Inhibition Of Actin Polymerization Research Articles

Short Communication: Goat Mastitis and the Formation of Neutrophil Extracellular Traps (NETs)

Mastitis, an inflammation of the mammary gland affecting milk production and quality in dairy herds, is often associated with Staphylococcus spp. in goats. Neutrophils are crucial in combating infections by migrating into milk and deploying various defense strategies, including the release of neutrophil extracellular traps (NETs) composed of DNA, histones, and bactericidal proteins. This study investigated whether NETs are released by goat neutrophils stimulated in vitro by Staphylococcus aureus and Staphylococcus warneri, two common pathogens of goat mastitis. PMNs were isolated from blood from healthy adult goats. We evaluated goat NET formation by stimulating cells with: phorbol 12-myristate 13-acetate (PMA) as a positive control, cytochalasin for inhibition of actin polymerization, S. aureus, and S. warneri. NET formation was observed in response to chemical stimulation and bacterial presence, effectively trapping pathogens. Variations in NET formation between S. aureus and S. warneri suggest pathogen-specific responses. These findings suggest that the formation of NETs may be an important complementary mechanism in the defense against mastitis in goats. In conclusion, this study unveils a novel defense mechanism in goats, indicating the role of NETs against S. aureus and S. warneri in mastitis.

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.

Mechanisms of ingested polystyrene micro-nanoplastics (MNPs) uptake and translocation in an in vitro tri-culture small intestinal epithelium

Micro and nanoplastics (MNPs) are now ubiquitous contaminants of food and water. Many cellular and animal studies have shown that ingested MNPs can breach the intestinal barrier to reach the circulation. To date however, the cellular mechanisms involved in intestinal absorption of MNPs have not been investigated with physiologically relevant models, and thus remain unknown. We employed in vitro simulated digestion, a tri-culture small intestinal epithelium model, and a panel of inhibitors to assess the contributions of the possible mechanisms to absorption of 26 nm carboxylated polystyrene (PS26C) MNPs. Inhibition of ATP synthesis reduced translocation by only 35 %, suggesting uptake by both active endocytic pathways and passive diffusion. Translocation was also decreased by inhibition of dynamin and clathrin, suggesting involvement of clathrin mediated endocytosis (CME) and fast endophilin-mediated endocytosis (FEME). Inhibition of actin polymerization also significantly reduced translocation, suggesting involvement of macropinocytosis or phagocytosis. However, inhibition of the Na+-H+ exchanger had no effect on translocation, thus ruling out macropinocytosis. Together these results suggest uptake by passive diffusion as well as by active phagocytosis, CME, and FEME pathways. Further studies are needed to assess uptake mechanisms for other environmentally relevant MNPs as a function of polymer, surface chemistry, and size.

Neonatal epithelial reparative capacity is enhanced by acellular placental extract in porcine models

Necrotizing enterocolitis (NEC) is a devastating condition with a ~30% mortality rate in pre-term, very low birth-weight infants. Premature infants lack vital in utero ingestion of amniotic fluid which accelerates intestinal maturation and reduces inflammation. Therefore, an acellular placental extract (PE) akin to amniotic fluid may be therapeutic for NEC. The objective of this study was to evaluate PE’s capacity to accelerate repair following or prevent the induction of NEC-like injury. We hypothesized that PE would enhance neonatal porcine intestinal epithelial cells’ capacity to repair in vitro and in vivo. NEC was induced in 12 piglets via pre-term cesarean delivery and hyperosmolar formula feeding. Prior to formula feeding, piglets were nil per os or enterally supplemented with PE. Tissue, collected at euthanasia, up to 72H after formula initiation, was evaluated grossly and histologically and with immunohistochemical staining for proliferation (Ki67), stem cell identity (Sox9), and apoptosis (CC3). Numerical outputs were assessed with Kolmogorov-Smirnov testing. PE’s effect on epithelial restitution was assessed with scratch assays performed on confluent monolayers derived from ~1-day old piglet ileum. Monolayers were pre-treated with either actin polymerization inhibitor, Latrunculin A, or a cell cycle inhibitor, hydroxyurea. After scratch, either media or PE in media was applied. Scratch-closure was monitored for 24H; percentage-closure was calculated and analyzed using 2-way ANOVA and Tukey multiple comparison. Scratch-wound leading-edge cells were examined for EdU and Ki67 immunofluorescent positivity which was evaluated using Kolmogorov-Smirnov analysis. Tight junction recovery was assessed using identically derived confluent monolayers grown on Transwell inserts and exposed to hypoxia (18H, 1%O2, 5%CO2). Following hypoxia, PE or media alone was added to the apical chamber. Transepithelial electrical resistance (TEER) was measured every 6-12H for 48H using a symmetrical disc electrode chamber. TEER rate of change was analyzed using a two-way ANOVA. For all analyses, significance was set at p<0.05. In vivo PE administration resulted in reduced gross and histological NEC damage in treated compared to untreated piglets (p=0.02). Ki67 and Sox9 expression increased (p=0.71; p=0.93) and CC3 expression decreased with PE treatment (p=0.65). Evaluation of these markers in additional animals is pending. In vitro, PE expedited scratch wound closure (p<0.05) and expanded proliferative cell number along scratch edges (EdU/DAPI, p<0.05; Ki67+EdU/DAPI, p<0.01). Other analyses are pending. PE application prevented gross NEC development in vivo and accelerated recovery in vitro. NIH #R44HD100243; #T32562967-39806. 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.

