Disruption Of Cytoskeleton Research Articles

Actin Dysregulation Induces Neuroendocrine Plasticity and Immune Evasion: A Vulnerability of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is aggressive with limited therapeutic options. Despite recent advances in targeted therapies and immunotherapies, therapy resistance is a recurring issue, which might be partly due to tumor cell plasticity, a change in cell fate. Nonetheless, the mechanisms underlying tumor cell plasticity and immune evasion in SCLC remain elusive. CRACD, a capping protein inhibitor that promotes actin polymerization, is frequently inactivated in SCLC. Cracd knockout (KO) transforms preneoplastic cells into SCLC tumor-like cells and promotes in vivo SCLC development driven by Rb1, Trp53, and Rbl2 triple KO. Cracd KO induces neuroendocrine (NE) plasticity and increases tumor cell heterogeneity of SCLC tumor cells via dysregulated NOTCH1 signaling by actin cytoskeleton disruption. CRACD depletion also reduces nuclear actin and induces EZH2-mediated H3K27 methylation. This nuclear event suppresses the MHC-I genes and thereby depletes intratumoral CD8+ T cells for accelerated SCLC tumorigenesis. Pharmacological blockade of EZH2 inhibits CRACD-negative SCLC tumorigenesis by restoring MHC-I expression and immune surveillance. Unsupervised single-cell transcriptomics identifies SCLC patient tumors with concomitant inactivation of CRACD and downregulated MHC-I pathway. This study defines CRACD, an actin regulator, as a tumor suppressor that limits cell plasticity and immune evasion and proposes EZH2 blockade as a viable therapeutic option for CRACD-negative SCLC.

Selection of Force Sensors for In Situ Measurement of Neotissue Microenvironments.

Mechanical forces are a critical stimulus in both native and engineered tissues. Direct measurement of these microenvironmental forces has been challenging, particularly for cell-dense models. To address this, we previously developed hydrogel-based force sensors that are approximately the size of a cell and can be imaged over time to computationally assess the forces exerted by surrounding cells and matrix. The goal of this project was to identify how the physical characteristics of force sensors impact measurements. Sensors were varied in size, elastic modulus, and surface coating before being included in stem cell suspensions that then spontaneously self-assembled into spheroidal neotissues. Using this model of early mesenchymal condensation, we hypothesized that larger, softer sensors would provide greater sensitivity and precision, whereas protein coatings would influence the directionality of applied forces (tensile vs. compressive). These experiments were conducted using a high-content imaging system that allowed analysis of over a thousand sensors to evaluate the various conditions. Results indicated that measurement fidelity was highest for force sensors that had a diameter >20 µm and modulus ∼0.2 kPa. Extremely soft sensors deformed too much, whereas stiffer sensors deformed too little. Collagen and N-cadherin coatings, which replicated cell-matrix or cell-cell binding, respectively, allowed for tensile forces to be exerted on the sensors, with greater forces being observed for N-cadherin sensors in these highly cellular neotissue constructs. Uncoated sensors were universally compressed due to the lack of cell-sensor adhesion. Disruption of the actin cytoskeleton lessened microenvironmental forces, whereas disruption of microtubules had no measurable effect. Potential future applications of the technology include studies of in situ forces in developing tissues as well as a real-time sensor for monitoring the growth of engineered constructs.

Lassa virus Z protein hijacks the autophagy machinery for efficient transportation by interrupting CCT2-mediated cytoskeleton network formation

