner. Our data identify excess actin polymerization as a hallmark of brain aging, which can be targeted to reverse brain aging phenotypes and prolong healthspan.

Actin dynamics switches two distinct modes of endosomal fusion in yolk sac visceral endoderm cells

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using mouse yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells.

Actin dynamics switches two distinct modes of endosomal fusion in yolk sac visceral endoderm cells.

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using mouse yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells.

Embryonic 6:2 Fluorotelomer Alcohol Exposure Disrupts the Blood‒Brain Barrier by Causing Endothelial‒to‒Mesenchymal Transition in the Male Mice.

6:2 Fluorotelomer alcohol (6:2 FTOH) is a raw material used in the manufacture of short-chain poly- and perfluoroalkyl substances. Our previous study revealed that gestational exposure to 6:2 FTOH can impair blood‒brain barrier (BBB) function in offspring, accompanied by anxiety-like behavior and learning memory deficits. The aim of this study was to further investigate the specific mechanism by which maternal exposure to 6:2 FTOH resulted in impaired BBB function in offspring mice. Pregnant mice were orally administered different doses of 6:2 FTOH (0, 5, 25, and 125mg/kg/day) from gestation day 8.5 until delivery. These results confirmed that maternal 6:2 FTOH exposure impaired BBB function and disrupted the brain immune microenvironment. Subsequent investigations revealed that endothelial-to-mesenchymal transition (EndMT) in the cerebral microvascular endothelium of offspring may be the mechanism mediating functional disruption of the BBB. Mechanistic studies revealed that exposure to 6:2 FTOH upregulated ETS proto-oncogene 1 (ETS1) expression via the tumor necrosis factor-α/extracellular signal-regulated kinase 1/2 signaling pathway, which mediated disturbances in energy metabolism, leading to impaired actin dynamics and subsequently triggering the EndMT phenotype. This is the first finding indicating that gestational 6:2 FTOH exposure caused functional impairment of the BBB through ETS1-mediated EndMT in cerebral microvascular endothelial cells, potentially providing novel insight into the environmental origins of neurodevelopmental disorders.

Single-vesicle imaging reveals actin-dependent spatial restriction of vesicles at the active zone, essential for sustained transmission

Synaptic-vesicle (SV) recruitment is thought to maintain reliable neurotransmitter release during high-frequency signaling. However, the mechanism underlying the SV reloading for sustained neurotransmission at central synapses remains unknown. To elucidate this, we performed direct observations of SV reloading and mobility at a single-vesicle level near the plasma membrane in cerebellar mossy fiber terminals using total internal reflection fluorescence microscopy, together with simultaneous recordings of membrane fusion by capacitance measurements. We found that actin disruption abolished the rapid SV recruitment and reduced sustained release. In contrast, induction of actin polymerization and stabilization did not affect vesicle recruitment and release, suggesting that the presence of actin filaments, rather than actin dynamics, was required for the rapid recruitment. Single-particle tracking experiments of quantum dot-labeled vesicles, which allows nanoscale resolution of vesicle mobility, revealed that actin disruption caused vesicles to diffuse more rapidly. Hidden Markov modeling with Bayesian inference revealed that SVs had two diffusion states under normal conditions: free-diffusing and trapped. After disruption of the actin filament, vesicles tended to have only the free-diffusing state. F-actin staining showed that actin filaments were localized outside the active zones (AZs) and surrounded some SV trajectories. Perturbation of SV mobility, possibly through interference with biomolecular condensates, also suggested that the restricted diffusion state determined the rate of SV recruitment. We propose that actin filaments confined SVs near the AZ to achieve rapid and efficient recruitment followed by priming and sustained synaptic transmission.