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

The implementation of antiretroviral therapy (ART) has effectively restricted the transmission of Human Immunodeficiency Virus (HIV) and improved overall clinical outcomes. However, a complete cure for HIV remains out of reach, as the virus persists in a stable pool of infected cell reservoir that is resistant to therapy and thus a main barrier towards complete elimination of viral infection. While the mechanisms by which host proteins govern viral gene expression and latency are well-studied, the emerging regulatory functions of non-coding RNAs (ncRNA) in the context of T cell activation, HIV gene expression and viral latency have not yet been thoroughly explored. Here, we report the identification of the Cytoskeleton Regulator (CYTOR) long non-coding RNA (lncRNA) as an activator of HIV gene expression that is upregulated following T cell stimulation. Functional studies show that CYTOR suppresses viral latency by directly binding to the HIV promoter and associating with the cellular positive transcription elongation factor (P-TEFb) to activate viral gene expression. CYTOR also plays a global role in regulating cellular gene expression, including those involved in controlling actin dynamics. Depletion of CYTOR expression reduces cytoplasmic actin polymerization in response to T cell activation. In addition, treating HIV-infected cells with pharmacological inhibitors of actin polymerization reduces HIV gene expression. We conclude that both direct and indirect effects of CYTOR regulate HIV gene expression.

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 β-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.

CdSe/ZnS Quantum Dots' Impact on In Vitro Actin Dynamics.

Quantum dots (QDs) are a novel type of nanomaterial that has unique optical and physical characteristics. As such, QDs are highly desired because of their potential to be used in both biomedical and industrial applications. However, the mass adoption of QDs usage has raised concerns among the scientific community regarding QDs' toxicity. Although many papers have reported the negative impact of QDs on a cellular level, the exact mechanism of the QDs' toxicity is still unclear. In this investigation, we study the adverse effects of QDs by focusing on one of the most important cellular processes: actin polymerization and depolymerization. Our results showed that QDs act in a biphasic manner where lower concentrations of QDs stimulate the polymerization of actin, while high concentrations of QDs inhibit actin polymerization. Furthermore, we found that QDs can bind to filamentous actin (F-actin) and cause bundling of the filament while also promoting actin depolymerization. Through this study, we found a novel mechanism in which QDs negatively influence cellular processes and exert toxicity.

Abstract 4141: Quantitative measurement of 3D tumor spheroid invasion using live cell time-lapse imaging