ABSTRACT The Lassa virus (LASV) is a widely recognized virulent pathogen that frequently results in lethal viral hemorrhagic fever (VHF). Earlier research has indicated that macroautophagy/autophagy plays a role in LASV replication, but, the precise mechanism is unknown. In this present study, we show that LASV matrix protein (LASV-Z) is essential for blocking intracellular autophagic flux. LASV-Z hinders actin and tubulin folding by interacting with CCT2, a component of the chaperonin-containing T-complexes (TRiC). When the cytoskeleton is disrupted, lysosomal enzyme transit is hampered. In addition, cytoskeleton disruption inhibits the merge of autophagosomes with lysosomes, resulting in autophagosome accumulation that promotes the budding of LASV virus-like particles (VLPs). Inhibition of LASV-Z-induced autophagosome accumulation blocks the LASV VLP budding process. Furthermore, it is found that glutamine at position 29 and tyrosine at position 48 on LASV-Z are important in interacting with CCT2. When these two sites are mutated, LASV-mut interacts with CCT2 less efficiently and can no longer inhibit the autophagic flux. These findings demonstrate a novel strategy for LASV-Z to hijack the host autophagy machinery to accomplish effective transportation. Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf-A1: bafilomycin A1; CCT2: chaperonin containing TCP1 subunit 2; co-IP: co-immunoprecipitation; CTSD: cathepsin D; DAPI: 4’,6-diamidino-2’-phenylindole; DMSO: dimethyl sulfoxide; EGFR: epidermal growth factor receptor; GFP: green fluorescent protein; hpi: hours post-infection; hpt: hours post-transfection; LAMP1: lysosomal-associated membrane protein 1; LASV: lassa virus; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry: red fluorescent protein; PM: plasma membrane; SQSTM1/p62: sequestosome 1; STX6: syntaxin 6; VLP: virus-like particle; TEM: transmission electron microscopy; TRiC: chaperonin-containing T-complex; WB: western blotting; μm: micrometer; μM: micromole.

Epithelial UNC-23 limits mechanical stress to maintain glia-neuron architecture in C. elegans

For an organ to maintain correct architecture and function, its diverse cellular components must coordinate their size and shape. Although cell-intrinsic mechanisms driving homotypic cell-cell coordination are known, it is unclear how cell shape is regulated across heterotypic cells. We find that epithelial cells maintain the shape of neighboring sense-organ glia-neuron units in adult Caenorhabditis elegans (C.elegans). Hsp co-chaperone UNC-23/BAG2 prevents epithelial cell shape from deforming, and its loss causes head epithelia to stretch aberrantly during animal movement. In the sense-organ glia, amphid sheath (AMsh), this causes progressive fibroblast growth factor receptor (FGFR)-dependent disruption of the glial apical cytoskeleton. Resultant glial cell shape alteration causes concomitant shape change in glia-associated neuron endings. Epithelial UNC-23 maintenance of glia-neuron shape is specific both spatially, within a defined anatomical zone, and temporally, in a developmentally critical period. As all molecular components uncovered are broadly conserved across central and peripheral nervous systems, we posit that epithelia may similarly regulate glia-neuron architecture cross-species.

Nicotinamide mononucleotide maintains cytoskeletal stability and fortifies mitochondrial function to mitigate oocyte damage induced by Triocresyl phosphate

Triocresyl phosphate (TOCP) was commonly used as flame retardant, plasticizer, lubricant, and jet fuel additive. Studies have shown adverse effects of TOCP on the reproductive system. However, the potential harm brought by TOCP, especially to mammalian female reproductive cells, remains a mystery. In this study, we employed an in vitro model for the first time to investigate the effects of TOCP on the maturation process of mouse oocytes. TOCP exposure hampered the meiotic division process, as evidenced by a reduction in the extrusion of the first polar body from oocytes. Subsequent research revealed the disruption of the oocyte cell cytoskeleton induced by TOCP, resulting in abnormalities in spindle organization, chromosome alignment, and actin filament distribution. This disturbance further extended to the rearrangement of organelles within oocytes, particularly affecting the mitochondria. Importantly, after TOCP treatment, mitochondrial function in oocytes was impaired, leading to oxidative stress, DNA damage, cell apoptosis, and subsequent changes of epigenetic modifications. Supplementation with nicotinamide mononucleotide (NMN) alleviated the harmful effects of TOCP. NMN exerted its mitigating effects through two fundamental mechanisms. On one hand, NMN conferred stability to the cell cytoskeleton, thereby supporting nuclear maturation. On the other hand, NMN enhanced mitochondrial function within oocytes, reducing the excess reactive oxygen species (ROS), restoring meiotic division abnormalities caused by TOCP, preventing oocyte DNA damage, and suppressing epigenetic changes. These findings not only enhance our understanding of the molecular basis of TOCP induced oocyte damage but also offer a promising avenue for the potential application of NMN in optimizing reproductive treatment strategies.