8381 The Pro-atrophic Effect of Vitamin D Binding Protein in Skeletal Muscle and Its Involvement in Cancer-Induced Muscle Wasting

Abstract Disclosure: N. Filigheddu: None. T. Raiteri: None. S. Reano: None. A. Scircoli: None. A. Antonioli: None. F. Prodam: None. Vitamin D binding protein (VDBP), encoded by the Gc gene, is a multifunctional serum glycoprotein synthesized by hepatocytes, whose primary function is the transport of vitamin D metabolites in the bloodstream. In addition, VDBP enhances the chemotactic activity of neutrophil chemoattractants and takes part in the actin-scavenger system by acting as a monomeric G-actin-binding protein. Several studies have reported a correlation between increased levels of VDBP and various pathologies often associated with muscle wasting, including different types of tumors. Given these findings, we hypothesized that VDBP may play a role in skeletal muscle homeostasis.In vitro, treatment of C2C12 myotubes with 100 mM VDBP for 24 h induced the perturbation of intracellular actin dynamics due to VDBP's ability to bind G-actin that, in turn, led to mitochondrial dysfunction (i.e., mitochondrial membrane potential dissipation, respiratory impairment, increased ROS production, induction of the fission machinery), exacerbation of autophagy, and, eventually, atrophy, seen as the reduction of myotube diameter. Remarkably, pharmacological intervention on myotubes with jasplakinolide (250 mM, 30 min pre-treatment) to counteract VDBP effects on intracellular actin dynamics was sufficient to prevent VDBP-induced atrophy. To assess if VDBP had a causative role in muscle atrophy in vivo, we injected VDBP (1.5 mg/Kg) every 48 h for one week in the tibialis anterior muscles of Gc knock-out mice (VDBP KO). At the end of the experimental period, we observed the induction of the mitophagic gene Bnip3, impairment of muscle performances (26% reduction of grip strength at the endpoint), and a 16% reduction of muscle mass compared to the saline-injected contralateral muscles, confirming the atrophic effect of VDBP in vivo.Coherently with the upregulation of VDBP observed in cancer patients, VDBP levels also increase in murine models of cancer cachexia. To test the hypothesis that VDBP could play a role in cancer-associated muscle wasting, we induced cancer cachexia in VDBP KO mice by inoculating 106 Lewis Lung Carcinoma (LLC) cells resuspended in 100 μl of saline on the back of mice. Tumor-bearing VDBP KO mice preserved their body weight and performances, and the muscle loss was lessened by more than 50% compared to cachectic WT mice. Notably, between the two groups, there were no differences in tumor growth or food intake, ruling out the possibility that the reduction in muscle wasting could depend on smaller tumors or differential development of anorexia in the two genotypes. In conclusion, we demonstrated that VDBP acts as a hormone per se, having a direct pro-atrophic activity on skeletal muscle. Our data suggest that VDBP could represent a potential therapeutic target to treat cancer cachexia and other pathologies in which the rise of VDBP could impinge muscle mass and functionality. Presentation: 6/1/2024

9268 The Pro-atrophic Effect of Vitamin D Binding Protein in Skeletal Muscle and Its Involvement in Cancer-Induced Muscle Wasting