Abstract 90% of the death of cancer individuals is related to cancer metastasis1. The invasion and migration of cancer cells into surrounding tissues and chambers is the first step of metastasis2. To have a better and more in-depth understanding of the pathophysiological activities involved in metastatic cancer, accurate and reliable methods for evaluating cell invasion are urgently needed. Compared to the 2D cell model, the 3D system mimics the natural physiological properties and conditions, such as structure, physiology, biological signals of living tissues, cell-matrix interactions4. In this study, we followed the commonly used protocol for 3D tumor invasion assay3 to generate spheroids in Ultra-Low-attachment (ULA) round bottom 96-well plate and subsequently embedded them in Matrigel® (5 mg/mL). After polymerization, the Matrigel that contains tumor spheroid was submerged in 100 μL cell culture media. The invasion of the 3D spheroid in the presence or absence of drug treatment was monitored and quantified using the brightfield and fluorescence live cell time-lapse image acquisition and image analysis functions of the Agilent xCELLigence RTCA eSight. The progress of spheroid invasion and the drug-inhibitory effects on tumor invasion were quantified by tracking and calculating the area of invasive protrusion from the spheroid. Our data show that 1) different types of cancer cells had different metastatic potentials demonstrated by the level of invadopodium extending into the Matrigel at desired time points; 2) the invasion of 3D HT1080 spheroid was inhibited by cytochalasin D (Cyto D), an inhibitor of actin polymerization, and GM6001, a potent broad-spectrum inhibitor of matrix metalloproteinases (MMP), in a dose-dependent manner; 3) Cyto D exhibited higher efficacy of inhibiting tumor invasion than GM6001. In summary, our approach by combining the 3D tumor spheroid invasion model with the RTCA eSight’s capability of live cell imaging and automatic quantification of the area of invasive protrusion can facilitate and enhance the development of new treatments at the preclinical stage in the future. Citation Format: Grace Yang, Tian Wang, Peifang Ye, Xiaoyu Zhang. Quantitative measurement of 3D tumor spheroid invasion using live cell time-lapse imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4141.

Abstract 5611: Unraveling the role of F-actin in resistant GBM: Insights into DNA repair, GSC phenotype, and therapeutic implications

Abstract Glioblastoma (GBM), a highly aggressive brain cancer, poses a formidable challenge marked by its resistance to conventional therapies. F-actin, a crucial cytoskeletal protein, influences DNA repair and impacts genomic stability. F-actin dynamics also shape the glioma stem cell (GSC) phenotype, affecting self-renewal and migration. Altered F-actin organization contributes to GBM resistance to conventional therapies. Understanding F-actin's multifaceted role is vital for developing targeted strategies to overcome treatment resistance and enhance outcomes for GBM patients. This study goes deeper into the intricate interplay between the F-actin cytoskeleton and the acquisition of resistance in GBM cells. Our primary goal was to explore the effects of pharmacological or genetic F-actin depolymerization on the reversal of acquired resistance to ionizing radiation (IR) and temozolomide (TMZ) in GBM cells, particularly focusing on the underlying mechanisms involving DNA repair, the glioma stem cell (GSC) phenotype, and therapeutic implications. To establish resistant sublines, GBM U87-MG cells underwent cycles of IR and TMZ exposure, revealing a significant increase in IC50 values for TMZ and ID50 values for IR through viability assays. Subsequent characterization of these sublines growth was in both adherent and spheroid cultures uncovering a distinct sphere-forming capacity. Notably, alterations in DNA repair and the Rho GTPase pathway were observed, with evident disorganized F-actin polymerization in resistant cells, especially at the periphery of spheroids. Elevated expression of stemness markers (Nestin, CD133, and CD44) highlighted a GSC-like profile in the resistant cells, a characteristic further accentuated in spheroid cultures. To assess the relevance of the actin cytoskeleton in maintaining the resistant phenotype, cells were treated with Rho-GTPase inhibitor (C3) or actin polymerization inhibitor (Cytochalasin D). Both treatments resulted in reduced TMZ IC50 and IR ID50 values, partially attributed to the negative modulation of DNA repair capacity - observed by alkaline comet and host-cell reactivation assays. Additionally, F-actin disassembly was found to impact the expression of stemness markers, further reinforcing the connection between cytoskeletal dynamics and the GSC phenotype. Overall, our data strongly suggest that F-actin cytoskeleton dynamics plays a pivotal role in the development and maintenance of resistance in GBM. Targeting these cytoskeletal components sees a promising pharmacological strategy for resensitizing recurrent and resistant GBM tumors, not only by diminishing DNA repair capabilities but also by influencing the GSC-like phenotype, thus opening new avenues for therapeutic interventions. Citation Format: Yuli Thamires Magalhães, Fábio Luís Forti. Unraveling the role of F-actin in resistant GBM: Insights into DNA repair, GSC phenotype, and therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5611.

