Tim4 enables large peritoneal macrophages to cross-present tumor antigens at early stages of tumorigenesis.

Receptors controlling the cross-presentation of tumor antigens by macrophage subsets in cancer tissues are poorly explored. Here, we show that TIM4+ large peritoneal macrophages efficiently capture and cross-present tumor-associated antigens at early stages of peritoneal infiltration by ovarian cancer cells. The phosphatidylserine (PS) receptor TIM4 promotes maximal uptake of dead cells or PS-coated artificial targets and triggers inflammatory and metabolic gene programs in combination with cytoskeletal remodeling and upregulation of transcriptional signatures related to antigen processing. At the cellular level, TIM4-mediated engulfment induces nucleation of F-actin around nascent phagosomes, delaying the recruitment of vacuolar ATPase, acidification, and cargo degradation. In vivo, TIM4 deletion blunts induction of early anti-tumoral effector CD8 T cells and accelerates the progression of ovarian tumors. We conclude that TIM4-mediated uptake drives the formation of specialized phagosomes that prolong the integrity of ingested antigens and facilitate cross-presentation, contributing to immune surveillance of the peritoneum.

Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

F-actin architecture determines the conversion of chemical energy into mechanical work.

Mechanical work serves as the foundation for dynamic cellular processes, ranging from cell division to migration. A fundamental driver of cellular mechanical work is the actin cytoskeleton, composed of filamentous actin (F-actin) and myosin motors, where force generation relies on adenosine triphosphate (ATP) hydrolysis. F-actin architectures, whether bundled by crosslinkers or branched via nucleators, have emerged as pivotal regulators of myosin II force generation. However, it remains unclear how distinct F-actin architectures impact the conversion of chemical energy to mechanical work. Here, we employ in vitro reconstitution of distinct F-actin architectures with purified components to investigate their influence on myosin ATP hydrolysis (consumption). We find that F-actin bundles composed of mixed polarity F-actin hinder network contraction compared to non-crosslinked network and dramatically decelerate ATP consumption rates. Conversely, linear-nucleated networks allow network contraction despite reducing ATP consumption rates. Surprisingly, branched-nucleated networks facilitate high ATP consumption without significant network contraction, suggesting that the branched network dissipates energy without performing work. This study establishes a link between F-actin architecture and myosin energy consumption, elucidating the energetic principles underlying F-actin structure formation and the performance of mechanical work.

Transcriptome and single-cell analysis reveal disulfidptosis-related modification patterns of tumor microenvironment and prognosis in osteosarcoma.

Osteosarcoma (OS) is the most common malignant bone tumor with high pathological heterogeneity. Our study aimed to investigate disulfidptosis-related modification patterns in OS and their relationship with survival outcomes in patients with OS. We analyzed the single-cell-level expression profiles of disulfidptosis-related genes (DSRGs) in both OS microenvironment and OS subclusters, and HMGB1 was found to be crucial for intercellular regulation of OS disulfidptosis. Next, we explored the molecular clusters of OS based on DSRGs and related immune cell infiltration using transcriptome data. Subsequently, the hub genes of disulfidptosis in OS were screened by applying multiple machine models. In vitro and patient experiments validated our results. Three main disulfidptosis-related molecular clusters were defined in OS, and immune infiltration analysis suggested high immune heterogeneity between distinct clusters. The in vitro experiment confirmed decreased cell viability of OS after ACTB silencing and higher expression of ACTB in patients with lower immune scores. Our study systematically revealed the underlying relationship between disulfidptosis and OS at the single-cell level, identified disulfidptosis-related subtypes, and revealed the potential role of ACTB expression in OS disulfidptosis.

A Novel Interaction of Slug (SNAI2) and Nuclear Actin.