Abstract Disclosure: N. Filigheddu: None. T. Raiteri: None. S. Reano: None. A. Scircoli: None. A. Antonioli: None. F. Prodam: None. Vitamin D binding protein (VDBP), encoded by the Gc gene, is a multifunctional serum glycoprotein synthesized by hepatocytes, whose primary function is the transport of vitamin D metabolites in the bloodstream. In addition, VDBP enhances the chemotactic activity of neutrophil chemoattractants and takes part in the actin-scavenger system by acting as a monomeric G-actin-binding protein. Several studies have reported a correlation between increased levels of VDBP and various pathologies often associated with muscle wasting, including different types of tumors. Given these findings, we hypothesized that VDBP may play a role in skeletal muscle homeostasis.In vitro, treatment of C2C12 myotubes with 100 mM VDBP for 24 h induced the perturbation of intracellular actin dynamics due to VDBP's ability to bind G-actin that, in turn, led to mitochondrial dysfunction (i.e., mitochondrial membrane potential dissipation, respiratory impairment, increased ROS production, induction of the fission machinery), exacerbation of autophagy, and, eventually, atrophy, seen as the reduction of myotube diameter. Remarkably, pharmacological intervention on myotubes with jasplakinolide (250 mM, 30 min pre-treatment) to counteract VDBP effects on intracellular actin dynamics was sufficient to prevent VDBP-induced atrophy. To assess if VDBP had a causative role in muscle atrophy in vivo, we injected VDBP (1.5 mg/Kg) every 48 h for one week in the tibialis anterior muscles of Gc knock-out mice (VDBP KO). At the end of the experimental period, we observed the induction of the mitophagic gene Bnip3, impairment of muscle performances (26% reduction of grip strength at the endpoint), and a 16% reduction of muscle mass compared to the saline-injected contralateral muscles, confirming the atrophic effect of VDBP in vivo.Coherently with the upregulation of VDBP observed in cancer patients, VDBP levels also increase in murine models of cancer cachexia. To test the hypothesis that VDBP could play a role in cancer-associated muscle wasting, we induced cancer cachexia in VDBP KO mice by inoculating 106 Lewis Lung Carcinoma (LLC) cells resuspended in 100 μl of saline on the back of mice. Tumor-bearing VDBP KO mice preserved their body weight and performances, and the muscle loss was lessened by more than 50% compared to cachectic WT mice. Notably, between the two groups, there were no differences in tumor growth or food intake, ruling out the possibility that the reduction in muscle wasting could depend on smaller tumors or differential development of anorexia in the two genotypes. In conclusion, we demonstrated that VDBP acts as a hormone per se, having a direct pro-atrophic activity on skeletal muscle. Our data suggest that VDBP could represent a potential therapeutic target to treat cancer cachexia and other pathologies in which the rise of VDBP could impinge muscle mass and functionality. Presentation: 6/1/2024

Mechanotransduction pathways regulating YAP nuclear translocation under Yoda1 and vibration in osteocytes

Yes-associated protein (YAP) is a mechanosensitive protein crucial for bone remodeling. Although research has identified pathways and components involved in YAP regulation, the precise mechanisms of its localization during Piezo1 activation or vibration remain unclear. Piezo1, a mechanosensitive ion channel, allows calcium ions to flow into cells upon activation. Recent studies suggest that combining Yoda1, a Piezo1 activator, with low-magnitude high-frequency (LMHF) vibration (>30 Hz, <1 g acceleration) enhances YAP nuclear translocation. This combination potentially improves the mechanoresponse and therapeutic efficacy of LMHF vibration in bone cells. This study aims to elucidate how Yoda1 and LMHF vibration regulate mechanosensitive structures and pathways, leading to YAP nuclear translocation in MLO-Y4 osteocyte like cells. We investigated the roles of the cytoskeleton and nuclear envelope (NE) in YAP activation under combined LMHF vibration and Yoda1 treatments. Additionally, we analyzed differentially expressed genes (DEGs) in MLO-Y4 cells subjected to these treatments and in Piezo1 knockdown MLO-Y4 cells exposed to vibration. Our findings indicated that increased YAP nuclear translocation with combined treatment may result from the distinct effects of Yoda1 and vibration. Specifically, Yoda1 influenced YAP through mechanisms involving actin and NE dynamics, while LMHF vibration may modulate YAP via the interleukin 6 (IL6)/signal transducer and activator of transcription 3 (STAT3) axis. This study provides new insights and potential therapeutic targets for osteocyte-related pathologies.

A human DCC variant causing mirror movement disorder reveals that the WAVE regulatory complex mediates axon guidance by netrin-1-DCC.

The axon guidance cue netrin-1 signals through its receptor DCC (deleted in colorectal cancer) to attract commissural axons to the midline. Variants in DCC are frequently associated with congenital mirror movements (CMMs). A CMM-associated variant in the cytoplasmic tail of DCC is located in a conserved motif predicted to bind to a regulator of actin dynamics called the WAVE (Wiskott-Aldrich syndrome protein-family verprolin homologous protein) regulatory complex (WRC). Here, we explored how this variant affects DCC function and may contribute to CMM. We found that a conserved WRC-interacting receptor sequence (WIRS) motif in the cytoplasmic tail of DCC mediated the interaction between DCC and the WRC. This interaction was required for netrin-1-mediated axon guidance in cultured rodent commissural neurons. Furthermore, the WIRS motif of Fra, the Drosophila DCC ortholog, was required for attractive signaling in vivo at the Drosophila midline. The CMM-associated R1343H variant of DCC, which altered the WIRS motif, prevented the DCC-WRC interaction and impaired axon guidance in cultured commissural neurons and in Drosophila. The findings reveal the WRC as a pivotal component of netrin-1-DCC signaling and uncover a molecular mechanism explaining how a human genetic variant in the cytoplasmic tail of DCC may lead to CMM.