Sustained activation of the FGF1-MEK-ERK pathway inhibits proliferation, invasion and migration and enhances radiosensitivity in mouse angiosarcoma cells.

Angiosarcoma is a rare refractory soft-tissue tumor with a poor prognosis and is treated by radiotherapy. The fibroblast growth factor 1 (FGF1) mutant, with enhanced thermostability due to several substituted amino acids, inhibits angiosarcoma cell metastasis, yet the mechanism of action is unclear. This study aims to clarify the FGF1 mutant mechanism of action using ISOS-1 mouse angiosarcoma cells. The wild-type FGF1 or FGF1 mutant was added to ISOS-1 cells and cultured, evaluating cell numbers over time. The invasive and migratory capacity of ISOS-1 cells was assessed by transwell analysis. ISOS-1 cell radiosensitivity was assessed by colony formation assay after X-ray irradiation. To examine whether mitogen-activated protein kinase (MEK) inhibitor counteracts the FGF1 mutant effects, a combination of MEK inhibitor and FGF1 mutant was added to ISOS-1 cells and cultured. The FGF1 mutant was observed to inhibit ISOS-1 cell proliferation, invasion and migration by sustained FGF1 signaling activation. A MEK inhibitor suppressed the FGF1 mutant-induced inhibition of proliferation, invasion and migration of ISOS-1 cells. Furthermore, the FGF1 mutant enhanced radiosensitivity of ISOS-1 cells, but MEK inhibition suppressed the increased radiosensitivity. In addition, we found that the FGF1 mutant strongly inhibits actin polymerization, suggesting that actin cytoskeletal dynamics are closely related to ISOS-1 cell radiosensitivity. Overall, this study demonstrated that in ISOS-1 cells, the FGF1 mutant inhibits proliferation, invasion and migration while enhancing radiosensitivity through sustained activation of the MEK-mediated signaling pathway.

Fluid shear stress induced-endothelial phenotypic transition contributes to cerebral ischemia-reperfusion injury and repair.

Long-term ischemia leads to insufficient cerebral microvascular perfusion and dysfunction. Reperfusion restores physiological fluid shear stress (FSS) but leads to serious injury. The mechanism underlying FSS-induced endothelial injury in ischemia-reperfusion injury (IRI) remains poorly understood. In this study, a rat model of middle cerebral artery occlusion was constructed to explore cerebrovascular endothelial function and inflammation in vivo. Additionally, the rat brain microvascular endothelial cells (rBMECs) were exposed to a laminar FSS of 0.5 dyn/cm2 for 6 h and subsequently restored to physiological fluid shear stress level (2 dyn/cm2) for 2 and 12 h, respectively. We found that reperfusion induced endothelial-to-mesenchymal transition (EndMT) in endothelial cells, leading to serious blood-brain barrier dysfunction and endothelial inflammation, accompanied by the nuclear accumulation of Yes-associated protein (YAP). During the later stage of reperfusion, cerebral endothelium was restored to the endothelial phenotype with a distinct change in mesenchymal-to-endothelial transition (MEndT), while YAP was translocated and phosphorylated in the cytoplasm. Knockdown of YAP or inhibition of actin polymerization markedly impaired the EndMT in rBMECs. These findings suggest that ischemia-reperfusion increased intensity of FSS triggered an EndMT process and, thus, led to endothelial inflammation and tissue injury, whereas continuous FSS induced a time-dependent reversal MEndT event contributing to the endothelial repair. This study provides valuable insight for therapeutic strategies targeting IRI.

Toward a Template for Synthetic Actin-Targeting Macrolide Analogues That Inhibit Cancer Cell Invasiveness.

Actin barbed end-binding macrolides have been shown to inhibit cancer cell motility and invasion of extracellular matrix (ECM), evoking their potential utility as therapies for metastatic cancers. Unfortunately, the direct use of these compounds in clinical settings is impeded by their limited natural abundance, challenging total synthesis, and detrimental effects on normal tissues. To develop potent analogues of these compounds that are simpler to synthesize and compatible with cell-specific targeting systems, such as antibodies, we designed over 20 analogues of the acyclic side chain (tail) of the macrolide Mycalolide B. These analogues probed the contributions of four distinct regions of the tail towards the inhibition of actin polymerization and ECM invasion by human lung cancer A549 cells. We observed that two of these regions tolerate considerable substituent variability, and we identified a specific combination of substituents that leads to the optimal inhibition of the ECM invasion activity of A549 cells.

