The microtubule protein βIII-tubulin is a prognostic, pro-survival, and chemoresistance factor in multiple malignancies, including pancreatic ductal adenocarcinoma (PDAC). However, the precise survival mechanisms controlled by βIII-tubulin in cancer remain unknown. Here, we discovered a link between βIII-tubulin and the activation of caspase 8-mediated extrinsic apoptosis. Silencing βIII-tubulin in PDAC cells activated caspase 8, leading to decreased cell viability and growth both in vitro and in vivo. βIII-tubulin knockdown also increased the sensitivity of PDAC cells to extrinsic cell death signals, including TNF-related apoptosis-inducing ligand (TRAIL), TNFα, and FasL. Furthermore, we demonstrated that βIII-tubulin knockdown in PDAC cells, in the absence or presence of TRAIL, increased diffusion and clustering of the TRAIL death receptor DR5 at the cell membrane, inducing extrinsic apoptosis. Nanoparticle delivery of βIII-tubulin siRNA to mouse PDAC tumours reduced tumour growth and increased responsiveness to TRAIL therapy. In patient-derived human PDAC explants, βIII-tubulin silencing reduced tumour cell frequency and improved sensitivity to TRAIL. Finally, we showed that high βIII-tubulin expression in the human PDAC stroma was independently prognostic for poor overall survival. Taken together, silencing βIII-tubulin represents an innovative strategy to activate a suicide signal in PDAC cells and render them more sensitive to microenvironment- and chemotherapy-derived death signals.

Genome-wide association studies (GWAS) link the Tropomyosin 1 (Tpm1) locus to quantitative blood trait variation, but related mechanisms are unclear. Tpm1 encodes an actin-binding protein that regulates actin filament diversity, cell adhesion, signaling, and actomyosin contractility. Murine Tpm1 deficiency enhances hemogenic endothelial cell (HEC) specification, but it was unclear if these effects extended to postnatal hematopoiesis. We used Cdh5Cre and VavCre models to conditionally knock out Tpm1 (Tpm1KO) in endothelial anor hematopoietic cells. Both models ablate Tpm1 in postnatal blood. Endothelial Tpm1KO increases HEC specification without altering hematopoietic progenitor cell production or adult blood counts, suggesting separate roles for Tpm1 in the embryonic and adult blood systems. Tpm1KO increases adult platelet lifespan and diminishes adhesion to fibronectin and fibrinogen. Chemical Tpm1 inhibition also reduces focal adhesion in murine and human platelets. Altered platelet morphology and reduced platelet spreading suggest perturbed actomyosin contractility underlies these findings. Platelet fibrin binding promotes blood clot contraction, which reduces occlusive thrombosis. Tpm1KO limits clot contraction and worsens vascular occlusion in ferric chloride-induced stroke models. In addition to offering a mechanistic explanation for why genetic variation at the TPM1 alters platelet traits in GWAS, our findings reveal novel roles for Tpm1 in clot contraction and thrombosis.

Abstract Apical cell extrusion is a mechanical process that allows epithelia to eliminate apoptotic cells and prevent inflammation. Mechanosensitive ion channels are often invoked for their capacity to mediate rapid mechanical responses in dynamic morphogenetic processes. Here we report an unexpected strategy for PIEZO1 to support apoptotic extrusion. PIEZO1 inhibition blocks extrusion in cultured cells and zebrafish larvae. However, although PIEZO1 mediates calcium signals during the extrusion process, we show that extrusion is instead antagonized by increase in the preexisting mechanical tension of the epithelium when PIEZO1 is disrupted. Correcting enhanced pre-stress in PIEZO1-disrupted epithelia is sufficient to rescue apoptotic extrusion, even though it does not restore dynamic calcium signals. PIEZO1 supports mechanical homeostasis through a calcium/calcineurin-dependent pathway that protects MYPT1/myosin phosphatase from degradation to limit Myosin II activation. Therefore, PIEZO1 support the morphogenetic process of apoptotic extrusion through mechanical homeostasis.

Tropomyosins (Tpm) are master regulators of actin dynamics through forming co-polymers with filamentous actin. Despite the well-understood function of muscle Tpms in the contractile apparatus of muscle cells, much less is known about the diverse physiological function of cytoplasmic Tpms in eukaryotic cells. Here, we investigated the role of the Tpm4.2 isoform in neuronal processes including signaling, neurite outgrowth, and receptor recycling using primary neurons from Tpm4.2 knock-out mice. Live imaging of calcium and electrophysiology data demonstrated increased frequency, yet reduced strength of single neuron spikes. Calcium imaging further showed an increase in neuronal networks. In vitro assays of Tpm4.2 knock-out neurons displayed impaired recycling of the AMPA neurotransmitter receptor subunit GluA1. Morphometric analysis of neurite growth showed increased dendritic complexity and altered dendritic spine morphology in Tpm4.2 knock-out primary neurons. Behavioral analysis of Tpm4.2 knock-out mice displayed heightened anxiety in the open field test, while the elevated plus maze displayed heightened anxiety only in females. Our study depicts the multi-faceted role of the Tpm4.2 isoform and its co-polymer F-actin population in neurons, with potential implications for better understanding diseases of the nervous system which involve actin cytoskeleton dysfunction.