Design, synthesis and biological evaluation of new 2,6-difluorinated phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates as new antimicrotubule agents

We previously discovered a novel family of antimicrotubule agents designated as phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs). In this study, we evaluated the effect of the difluorination of the aromatic ring bearing the imidazolidin-2-one moiety (ring A) at positions 3, 5 and 2, 6 on their antiproliferative activity on four cancer cell lines, their ability to disrupt the microtubules and their toxicity toward chick embryos. We thus synthesized, characterized and biologically evaluated 24 new difluorinated PIB-SO derivatives designated as phenyl 3,5-difluoro-4-(2-oxoimidazolidin-1-yl)benzenesulfonates (3,5-PFB-SOs, 4–15) and phenyl 2,6-difluoro-4-(2-oxoimidazolidin-1-yl)benzenesulfonates (2,6-PFB-SOs, 16–27). The concentration of the drug required to inhibit cell growth by 50% (IC50) of 3,5-PFB-SOs is over 1000 nM while most of 2,6-PFB-SOs exhibit IC50 in the nanomolar range (23–900 nM). Furthermore, the most potent 2,6-PFB-SOs 19, 26 and 27 arrest the cell cycle progression in G2/M phase, induce cytoskeleton disruption and impair microtubule polymerization. Docking studies also show that the most potent 2,6-PFB-SOs 19, 21, 24, 26 and 27 have binding affinity toward the colchicine-binding site (C-BS). Moreover, their antiproliferative activity is not affected by antimicrotubule- and multidrug-resistant cell lines. Besides, they exhibit improved in vitro hepatic stability in the mouse, rat and human microsomes compared to their non-fluorinated counterparts. They also showed theoretical pharmacokinetic, physicochemical and drug-like properties suited for further in vivo assays. In addition, they exhibit low to no systemic toxicity toward chick embryos. Finally, our study evidences that PIB-SOs must be fluorinated in specific positions on ring A to maintain both their antiproliferative activity and their biological activity toward microtubules.

The redox-sensitive GSK3β is a key regulator of glomerular podocyte injury in type 2 diabetic kidney disease

Emerging evidence suggests that GSK3β, a redox-sensitive transducer downstream of insulin signaling, acts as a convergent point for myriad pathways implicated in kidney injury, repair, and regeneration. However, its role in diabetic kidney disease remains controversial. In cultured glomerular podocytes, exposure to a milieu of type 2 diabetes elicited prominent signs of podocyte injury and degeneration, marked by loss of homeostatic marker proteins like synaptopodin, actin cytoskeleton disruption, oxidative stress, apoptosis, and stress-induced premature senescence, as shown by increased staining for senescence-associated β-galactosidase activity, amplified formation of γH2AX foci, and elevated expression of mediators of senescence signaling, like p21 and p16INK4A. These degenerative changes coincided with GSK3β hyperactivity, as evidenced by GSK3β overexpression and reduced inhibitory phosphorylation of GSK3β, and were averted by tideglusib, a highly-selective small molecule inhibitor of GSK3β. In agreement, post-hoc analysis of a publicly-available glomerular transcriptomics dataset from patients with type 2 diabetic nephropathy revealed that the curated diabetic nephropathy-related gene set was enriched in high GSK3β expression group. Mechanistically, GSK3β-modulated nuclear factor Nrf2 signaling is involved in diabetic podocytopathy, because GSK3β knockdown reinforced Nrf2 antioxidant response and suppressed oxidative stress, resulting in an improvement in podocyte injury and senescence. Conversely, ectopic expression of the constitutively active mutant of GSK3β impaired Nrf2 antioxidant response and augmented oxidative stress, culminating in an exacerbated diabetic podocyte injury and senescence. Moreover, IRS-1 was found to be a cognate substrate of GSK3β for phosphorylation at IRS-1S332, which negatively regulates IRS-1 activity. GSK3β hyperactivity promoted IRS-1 phosphorylation, denoting a desensitized insulin signaling. Consistently, in vivo in db/db mice with diabetic nephropathy, GSK3β was hyperactive in glomerular podocytes, associated with IRS-1 hyperphosphorylation, impaired Nrf2 response and premature senescence. Our finding suggests that GSK3β is likely a novel therapeutic target for treating type 2 diabetic glomerular injury.