Inhibiting F-Actin Polymerization Impairs the Internalization of Moraxella catarrhalis.

Moraxella catarrhalis, a commensal in the human nasopharynx, plays a significant role in the acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Its pathogenicity involves adherence to respiratory epithelial cells, leading to infection through a macropinocytosis-like mechanism. Previous investigations highlighted the diverse abilities of M. catarrhalis isolates with different phenotypes to adhere to and invade respiratory epithelial cells. This study used a murine COPD model and in vitro experiments to explore the factors influencing the pathogenicity of distinct phenotypes of M. catarrhalis. Transcriptome sequencing suggested a potential association between actin cytoskeleton regulation and the infection of lung epithelial cells by M. catarrhalis with different phenotypes. Electron microscopy and Western blot analyses revealed a decrease in filamentous actin (F-actin) expression upon infection with various M. catarrhalis phenotypes. Inhibition of actin polymerization indicated the involvement of F-actin dynamics in M. catarrhalis internalization, distinguishing it from the adhesion process. Notably, hindering F-actin polymerization impaired the internalization of M. catarrhalis. These findings contribute vital theoretical insights for developing preventive strategies and individualized clinical treatments for AECOPD patients infected with M. catarrhalis. The study underscores the importance of understanding the nuanced interactions between M. catarrhalis phenotypes and host lung epithelial cells, offering valuable implications for the management of AECOPD infections.

SARS-CoV-2 NSP2 as a Potential Delivery Vehicle for Proteins.

The development of biomolecule delivery systems is essential for the treatment of various diseases such as cancer, immunological diseases, and metabolic disorders. For the first time, we found that SARS-CoV-2-encoded nonstructural protein 2 (NSP2) can be secreted from the cells, where it is synthesized. Brefeldin A and H89, inhibitors of ER/Golgi secretion pathways, did not inhibit NSP2 secretion. NSP2 is likely secreted via an unconventional secretory pathway. Moreover, both secreted and purified NSP2 proteins were able to traverse the plasma membrane barrier and enter both immortalized human umbilical vein endothelial cells and tumor cell lines. After entry, the NSP2 protein was localized in only the cytoplasm. Cytochalasin D, a potent inhibitor of actin polymerization, inhibited the entry of NSP2. NSP2 can carry other molecules into cells. Burkholderia lethal factor 1, a monomeric toxin from the intracellular pathogen Burkholderia pseudomallei, has demonstrated antitumor activity by targeting host eukaryotic initiation translation factor 4A. An NSP2-BLF1 fusion protein was translocated across the cellular membranes of Huh7 cells and mediated cell killing. By using different approaches, including protein purification, chemical inhibition, and cell imaging, we confirm that NSP2 is able to deliver heterologous proteins into cells. NSP2 can act as a potential delivery vehicle for proteins.

Dual Control of Host Actin Polymerization by a Legionella Effector Pair

Host actin cytoskeleton is often targeted by pathogenic bacteria through the secretion of effectors. Legionella pneumophila virulence relies on the injection of the largest known arsenal of bacterial proteins, over 300 Dot/Icm type 4 secretion system effectors, into the host cytosol. Here, we define the functional interactions between VipA and LegK2, two effectors with antagonistic activities towards actin polymerization that have been proposed to interfere with the endosomal pathway. We confirmed the prominent role of LegK2 effector in Legionella infection, as the deletion of legK2 results in defects in the inhibition of actin polymerization at the Legionella-containing vacuole, as well as in endosomal escape of bacteria and subsequent intracellular replication. More importantly, we observed the restoration of the ΔlegK2 mutant defects, upon deletion of vipA gene, making LegK2/VipA a novel example of effector-effector suppression pair that targets the actin cytoskeleton and whose functional interaction impacts L. pneumophila virulence. We demonstrated that LegK2 and VipA do not modulate each other’s activity in a “metaeffector” relationship. Instead, the antagonistic activities of the LegK2/VipA effector pair would target different substrates, Arp2/3 for LegK2 and G-actin for VipA, to temporally control actin polymerization at the LCV and interfere with phagosome maturation and endosome recycling, thus contributing to the intracellular life cycle of the bacterium. Strikingly, the functional interaction between LegK2 and VipA is consolidated by an evolutionary history that has refined the best effector repertoire for the benefit of L. pneumophila virulence.