α-Parvin, an Integrin-Related Scaffold Protein, Regulates Actin Dynamics to Facilitate Kidney Ureteric Bud Branching Morphogenesis

α-Parvin, an Integrin-Related Scaffold Protein, Regulates Actin Dynamics to Facilitate Kidney Ureteric Bud Branching Morphogenesis

Gelsolin controls the release of phosphatidylserine (PS)-positive microvesicles (MVs) from platelets

Upon activation by vascular injury or extracellular agonists, platelets undergo rapid change shape, a process regulated by the actin cytoskeleton and accessory proteins. Platelet shape change is accompanied by the secretion of hemostatic factors and immunomodulatory cytokines from their intracellular granules, as well as the release of microvesicles (MVs) containing pro-inflammatory cytokines and procoagulant phosphatidylserine (PS). However, the role of actin dynamics in MV generation remains unclear. In this study, we found that blocking actin polymerization with cytochalasin D attenuated the release of PS-positive MVs in human platelets stimulated by thrombin or the calcium ionophore A23187. The actin-severing protein gelsolin (Gsn) facilitates normal actin filament turnover in activated platelets. Platelets from Gsn-deficient (Gsn−/−) mice showed reduced MV release compared to platelets from control mice. These findings indicate that the proper dynamics of the actin cytoskeleton are essential for MV generation in platelets, which has implications for their pro-inflammatory and procoagulant functions.

SUN2 mediates calcium-triggered nuclear actin polymerization to cluster active RNA polymerase II

The nucleoskeleton is essential for nuclear architecture as well as genome integrity and gene expression. In addition to lamins, titin or spectrins, dynamic actin filament polymerization has emerged as a potential intranuclear structural element but its functions are less well explored. Here we found that calcium elevations trigger rapid nuclear actin assembly requiring the nuclear membrane protein SUN2 independently of its function as a component of the LINC complex. Instead, SUN2 colocalized and associated with the formin and actin nucleator INF2 in the nuclear envelope in a calcium-regulated manner. Moreover, SUN2 is required for active RNA polymerase II (RNA Pol II) clustering in response to calcium elevations. Thus, our data uncover a SUN2-formin module linking the nuclear envelope to intranuclear actin assembly to promote signal-dependent spatial reorganization of active RNA Pol II.

Gliding motility of the diatom Craspedostauros australis coincides with the intracellular movement of raphid-specific myosins

Raphid diatoms are one of the few eukaryotes capable of gliding motility, which is remarkably fast and allows for quasi-instantaneous directional reversals. Besides other mechanistic models, it has been suggested that an actomyosin system provides the force for diatom gliding. However, in vivo data on the dynamics of actin and myosin in diatoms are lacking. In this study, we demonstrate that the raphe-associated actin bundles required for diatom movement do not exhibit a directional turnover of subunits and thus their dynamics do not contribute directly to force generation. By phylogenomic analysis, we identified four raphid diatom-specific myosins in Craspedostauros australis (CaMyo51A-D) and investigated their in vivo localization and dynamics through GFP-tagging. Only CaMyo51B-D but not CaMyo51A exhibited coordinated movement during gliding, consistent with a role in force generation. The characterization of raphid diatom-specific myosins lays the foundation for unraveling the molecular mechanisms that underlie the gliding motility of diatoms.

The spinal muscular atrophy gene product regulates actin dynamics.