Inhibitory effects of kaempferol, quercetin and luteolin on the replication of human parainfluenza virus type 2 in vitro.

The eight flavonoids, apigenin, chrysin, hesperidin, kaempferol, myricetin, quercetin, rutin and luteolin were tested for the inhibition of human parainfluenza virus type 2 (hPIV-2) replication. Three flavonoids out of the eight, kaempferol, quercetin and luteolin inhibited hPIV-2 replication. Kaempferol reduced the virus release (below 1/10,000), partly inhibited genome and mRNA syntheses, but protein synthesis was observed. It partly inhibited virus entry into the cells and virus spreading, and also partly disrupted microtubules and actin microfilaments, indicating that the virus release inhibition was partly caused by the disruption of cytoskeleton. Quercetine reduced the virus release (below 1/10,000), partly inhibited genome, mRNA and protein syntheses. It partly inhibited virus entry and spreading, and also partly destroyed microtubules and microfilaments. Luteolin reduced the virus release (below 1/100,000), largely inhibited genome, mRNA and protein syntheses. It inhibited virus entry and spreading. It disrupted microtubules and microfilaments. These results indicated that luteolin has the most inhibitory effect on hPIV-2 relication. In conclusion, the three flavonoids inhibited virus replication by the inhibition of genome, mRNA and protein syntheses, and in addition to those, by the disruption of cytoskeleton in vitro.

Immortalised murine R349P desmin knock-in myotubes exhibit a reduced proton leak and decreased ADP/ATP translocase levels in purified mitochondria

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.

Chemical Proteomics Reveals Nε-Fatty-Acylation of Septins by Rho Inactivation Domain (RID) of the Vibrio MARTX Toxin to Alter Septin Localization and Organization

Vibrio species, the Gram-negative bacterial pathogens causing cholera and sepsis, produce multiple secreted virulence factors for infection and pathogenesis. Among these is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin that releases several critical effector domains with distinct functions inside eukaryotic host cells. One such effector domain, the Rho inactivation domain (RID), has been discovered to catalyze long-chain Nε-fatty-acylation on lysine residues of Rho GTPases, causing inactivation of Rho GTPases and disruption of the host actin cytoskeleton. However, whether RID modifies other host proteins to exert additional functions remains to be determined. Herein, we describe the integration of bioorthogonal chemical labeling and quantitative proteomics to globally profile the target proteins modified by RID in living cells. More than 246 proteins are identified as new RID substrates, including many involved in GTPase regulation, cytoskeletal organization, and cell division. We demonstrate that RID extensively Nε-fatty-acylates septin proteins, the fourth cytoskeletal component of mammalian cells with important roles in diverse cellular processes. While affinity purification and mass spectrometry analysis show that RID-mediated Nε-fatty-acylation does not affect septin-septin interactions, this modification increases the membrane association of septins and confers localization to detergent-resistant membrane rafts. As a result, the filamentous assembly and organization of septins are disrupted by RID-mediated Nε-fatty-acylation, further contributing to cytoskeletal and mitotic defects that phenocopy the effects of septin depletion. Overall, our work greatly expands the substrate scope and function of RID and demonstrates the role of RID-mediated Nε-fatty-acylation in manipulating septin localization and organization.

F-actin coordinates spindle morphology and function in Drosophila meiosis.

Meiosis is a highly conserved feature of sexual reproduction that ensures germ cells have the correct number of chromosomes prior to fertilization. A subset of microtubules, known as the spindle, are essential for accurate chromosome segregation during meiosis. Building evidence in mammalian systems has recently highlighted the unexpected requirement of the actin cytoskeleton in chromosome segregation; a network of spindle actin filaments appear to regulate many aspects of this process. Here we show that Drosophila oocytes also have a spindle population of actin that appears to regulate the formation of the microtubule spindle and chromosomal movements throughout meiosis. We demonstrate that genetic and pharmacological disruption of the actin cytoskeleton has a significant impact on spindle morphology, dynamics, and chromosome alignment and segregation during maturation and the metaphase-anaphase transition. We further reveal a role for calcium in maintaining the microtubule spindle and spindle actin. Together, our data highlights potential conservation of morphology and mechanism of the spindle actin during meiosis.