The unbranched actin filaments in mammalian cells are usually composed of co-polymers of a specific tropomyosin isoform with actin. Genetic manipulation has revealed that the tropomyosins largely define the functional properties of actin filaments in an isoform-specific, non-redundant manner. Tropomyosin isoforms play a role in human diseases including cancers, thrombocytopenia and thrombocythaemia, endometrial decidualisation resistance, and ulcerative colitis. Hence, the development of compounds that target different tropomyosins are potentially valuable tools for cell biology as well as potential therapeutics. We have recently identified compounds that target Tpm1.8/1.9 and now address the isoform specificity of these compounds. Tpm1.8/1.9 is primarily enriched in the lamellipodium of migrating cells but not in stress fibre bundles unlike Tpm3.1/3.2 and Tpm4.2, which are enriched in stress fibres. Human fibroblasts also incorporate Tpm1.8/1.9 into fine filaments emanating from the perinuclear region. Exposure of human fibroblasts and SK-N-SH cells to the compounds 189-1 and 189-3 results in dispersal of Tpm1.8/1.9 from the lamellipodium and fine filaments to a diffuse organisation in the cytoplasm. In contrast, at doses that disperse Tpm1.8/1.9, 189-3 has no impact on the association of either Tpm3.1/3.2 or Tpm4.2 with actin filament bundles, whereas 189-1 also targets Tpm4.2. Tpm1.8/1.9 organisation becomes dispersed between 12- and 18-hour exposure to 189-3, and the organisation of Tpm1.8/1.9 returns within 4 h of drug washout. We conclude that the amino acid sequence differences located at 7 positions in the first 19 residues of these isoforms provide sufficient specificity to generate compounds that target Tpm1.8/1.9 alone.

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes.

Tissue tension encompasses the mechanical forces exerted on solid tissues within animal bodies, originating from various sources such as cellular contractility, interactions with neighboring cells and the extracellular matrix. Emerging evidence indicates that an imbalance in such forces can influence structural organization, homeostasis, and potentially contribute to disease. For instance, heightened tissue tension can impede apical cell extrusion, leading to the retention of apoptotic or transformed cells. In this study, we investigate the potential role of adenomatous polyposis coli (APC) in modulating tissue tension. Our findings reveal that expression of an APC truncation mutant elevates epithelial tension via the RhoA/ROCK pathway. This elevation induces morphological alterations and hampers apoptotic cell extrusion in cultured epithelial cells and organoids, both of which could be mitigated by pharmacologically restoring the tissue tension. This raises the possibility that APC mutations may exert pathogenetic effects by altering tissue mechanics.

Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a novel cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes in the cell by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. We have developed a protocol where cells can be frozen, imaged by cryo-fluorescence microscopy and ready for batch cryo-ET within a day.

Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET). By taking advantage of the co-existence of epithelial and quasi-mesenchymal cells within immortalized cancer cell lines, we have analyzed the role of EMT-related gene isoforms in the regulation of epithelial mesenchymal plasticity (EMP) in high grade serous ovarian cancer. When compared with colon cancer, a distinct spectrum of downstream targets characterizes quasi-mesenchymal ovarian cancer cells, likely to reflect the different modalities of metastasis formation between these two types of malignancy, i.e. hematogenous in colon and transcoelomic in ovarian cancer. Moreover, upstream RNA-binding proteins differentially expressed between epithelial and quasi-mesenchymal subpopulations of ovarian cancer cells were identified that underlie differential regulation of EMT-related isoforms. In particular, the up- and down-regulation of RBM24 and ESRP1, respectively, represent a main regulator of EMT in ovarian cancer cells. To validate the functional and clinical relevance of our approach, we selected and functionally analyzed the Tropomyosin 1 gene (TPM1), encoding for a protein that specifies the functional characteristics of individual actin filaments in contractile cells, among the ovarian-specific downstream AS targets. The low-molecular weight Tpm1.8/9 isoforms are specifically expressed in patient-derived ascites and promote invasion through activation of EMT and Wnt signaling, together with a broad spectrum of inflammation-related pathways. Moreover, Tpm1.8/9 expression confers resistance to taxane- and platinum-based chemotherapy. Small molecule inhibitors that target the Tpm1 isoforms support targeting Tpm1.8/9 as therapeutic targets for the development of future tailor-made clinical interventions.