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by low levels of the Survival of Motoneuron (SMN) protein. SMN interacts with and regulates the actin-binding protein profilin2a, thereby influencing actin dynamics. Dysfunctional actin dynamics caused by SMN loss disrupts neurite outgrowth, axonal pathfinding, and formation of functional synapses in neurons. Whether the SMN protein directly interacts with and regulates filamentous (F-) and monomeric globular (G-) actin is still elusive. In a quantitative single cell approach, we show that SMN loss leads to dysregulated F-/G-actin fractions. Furthermore, quantitative assessment of cell morphology suggests an F-actin organizational defect. Interestingly, this is mediated by an interaction of SMN with G- and F-actin. In co-immunoprecipitation, in-vitro pulldown and co-localization assays, we elucidated that this interaction is independent of the SMN-profilin2a interaction. Therefore, we suggest two populations being relevant for functional actin dynamics in healthy neurons: SMN-profilin2a-actin and SMN-actin. Additionally, those two populations may influence each other and therefore regulate binding of SMN to actin. In SMA, we showed a dysregulated co-localization pattern of SMN-actin which could only partially rescued by SMN restoration. However, dysregulation of F-/G-actin fractions was reduced by SMN restoration. Taken together, our results suggest a novel molecular function of SMN in binding to actin independent from SMN-profilin2a interaction.

TBK1-Zyxin signaling controls tumor-associated macrophage recruitment to mitigate antitumor immunity

Mechanical control is fundamental for cellular localization within a tissue, including for tumor-associated macrophages (TAMs). While the innate immune sensing pathways cGAS-STING and RLR-MAVS impact the pathogenesis and therapeutics of malignant diseases, their effects on cell residency and motility remain incompletely understood. Here, we uncovered that TBK1 kinase, activated by cGAS-STING or RLR-MAVS signaling in macrophages, directly phosphorylates and mobilizes Zyxin, a key regulator of actin dynamics. Under pathological conditions and in STING or MAVS signalosomes, TBK1-mediated Zyxin phosphorylation at S143 facilitates rapid recruitment of phospho-Zyxin to focal adhesions, leading to subsequent F-actin reorganization and reduced macrophage migration. Intratumoral STING-TBK1-Zyxin signaling was evident in TAMs and critical in antitumor immunity. Furthermore, myeloid-specific or global disruption of this signaling decreased the population of CD11b+ F4/80+ TAMs and promoted PD-1-mediated antitumor immunotherapy. Thus, our findings identify a new biological function of innate immune sensing pathways by regulating macrophage tissue localization, thus providing insights into context-dependent mitigation of antitumor immunity.

Actin cytoskeleton and plasma membrane aquaporins are involved in different drought response of Arabidopsis rhd2 and der1 root hair mutants

Actin cytoskeleton and reactive oxygen species are principal determinants of root hair polarity and tip growth. Loss of function in RESPIRATORY BURST OXIDASE HOMOLOG C/ROOT HAIR DEFECTIVE 2 (AtRBOHC/RHD2), an NADPH oxidase emitting superoxide to the apoplast, and in ACTIN 2, a vegetative actin isovariant, in rhd2-1 and der1-3 mutants, respectively, lead to similar defects in root hair formation and elongation Since early endosome-mediated polar localization of AtRBOHC/RHD2 depends on actin cytoskeleton, comparing the proteome-wide consequences of both mutations might be of eminent interest. Therefore, we employed a differential proteomic analysis of Arabidopsis rhd2-1 and der1-3 mutants. Both mutants exhibited substantial alterations in abundances of stress-related proteins. Notably, plasma membrane (PM)-localized PIP aquaporins showed contrasting abundance patterns in the mutants compared to wild-types. Drought-responsive proteins were mostly downregulated in rhd2-1 but upregulated in der1-3. Proteomic data suggest that opposite to der1-3, altered vesicular transport in rhd2-1 mutant likely contributes to the deregulation of PM-localized proteins, including PIPs. Moreover, lattice light sheet microscopy revealed reduced actin dynamics in rhd2-1 roots, a finding contrasting with previous reports on der1-3 mutant. Phenotypic experiments demonstrated a drought stress susceptibility in rhd2-1 and resistance in der1-3. Thus, mutations in AtRBOHC/RHD2 and ACTIN2 cause similar root hair defects, but they differently affect the actin cytoskeleton and vesicular transport. Reduced actin dynamics in rhd2-1 mutant is accompanied by alteration of vesicular transport proteins abundance, likely leading to altered protein delivery to PM, including aquaporins, thereby significantly affecting drought stress responses.