Actin is a protein of central importance to many cellular functions. Its localization and activity are regulated by interactions with a high number of actin-binding proteins. In a yeast two-hybrid (Y2H) screening system, snail family transcriptional repressor 2 (SNAI2 or slug) was identified as a yet unknown potential actin-binding protein. We validated this interaction using immunoprecipitation and analyzed the functional relation between slug and actin. Since both proteins have been reported to be involved in DNA double-strand break (DSB) repair, we focused on their interaction during this process after treatment with doxorubicin or UV irradiation. Confocal microscopy elicits that the overexpression of actin fused to an NLS stabilizes complexes of slug and γH2AX, an early marker of DNA damage repair.

TMC7 functions as a suppressor of Piezo2 in primary sensory neurons blunting peripheral mechanotransduction.

The transmembrane channel-like (TMC) protein family comprises eight members, with TMC1 and TMC2 being extensively studied. This study demonstrates substantial co-expression of TMC7 with the mechanosensitive channel Piezo2 in somatosensory neurons. Genetic deletion of TMC7 in primary sensory ganglia neurons in vivo enhances sensitivity in both physiological and pathological mechanosensory transduction. This deletion leads to an increase in proportion of rapidly adapting (RA) currents conducted by Piezo2 in dorsal root ganglion (DRG) neurons and accelerates RA deactivation kinetics. In HEK293 cells expressing both proteins, TMC7 significantly suppresses the current amplitudes of co-expressed Piezo2. Our findings reveal that TMC7 and Piezo2 exhibit physical interactions, and both proteins also physically interact with cytoskeletal ß-actin. We hypothesize that TMC7 functions as an inhibitory modulator of Piezo2 in DRG neurons, either through direct inhibition or by disrupting the transmission of mechanical forces from the cytoskeleton to the channel.

Molecular mechanism of actin filament elongation by formins.

Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.

Clutch model for focal adhesions predicts reduced self-stabilization under oblique pulling.

Cell-matrix adhesions connect the cytoskeleton to the extracellular environment and are essential for maintaining the integrity of tissue and whole organisms. Remarkably, cell adhesions can adapt their size and composition to an applied force such that their size and strength increases proportionally to the load. Mathematical models for the clutch-like force transmission at adhesions are frequently based on the assumption that mechanical load is applied tangentially to the adhesion plane. Recently, we suggested a molecular mechanism that can explain adhesion growth under load for planar cell adhesions. The mechanism is based on conformation changes of adhesion molecules that are dynamically exchanged with a reservoir. Tangential loading drives the occupation of some states out of equilibrium, which for thermodynamic reasons, leads to the association of further molecules with the cluster, which we refer to as self-stabilization. Here, we generalize this model to forces that pull at an oblique angle to the plane supporting the cell, and examine if this idealized model also predicts self-stabilization. We also allow for a variable distance between the parallel planes representing cytoskeletal F-actin and transmembrane integrins. Simulation results demonstrate that the binding mechanism and the geometry of the cluster have a strong influence on the response of adhesion clusters to force. For oblique angles smaller than about 40∘, we observe a growth of the adhesion site under force. However this self-stabilization is reduced as the angle between the force and substrate plane increases, with vanishing self-stabilization for normal pulling. Overall, these results highlight the fundamental difference between the assumption of pulling and shearing forces in commonly used models of cell adhesion.

Early embryonic polarity establishment and implications for lineage differentiation.

Polarity establishment is one of the key factors affecting early embryonic development. Polarity establishment begins with myosin phosphorylation in the 8-cell embryo, and phosphorylation activates actin leading to its initiation of contractility. Subsequently, actin undergoes reorganization to form an apical domain rich in microvilli on the non-contacting surface of each blastomere, and form the actomyosin ring that marks the maturation of the apical domain in conjunction with polar protein complexes and others. From the process of polarity establishment, it can be seen that the formation of the apical domain is influenced by actin-related proteins and polar protein complexes. Some zygote genome activation (ZGA) and lineage-specific genes also regulate polarity establishment. Polarity establishment underlies the first cell lineage differentiation during early embryonic development. It regulates lineage segregation and morphogenesis by affecting asymmetric cell division, asymmetric localization of lineage differentiation factors, and activity of the Hippo signaling pathway. In this review, we systematically summarize the mechanisms of early embryonic polarity establishment and its impact on lineage differentiation in mammals, and discuss the shortcomings of the currently available studies in terms of regulatory mechanisms and species, thereby providing clues and systematic perspectives for elucidating early embryonic polarity establishment.