Microtubule-Targeting Agents: Disruption of the Cellular Cytoskeleton as a Backbone of Ovarian Cancer Therapy.

Microtubules are dynamic polymers composed of α- and β-tubulin heterodimers. Microtubules are universally conserved among eukaryotes and participate in nearly every cellular process, including intracellular trafficking, replication, polarity, cytoskeletal shape, and motility. Due to their fundamental role in mitosis, they represent a classic target of anti-cancer therapy. Microtubule-stabilizing agents currently constitute a component of the most effective regimens for ovarian cancer therapy in both primary and recurrent settings. Unfortunately, the development of resistance continues to present a therapeutic challenge. An understanding of the underlying mechanisms of resistance to microtubule-active agents may facilitate the development of novel and improved approaches to this disease.

Cellular Uptake and Cytotoxicity of Clostridium perfringens Iota-Toxin.

Clostridium perfringens iota-toxin is composed of two separate proteins: a binding protein (Ib) that recognizes a host cell receptor and promotes the cellular uptake of a catalytic protein and (Ia) possessing ADP-ribosyltransferase activity that induces actin cytoskeleton disorganization. Ib exhibits the overall structure of bacterial pore-forming toxins (PFTs). Lipolysis-stimulated lipoprotein receptor (LSR) is defined as a host cell receptor for Ib. The binding of Ib to LSR causes an oligomer formation of Ib in lipid rafts of plasma membranes, mediating the entry of Ia into the cytoplasm. Ia induces actin cytoskeleton disruption via the ADP-ribosylation of G-actin and causes cell rounding and death. The binding protein alone disrupts the cell membrane and induces cytotoxicity in sensitive cells. Host cells permeabilized by the pore formation of Ib are repaired by a Ca2+-dependent plasma repair pathway. This review shows that the cellular uptake of iota-toxin utilizes a pathway of plasma membrane repair and that Ib alone induces cytotoxicity.

High-Glucose-Induced Injury to Proximal Tubules of the Renal System Is Alleviated by Netrin-1 Suppression of Akt/mTOR.

The kidneys have a high level of Netrin-1 expression, which protects against some acute and chronic kidney disorders. However, it is yet unknown how Netrin-1 affects renal proximal tubule cells in diabetic nephropathy (DN) under pathological circumstances. Research has shown that autophagy protects the kidneys in animal models of renal disease. In this study, we looked at the probable autophagy regulation mechanism of Netrin-1 and its function in the pathogenesis of DN. We proved that in HK-2 cell, high blood sugar levels caused Netrin-1 to be downregulated, which then triggered the Akt/mTOR signaling pathway and enhanced cell death and actin cytoskeleton disruption. By adding Netrin-1 or an autophagy activator in vitro, these pathogenic alterations were reverted. Our results indicate that Netrin-1 stimulates autophagy by blocking the Akt/mTOR signaling pathway, which underlies high-glucose-induced malfunction of the renal proximal tubules. After HK-2 cells were incubated with Netrin-1 recombination protein and rapamycin under HG conditions for 24 h, the apoptosis was significantly reduced, as shown by the higher levels of Bcl-2, as well as lower levels of Bax and cleaved caspase-3 (P = 0.012, Cohen's d = 0.489, Glass's delta = 0.23, Hedges' g = 0.641). This study reveals that targeting Netrin-1-related signaling has therapeutic potential for DN and advances our knowledge of the processes operating in renal proximal tubules in DN.

Mechanism of synergistic inhibitory effect of benzyl isothiocyanate and zoledronic acid combination on breast cancer induction of osteoclast differentiation.