Myeloid leukemia-derived galectin-1 downregulates CAR expression to hinder cytotoxicity of CAR T cells

BackgroundChimeric antigen receptor (CAR) T cells have shown significant activity in B-lineage malignancies. However, their efficacy in myeloid leukemia has not been successful due to unclear molecular mechanisms.MethodsWe conducted in vitro and in vivo experiments to investigate whether myeloid leukemia cells directly induce CAR down-regulation. Furthermore, we designed a CD33 CARKR in which all lysines in the cytoplasmic domain of CAR were mutated to arginine and verified through in vitro experiments that it could reduce the down-regulation of surface CARs and enhance the killing ability. Transcriptome sequencing was performed on various AML and ALL cell lines and primary samples, and the galectin-1-specific inhibitory peptide (anginex) successfully rescued the killing defect and T-cell activation in in vitro assays.ResultsCAR down-regulation induced by myeloid leukemia cells under conditions of low effector-to-tumor ratio, which in turn impairs the cytotoxicity of CAR T cells. In contrast, lysosomal degradation or actin polymerization inhibitors can effectively alleviate CAR down-regulation and restore CAR T cell-mediated anti-tumor functions. In addition, this study identified galectin-1 as a critical factor used by myeloid leukemia cells to induce CAR down-regulation, resulting in impaired T-cell activation.ConclusionThe discovery of the role of galectin-1 in cell surface CAR down-regulation provides important insights for developing strategies to restore anti-tumor functions.

Synthesis of cytochalasan analogues with aryl substituents at position 10.

Cytochalasans are fungal metabolites that are known to inhibit actin polymerization. Despite their remarkable bioactivity, there are few studies on the structure-activity relationship (SAR) of the cytochalasan scaffold. The full potential of structural modifications remains largely unexplored. The substituent at position 10 of the cytochalasan scaffold is derived from an amino acid incorporated into the cytochalasan core, thus limiting the structural variability at this position in natural products. Additionally, modifications at this position have only been achieved through semisynthetic or mutasynthetic approaches using modified amino acids. This paper introduces a modular approach for late-stage modifications at position 10 of the cytochalasan scaffold. Iron-mediated cross-coupling reactions with corresponding Grignard reagents were used to introduce aryl or benzyl groups in position 10, resulting in the synthesis of six new cytochalasan analogues bearing non-natural aromatic residues. This methodology enables further exploration of modifications at this position and SAR studies among cytochalasan analogues.

A risk variant in TTC3 modifies the actin cytoskeleton organization and PI3K‐Akt signaling in iPSC‐derived forebrain neurons

AbstractBackgroundWe identified a rare, nonsynonymous variant in the tetratricopeptide repeat domain 3 (TTC3) gene that segregated in a non‐Hispanic white late onset Alzheimer disease (LOAD) family (Kohli, et al, 2016). This missense alteration, rs377155188 (p.S1038C), is predicted to be deleterious and is extremely rare. Studies have reported that cortical TTC3 expression is reduced in LOAD patients and negatively correlated with AD neuropathology.MethodTo understand the mechanism by which the TTC3 p.S1038C may contribute to LOAD risk, CRISPR/Cas9 genome edited induced pluripotent stem cells (iPSCs) were developed that were homozygous for the variant to examine cellular and transcriptional consequences in iPSC‐derived neuronal cells (Laverde‐Paz, et al, 2021).ResultQuantitative PCR and western blot analysis demonstrated that TTC3 levels were decreased in edited compared to unedited iPSCs, as well as differentiated neurons. In growing neuronal precursor cells (NPCs), cells with the TTC3 variant recovered more quickly from a scratch wound. Since there is evidence that modulation of TTC3 affects neurite growth, morphological measures of axon formation were assessed using the Incucyte Zoom. Studies demonstrate an increase in neurite outgrowth, which phenotypically corresponds with previous studies of a decrease in TTC3 function. This phenotype was tempered by treatment with Cytochalasin D, an inhibitor of actin polymerization. Additionally, TTC3 ubiquitinates phosphorylated AKT and regulates AKT signaling. The edited cells were found to have an increase in phosphorylated AKT (pAKT) relative to total AKT. RNA‐seq of day 70 neurons identified 979 genes that were differentially expressed (FDR<0.05). This included known AD genes (BACE1) and genes in AD GWAS loci (ADAMTS1, MAF, NCK2). KEGG pathway analysis identified differential expression in PI3K‐Akt signaling pathway components, in which TTC3 has been previously implicated, as well as the axon guidance pathway, the GABAergic synapse pathway, and the Wnt signaling pathway.ConclusionCombined, these results suggest that the TTC3 p.S1038C variant causes a loss of function. Utilizing a CRISPR genome edited iPSC carrying a homozygous alteration in TTC3, we were able to identify potential mechanisms by which TTC3 may contribute to LOAD risk.