Potential small effector molecules restoring cellular defects due to sialic acid biosynthetic enzyme deficiency: Pathological relevance to GNE myopathy.

GNEM (GNE Myopathy) is a rare neuromuscular disease caused due to biallelic mutations in sialic acid biosynthetic GNE enzyme (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase). Recently direct or indirect role of GNE in other cellular functions have been elucidated. Hyposialylation of IGF-1R leads to apoptosis due to mitochondrial dysfunction while hyposialylation of ß1 integrin receptor leads to altered F-actin assembly, disrupted cytoskeletal organization and slow cell migration. Other cellular defects in presence of GNE mutation include altered ER redox state and chaperone expression such as HSP70 or PrdxIV. Currently, there is no cure to treat GNEM. Possible therapeutic trials focus on supplementation with sialic acid, ManNAc, sialyllactose and gene therapy that slows the disease progression. In the present study, we analyzed the effect of small molecules like BGP-15 (HSP70 modulator), IGF-1 (IGF-1R ligand) and CGA (cofilin activator) on cellular phenotypes of GNE heterozygous knock out L6 rat skeletal muscle cell line (SKM­GNEHz). Treatment with BGP-15 improved GNE epimerase activity by 40 % and reduced ER stress by 45 % for SKM­GNEHz. Treatment with IGF-1 improved epimerase activity by 37.5 %, F-actin assembly by 100 %, cell migration upto 36 % (36 h) and atrophy by 0.44-fold for SKM­GNEHz. Treatment with CGA recovered epimerase activity by 49 %, F-actin assembly by 132 % and cell migration upto 41 % (24 h) in SKM­GNEHz. Our study shows that treatment with these small effector molecules reduces the detrimental phenotype observed in SKM­GNEHz, thereby, providing insights into potential therapeutic targets for GNEM.

Nuclear actin dynamics and functions at a glance.

Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development.

The Shot CH1 domain recognises a distinct form of F-actin during Drosophila oocyte determination.

In Drosophila, only one cell in a multicellular female germline cyst is specified as an oocyte and a similar process occurs in mammals. The symmetry-breaking cue for oocyte selection is provided by the fusome, a tubular structure connecting all cells in the cyst. The Drosophila spectraplakin Shot localises to the fusome and translates its asymmetry into a polarised microtubule network that is essential for oocyte specification, but how Shot recognises the fusome is unclear. Here, we demonstrate that the actin-binding domain (ABD) of Shot is necessary and sufficient to localise Shot to the fusome and mediates Shot function in oocyte specification together with the microtubule-binding domains. The calponin homology domain 1 of the Shot ABD recognises fusomal F-actin and requires calponin homology domain 2 to distinguish it from other forms of F-actin in the cyst. By contrast, the ABDs of utrophin, Fimbrin, Filamin, Lifeact and F-tractin do not recognise fusomal F-actin. We therefore propose that Shot propagates fusome asymmetry by recognising a specific conformational state of F-actin on the fusome.

Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

Map-1a regulates Sertoli cell BTB dynamics through the cytoskeletal organization of microtubule and F-actin.