Bone is the most favored site for metastasis for each major subtype of breast cancer. Therapeutic modalities for alleviation of clinical symptoms associated with bone metastasis include surgical resection, radiation, and bone-targeted therapies, including bisphosphonates (e.g., zoledronic acid; ZA) and a humanized antibody against receptor activator of nuclear factor-κB ligand (denosumab). However, the bone-targeted therapies are expensive, and have poor pharmacokinetic attributes and/or serious adverse effects. Therefore, novel strategies are needed for treatment of bone metastasis or to increase effectiveness of existing bone-targeted therapies. We have shown previously that benzyl isothiocyanate (BITC) is a novel inhibitor of osteoclast differentiation in vitro and bone metastasis in vivo. The present study shows that BITC + ZA combination synergistically inhibits osteoclast differentiation induced by addition of conditioned media from breast cancer cells. These effects were associated with a significant increase in levels of several antiosteoclastogenic cytokines, including interferons, interleukin (IL)-3, IL-4, and IL-27. Kyoto Encyclopedia of Genes and Genomes pathway analysis of RNA-seq data from BITC and/or ZA-treated cells revealed downregulation of genes of many pathways (e.g., actin cytoskeleton, Hippo signaling, etc.) by treatment with BITC + ZA combination, but not by BITC alone or ZA alone. Confocal microscopy confirmed severe disruption of actin cytoskeleton upon treatment of MCF-7 and MDA-MB-231 cells with the BITC + ZA combination. This combination also decreased the nuclear level of yes-associated protein, a core component of Hippo signaling. In conclusion, the present study offers a novel combination for prevention or treatment of bone metastasis of breast cancer.

Retinoic acid and proteotoxic stress induce AML cell death overcoming stromal cell protection

BackgroundAcute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A).MethodsWe treated FLT3-ITD+ AML cells with low doses of the aforementioned drugs, used alone or in combinations and we investigated the induction of ER and oxidative stress. We then performed the same experiments in an in vitro co-culture system of FLT3-ITD+ AML cells and bone marrow stromal cells (BMSCs) to assess the protective role of the niche on AML blasts. Eventually, we tested the combination of drugs in an orthotopic murine model of human AML.ResultsThe combination RBA exerts strong cytotoxic activity on FLT3-ITD+ AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with BMSCs. Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD+ AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML and BMSCs upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSCs.ConclusionsOur findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD+ AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML and BMSCs, involving mechanotransduction and YAP signaling.

Evaluation of trabecular meshwork-specific promoters in vitro and in vivo using scAAV2 vectors expressing C3 transferase.

To evaluate the potential of two trabecular meshwork (TM)-specific promoters, Chitinase 3-like 1 (Ch3L1) and matrix gla protein (MGP), for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2 (scAAV2) vector technologies. An scAAV2 vector with C3 transferase (C3) as the reporter gene (scAAV2-C3) was selected. The scAAV2-C3 vectors were driven by Ch3L1 (scAAV2-Ch3L1-C3), MGP (scAAV2-MGP-C3), enhanced MGP (scAAV2-eMGP-C3) and cytomegalovirus (scAAV2-CMV-C3), respectively. The cultured primary human TM cells were treated with each vector at different multiplicities of infections. Changes in cell morphology were observed by phase contrast microscopy. Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining, real-time quantitative polymerase chain reaction and Western blot. Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively. In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope. Intraocular pressure (IOP) was monitored using a rebound tonometer. Ocular responses were evaluated by slit-lamp microscopy. Ocular histopathology analysis was examined by hematoxylin and eosin staining. In TM cell culture studies, the vector-mediated C3 expression induced morphologic changes, disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rho-associated protein kinase signaling pathway. At the same dose, these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3, but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3. At low-injected dose, the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGP-C3-injected and scAAV2-eMGP-C3-injected eyes. At high-injected dose, significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes. Similar to scAAV2-CMV-C3, scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium. In anterior segment tissues of scAAV2-eMGP-C3-injected eyes, no obvious morphological changes were found except for the TM. Inflammation was absent. In scAAV2-transduced TM cells, the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus, but obviously higher than that of MGP. In the anterior chamber of rat eye, the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter, but not by Ch3L1 promoter. These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.