Mechanobiology of Megakaryocyte-Driven Contraction of Plasma Clots

Background: Contractility of non-muscle cells plays a crucial role in various cellular processes including motility, morphogenesis, division, genome replication, intracellular transport, and secretion. Platelet-induced blood clot contraction is an essential process which plays a role in preventing bleeding and in thrombotic disorders. Megakaryocytes (MKs), which are the precursor cells of platelets, share many proteins and structures with platelets. However, little is known about the ability of MKs to contract and interact with extracellular matrix proteins such as fibrin. Previously, the non-muscle myosin II contractility was shown to be essential for MK migration within the bone marrow, to avoid premature proplatelet formation, and to allow branching of proplatelets. Recent studies have identified MKs in lung and brain tissues, as well as associated with thrombi in deceased COVID-19 patients. Notably, patients with severe COVID-19 exhibit elevated MK counts in their blood, indicating potential involvement in hemostasis and thrombosis. Nevertheless, the specific mechanisms by which MKs can contribute to clot contraction remain unknown. Methods: Human induced pluripotent stem cells (iPSC) were differentiated into HPCs followed by expansion to megakaryocytes (iMKs) and analyzed by flowcytometry. Clot formation and contraction were initiated by adding thrombin and calcium to platelet-free-plasma with resuspended iMKs. A combination of real-time macroscale optical tracking, high-resolution confocal microscopy and biomechanical measurements were applied to elucidate the contractile cellular mechanisms and mechanotransduction pathways in iMKs, and ultimately comparing them with platelets. Results: Flowcytometry analysis of unstimulated and thrombin-stimulated iMKs revealed that the major fraction of unstimulated cells expressed αIIbβ3 and GPIbα (~78%), but did not bind PAC-1, a monoclonal antibody specific for the activated conformation of αIIbβ3. Stimulation of iMKs with 1U/ml of thrombin resulted in PAC-1 binding (~68%), indicating activation of aIIbb3. The optical tracking of clot size change over time revealed that thrombin-stimulated iMKs were able to shrink macroscopic plasma clots with biphasic kinetics, similar to platelets. iMK-containing clots and platelet-reach-plasma clots equalized by the total cell surface area demonstrated comparable contraction rates and extent of contraction. The average maximal contractile force per individual iMK in the clot was found to be 145±98nN. In the presence of blebbistatin, the mean final extent and velocity of clot contraction of iMK-containing clots reduced in a dose-dependent manner, in comparison to untreated clots. Pretreatment of iMKs with 1μM latrunculin A, an inhibitor of actin polymerization, completely prevented contraction of plasma clots. In addition, contraction of plasma clots by activated iMKs was abrogated by 10μg/ml abciximab, that prevented binding of fibrin(ogen) to integrin a IIbb 3 receptors. The structural mechanisms of iMK contractility involved formation of filopodia and larger cytoplasmic protrusions undergoing extension-retraction cycles after being attached to surrounding fibrin fibers. Contraction of iMKs caused remodeling of the extracellular fibrin matrix by inducing spatial reorientation of fibrin fibers, accumulation of the fibrin mass on the iMK surface, and compaction of the entire fibrin network. Conclusions: We have demonstrated a hitherto unknown ability of individual MKs to shrink fibrin clots and deciphered the cellular mechanisms of contractility of iPSC-derived MKs. The molecular mechanisms of MK-driven fibrin clot shrinkage were shown to be similar or identical to those of activated platelets, involving non-muscle myosin II activity, actin polymerization, and integrin αIIbβ3-fibrin interactions. Our findings provide a novel mechanistic insight into mechanobiology of MKs that may play a role in modulating the properties of hemostatic blood clots and thrombi. In addition, iMKs can be used as model cells with a potential of genetic modifications to study platelet structure and function, including their contractility.