Microtubule-associated protein 1a (Map1a) is a microtubule (MT) regulatory protein that binds to the MT protofilaments in mammalian cells to promote MT stabilization. Maps work with MT cleavage proteins and other MT catastrophe-inducing proteins to confer MT dynamics to support changes in the Sertoli cell shape to sustain spermatogenesis. However, no functional studies are found in the literature to probe its role in spermatogenesis. Using an RNAi approach, coupled with the use of toxicant-induced testis (in vivo)- and Sertoli cell (in vitro)-injury models, RNA-Seq analysis, transcriptome profiling, and relevant bioinformatics analysis, immunofluorescence analysis, and pertinent biochemical assays for cytoskeletal organization, we have delineated the functional role of Map1a in Sertoli cells and testes. Map1a was shown to support MT structural organization, and its knockdown (KD) also perturbed the structural organization of actin, vimentin, and septin cytoskeletons as these cytoskeletons are intimately related, working in concert to support spermatogenesis. More importantly, cadmium-induced Sertoli cell injury that perturbed the MT structural organization across the cell cytoplasm was associated with disruptive changes in the distribution of Map1a and a surge in p-p38-MAPK (phosphorylated p38-mitogen-activated protein kinase) expression but not total p38-MAPK. These findings thus support the notion that p-p38-MAPK activation is involved in cadmium-induced Sertoli cell injury. This conclusion was supported by studies using doramapimod, a specific p38-MAPK phosphorylation (activation) inhibitor, which was capable of restoring the cadmium-induced disruptive structural organization of MTs across the Sertoli cell cytoplasm. In summary: this study provides mechanistic insights regarding restoration of toxicant-induced Sertoli cell and testis injury and male infertility.

[Advances in the study of the actin nucleation factor Spire].

There are three main classes of actin nucleation factors: Arp2/3 complexes, Spire and Formin. Spire assembles microfilaments by nucleating stable longitudinal tetramers and binding actin to the growing end of the microfilament. As early as 1999, Wellington et al. identified Spire as an actin nucleating agent, however, over the years, most studies have focused on Arp2/3 and Formin proteins; there has been relatively less research on Spire as a member of the actin nucleating factors. Recent studies have shown that Spire is involved in the vesicular transport through the synthesis of actin and plays an important role in neural development. In this paper, we reviewed the structure, expression and function of Spire, and its association with disease in order to identify meaningful potential directions for studies on Spire.

Attenuating Renal Interstitial Fibrosis by Shenqi Pill via Reducing Inflammation Response Regulated by NF-κB Pathway In Vitro and In Vivo.

INTRODUCTION: One of the most significant clinical features of chronic  kidney disease is renal interstitial fibrosis (RIF). This study aimed  to investigate the role and mechanism of Shenqi Pill (SQP) on RIF. METHODS: RIF model was established by conducting unilateral  ureteral obstruction (UUO) surgery on rat or stimulating human  kidney-2 (HK-2) cell with transforming growth factor ß1 (TGFß1).  After modeling, the rats in the SQP low dose group (SQP-L), SQP  middle dose group (SQP-M) and SQP high dose group (SQP-H)  were treated with SQP at 1.5, 3 or 6 g/kg/d, and the cells in the  TGFß1+SQP-L/M/H were treated with 2.5%, 5%, 10% SQP-containing  serum. In in vivo assays, serum creatinine (SCr) and blood urea  nitrogen (BUN) content were measured, kidney histopathology  was evaluated., and α-smooth muscle actin (α-SMA) expression  was detected by immunohistochemistry. Interleukin-1ß (IL-1ß),  interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) content,  inhibitor of kappa B alpha (IKBα) and P65 phosphorylation were  assessed. Meanwhile, cell viability, inflammatory cytokines content,  α-SMA expression, IKBα and P65 phosphorylation were detected  in vitro experiment.  Results. SQP exhibited reno-protective effect by decreasing SCr  and BUN content, improving renal interstitial damage, blunting  fibronectin (FN) and α-SMA expression in RIF rats. Similarly, after  the treatment with SQP-containing serum, viability and α-SMA  expression were remarkably decreased in TGFß1-stimulated HK-2  cell. Furthermore, SQP markedly down-regulated IL-1ß, IL-6, and  TNF-α content, IKBα and RelA (P65) phosphorylation both in vivo and in vitro.  Conclusion. SQP has a reno-protective effect against RIF in vivo and in vitro, and the effect is partly linked to nuclear factor-kappa  B (NF-κB) pathway related inflammatory response, which indicates  that SQP may be a candidate drug for RIF. DOI: 10.52547/ijkd.7546.

Effects of cytochalasin D on relaxation process of skinned taenia cecum and carotid artery from guinea pig.