HPV disrupt the cytoskeleton in oral squamous cell carcinomas from non-oropharyngeal sites via the E-cadherin/Mena/SMA pathway

In this paper, we aimed to evaluate the mechanism of actin cytoskeleton disruption, in oral squamous cell carcinoma (OSCC). A total of 43 patients with surgically resected OSCCs located in non-oropharyngeal regions were randomly selected. The expression of E-cadherin, β-catenin, smooth muscle actin (SMA), Mena, maspin, V-set and immunoglobulin domain containing 1 (VSIG1), β human chorionic gonadotropin (βhCG), and angiotensin-converting enzyme (ACE) was assessed via immunohistochemistry (IHC) and evaluated in association with the prevalence of high-risk human papillomavirus (HPV). Mena positivity (n = 30; 69.77%) was more frequent in poorly differentiated OSCC of the tongue and lips with high-risk HPV viral DNA and a lymph node ratio (LNR) ≤ 2.5. Loss of E-cadherin was more prevalent among poorly differentiated stage pT4N1 tumors with an LNR ≤ 2.5 and perineural invasion. These cases were classified as SMA-high tumors. Independent negative prognostic factors included high Mena expression, loss of E-cadherin, high SMA expression, and the presence of high-risk HPV. No VSIG1 positivity was observed. In conclusion, in non-oropharyngeal OSCC, cytoskeleton activity might be driven by the Mena/E-cadherin/SMA axis, reflecting active epithelial-mesenchymal interaction. High Mena intensity is an indicator of poorly differentiated carcinomas with high-risk HPV and unfavorable prognosis.

Aromatic amino acid biosynthesis impacts root hair development and symbiotic associations in Lotus japonicus.

Legume roots can be symbiotically colonized by arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria. In Lotus japonicus, the latter occurs intracellularly by the cognate rhizobial partner Mesorhizobium loti or intercellularly with the Agrobacterium pusense strain IRBG74. Although these symbiotic programs show distinctive cellular and transcriptome signatures, some molecular components are shared. In this study, we demonstrate that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase 1 (DAHPS1), the first enzyme in the biosynthetic pathway of aromatic amino acids (AAAs), plays a critical role in root hair development and for AM and rhizobial symbioses in Lotus. Two homozygous DAHPS1 mutants (dahps1-1 and dahps1-2) showed drastic alterations in root hair morphology, associated with alterations in cell wall dynamics and a progressive disruption of the actin cytoskeleton. The altered root hair structure was prevented by pharmacological and genetic complementation. dahps1-1 and dahps1-2 showed significant reductions in rhizobial infection (intracellular and intercellular) and nodule organogenesis and a delay in AM colonization. RNAseq analysis of dahps1-2 roots suggested that these phenotypes are associated with downregulation of several cell wall-related genes, and with an attenuated signaling response. Interestingly, the dahps1 mutants showed no detectable pleiotropic effects, suggesting a more selective recruitment of this gene in certain biological processes. This work provides robust evidence linking AAA metabolism to root hair development and successful symbiotic associations.

Measuring (biological) materials mechanics with atomic force microscopy. 5. Traction force microscopy (cell traction forces).

Cells generate traction forces to probe the mechanical properties of the surroundings and maintain a basal equilibrium state of stress. Traction forces are also implicated in cell migration, adhesion and ECM remodeling, and alteration of these forces is often observed in pathologies such as cancer. Thus, analyzing the traction forces is important for studies of cell mechanics in cancer and metastasis. In this primer, the methodology for conducting two-dimensional traction force microscopy (2D-TFM) experiments is reported. As a practical example, we analyzed the traction forces generated by three human breast cancer cell lines of different metastatic potential: MCF10-A, MCF-7 and MDA-MB-231 cells, and studied the effects of actin cytoskeleton disruption on those traction forces. Contrary to what is often reported in literature, lower traction forces were observed in cells with higher metastatic potential (MDA-MB-231). Implications of substrate stiffness and concentration of extracellular matrix proteins in such findings are discussed in the text. RESEARCH HIGHLIGHTS: Traction force microscopy (TFM) is suitable for studying and quantifying cell-substrate and cell-cell forces. TFM is suitable for investigating the relationship between chemical to mechanical signal transduction and vice versa. TFM can be combined with classical indentation studies providing a compact picture of cell mechanics. TFM still needs new physico-chemical (sample preparation) and computational approaches for more accurate data evaluation.