Trogocytosis-Resistant CAR T Cells Exhibit Increased Persistence and Prevent Antigen Escape

Chimeric Antigen Receptor (CAR) T cell therapy is a highly effective treatment for cancer patients in which T cells are genetically modified to express a CAR targeting an antigen expressed on the surface of cancer cells. While CAR T cells have shown remarkable efficacy, many patients receiving CAR T cell therapy relapse within five years of treatment. It has recently been shown that tumor cells can escape killing by CAR T cells using a mechanism called trogocytosis. CAR-mediated trogocytosis (CMT) involves the transfer of cell surface proteins from tumor cells to CAR T cells. Antigen transfer by CMT results in antigen-negative tumor cells and reduced CAR T cell persistence due to increased CAR T cell exhaustion and fratricide. To date, the molecular processes causing CMT remain poorly understood and there are currently no approaches to therapeutically target CMT. To track CMT in real-time, we first developed a custom luciferase complementation assay with a detection limit of approximately 100 CAR T cells displaying the target antigen (CompLuc). Using this assay, we found that CMT is effector-target ratio and time-dependent and can be modulated by inhibiting actin polymerization, a process essential for key T cell functions including vesicle formation, protein transport, and target cell killing. To develop a universal approach to inhibit CMT in CAR T cells without altering tumor cell killing, we investigated the effect of inhibiting adhesion, endocytosis, vesicle formation, and proteolytic degradation using various small-molecule inhibitors. We found that inhibition of the cysteine protease Cathepsin B with the small molecule inhibitor Ca-074-Me significantly reduced CMT as determined by CompLuc assay and flow cytometry. Performing a luciferase-based cytotoxicity assay, we found that Cathepsin B inhibition does not affect tumor cell killing. Based on these data, we hypothesized that Cathepsin B inhibition could be a potential therapeutic approach to limit CMT. Cathepsin B activity in human cells is regulated by the human proteins Cystatin A and Cystatin B. Structural data of Cathepsin B in complex with Ca-074-Me or Cystatin A show that both molecules appear to sterically block access of substrates to the active site cysteine of Cathepsin B. We therefore explored if overexpression of cystatins in CAR T cells could be an efficient approach to inhibit CMT. We found that overexpression of Cystatin A significantly reduced Cathepsin B activity in CAR T cells and limited transfer of tumor antigen to CAR T cells. We also observed that cystatin overexpression reduced loss of antigen from the tumor cell surface, indicating that Cathepsin B inhibition blocks CMT at an early stage of the trogocytic process. Importantly, Cystatin A overexpression did not alter CAR T cell expansion, tumor cell killing, or CAR T cell phenotype, indicating that this approach selectively inhibits CMT and that key CAR T cell functions remain intact. Cystatin overexpression also led to significantly increased tumor-specific CAR T cell numbers (78% of non-targeting CAR T cells) compared to wild-type CAR T cells (61% of non-targeting CAR T cells, p = 0.0298) after 24 h exposure to tumor cells, indicating that selective inhibition of CMT prevents CAR T cell fratricide and/or exhaustion. To further reduce presence of antigen on CAR T cells following CMT, we next developed a system to actively degrade the trogocytosed protein in CAR T cells. Specifically, we developed a fusion protein combining the SH2 domain of the Src kinase Lyn, which binds with high affinity to the intracellular domain of CD19, with the von Hippel-Lindau E3 ubiquitin ligase. We show that expression of this fusion protein within CAR T cells rapidly targets trogocytosed CD19 for degradation and eliminates its presence on the surface of CAR T cells. Trogocytosed antigen degradation also increased CAR T cell survival at the end of in vitro co-culture, indicating reduced CAR T cell fratricide and/or exhaustion. Taken together, we provide the first preclinical proof-of-concept that, using a fully human genetic engineering approach, CMT can be targeted without compromising essential CAR T cell functions. This represents a promising approach to improve CAR T cell efficacy and limit the occurrence of relapse in patients receiving CAR T cell therapy.