Actin linked regulatory mechanisms are known to contribute contraction/relaxation in smooth muscle. In order to clarify whether modulation of polymerization/depolymerization of actin filaments affects relaxation process, we examined the effects of cytochalasin D on relaxation process by Ca2+ removal after Ca2+-induced contraction of ß-escin skinned (cell membrane permeabilized) taenia cecum and carotid artery preparations from guinea pigs. Cytochalasin D, an inhibitor of actin polymerization, significantly suppressed the force during relaxation both in skinned taenia cecum and carotid artery. The data fitting analysis of the relaxation processes indicates that cytochalasin D accelerates slow (latch-like) bridge dissociation. Cytochalasin D seems to directly disrupts actin filament organization or its length, resulting in modulation of actin filament structure that prevents myosin binding.

BioID Analysis of Actin-Binding Proteins.

Proteins often exist and function as part of higher-order complexes or networks. A challenge is to identify the universe of proximal and interacting partners for a given protein. We describe how the high-activity promiscuous biotin ligase called TurboID is fused to the actin-binding peptide LifeAct to label by biotinylation proteins that bind, or are in close proximity, to actin. The rapid enzyme kinetics of TurboID allows the profiles of actin-binding proteins to be compared under different conditions, such as acute disruption of filamentous actin structures with cytochalasin D.

Insights into the Mechanism of Action of the Degraded Limonoid Prieurianin.

Limonoids are extremely diversified in plants, with many categories of products bearing an intact, rearranged or fragmented oxygenated scaffold. A specific subgroup of fragmented or degraded limonoids derives from the tetranortriterpenoid prieurianin, initially isolated from the tree Trichilia prieuriana but also found in other plants of the Meliaceae family, including the more abundant species Aphanamixis polystachya. Prieurianin-type limonoids include about seventy compounds, among which are dregeanin and rohitukin. Prieurianin and analogs exhibit insecticidal, antimicrobial, antiadipogenic and/or antiparasitic properties but their mechanism of action remains ill-defined at present. Previous studies have shown that prieurianin, initially known as endosidin 1, stabilizes the actin cytoskeleton in plant and mammalian cells via the modulation of the architecture and dynamic of the actin network, most likely via interference with actin-binding proteins. A new mechanistic hypothesis is advanced here based on the recent discovery of the targeting of the chaperone protein Hsp47 by the fragmented limonoid fraxinellone. Molecular modeling suggested that prieurianin and, to a lesser extent dregeanin, can form very stable complexes with Hsp47 at the protein-collagen interface. Hsp-binding may account for the insecticidal action of the product. The present review draws up a new mechanistic portrait of prieurianin and provides an overview of the pharmacological properties of this atypical limonoid and its chemical family.

Oleate Promotes Triple-Negative Breast Cancer Cell Migration by Enhancing Filopodia Formation through a PLD/Cdc42-Dependent Pathway.

Breast cancer, particularly triple-negative breast cancer (TNBC), poses a global health challenge. Emerging evidence has established a positive association between elevated levels of stearoyl-CoA desaturase 1 (SCD1) and its product oleate (OA) with cancer development and metastasis. SCD1/OA leads to alterations in migration speed, direction, and cell morphology in TNBC cells, yet the underlying molecular mechanisms remain elusive. To address this gap, we aim to investigate the impact of OA on remodeling the actin structure in TNBC cell lines, and the underlying signaling. Using TNBC cell lines and bioinformatics tools, we show that OA stimulation induces rapid cell membrane ruffling and enhances filopodia formation. OA treatment triggers the subcellular translocation of Arp2/3 complex and Cdc42. Inhibiting Cdc42, not the Arp2/3 complex, effectively abolishes OA-induced filopodia formation and cell migration. Additionally, our findings suggest that phospholipase D is involved in Cdc42-dependent filopodia formation and cell migration. Lastly, the elevated expression of Cdc42 in breast tumor tissues is associated with a lower survival rate in TNBC patients. Our study outlines a new signaling pathway in the OA-induced migration of TNBC cells, via the promotion of Cdc42-dependent filopodia formation, providing a novel insight for therapeutic strategies in TNBC treatment.