The actin cytoskeleton is composed of both branched and unbranched actin filaments. In mammals, the unbranched actin filaments are primarily copolymers of actin and tropomyosin. Biochemical and imaging studies indicate that different tropomyosin isoforms are segregated to different actin filament populations in cells and tissues, providing isoform-specific functionality to the actin filament. Intrinsic to this model is the prediction that single-molecule imaging of tropomyosin isoforms would confirm homopolymer formation along the length of single actin filaments, a knowledge gap that remains unaddressed in the cellular environment. We combined chemical labeling of genetically engineered tropomyosin isoforms with electron tomography to locate individual tropomyosin molecules in fibroblasts. We find that the organization of two non-muscle tropomyosins, Tpm3.1 with Tpm4.2, can be distinguished from each other using light and electron microscopy. Visualization of single tropomyosin molecules associated with actin filaments supports the hypothesis that tropomyosins form continuous homopolymers, instead of heteropolymers, in the presence of all physiologically native actin-binding proteins. This is true for both isoforms tested. Furthermore, the data suggest that the tropomyosin molecules on one side of an actin filament may not be in register with those on the opposite side, indicating that each tropomyosin polymer may assembly independently.

The negative impact of irradiation or diet on the metabolic and immune profiles of cancer survivors have been previously demonstrated. The gut microbiota plays a critical role in regulating these functions and is highly sensitive to cancer therapies. The aim of this study was to investigate the effect of irradiation and diet on the gut microbiota and metabolic or immune functions. We exposed C57Bl/6J mice to a single dose of 6 Gy radiation and after 5 weeks, fed them a chow or high-fat diet (HFD) for 12 weeks. We characterised their faecal microbiota, metabolic (whole body and adipose tissue) functions, and systemic (multiplex cytokine, chemokine assay, and immune cell profiling) and adipose tissue inflammatory profiles (immune cell profiling). At the end of the study, we observed a compounding effect of irradiation and diet on the metabolic and immune profiles of adipose tissue, with exposed mice fed a HFD displaying a greater inflammatory signature and impaired metabolism. Mice fed a HFD also showed altered microbiota, irrespective of irradiation status. An altered diet may exacerbate the detrimental effects of irradiation on both the metabolic and inflammatory profiles. This could have implications for the diagnosis and prevention of metabolic complications in cancer survivors exposed to radiation.

The combinations of anti-Tpm3.1 compounds and VCR synergistically arrest HeLa cells in mitosis.

The measurement of spindle length and cell vertical length.

The synergistic anti-cancer efficiency of the drug combinations is well correlated with the combinational impact on mitosis.

ACTA1 encodes skeletal muscle-specific a-actin, which polymerizes to form the thin filament of the sarco-mere. Mutations in ACTA1 are responsible for approximately 30% of nemaline myopathy (NM) cases. Previous studies of weakness in NM have focused on muscle structure and contractility, butgenetic issues alone do not explain the phenotypic heterogeneity observed in patients with NM or NM mouse models. To identify additional biological processes related to NM phenotypic severity, proteomic analysis was performed using muscle protein isolates from wild-type mice in comparison to moderately affected knock-in (KI) Acta1H40Y and the minimally affected transgenic (Tg) ACTA1D286G NM mice. This analysis revealed abnormalities in mitochondrial function and stress-related pathways in both mouse models, supporting an in-depth assessment of mitochondrial biology. Interestingly, evaluating each model in comparison to its wild-type counterpart identified different degrees of mitochondrial abnormality that correlated well with the phenotypic severity of the mouse model. Muscle histology, mitochondrial respiration, electron transport chain function, and mitochondrial transmembrane potential were all normal or minimally affected in the TgACTA1D286G mouse model. In contrast, the more severely affected KI.Acta1H40Y mice displayed significant abnormalities in relation to muscle histology, mitochondrial respirometry, ATP, ADP, and phosphate content, and mitochondrial transmembrane potential. These findings suggest that abnormal energy metabolism is related to symptomatic severity in NM and may constitute a contributor to phenotypic variability and a novel treatment target.

Platelets, small hemostatic blood cells, are derived from megakaryocytes. Both bone marrow and lung are principal sites of thrombopoiesis although underlying mechanisms remain unclear. Outside the body, however, our ability to generate large number of functional platelets is poor. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates substantial platelet numbers, up to 3000 per megakaryocyte. Despite their large size, megakaryocytes are able repeatedly to passage through the lung vasculature, leading to enucleation and subsequent platelet generation intravascularly. Using ex vivo lung and an in vitro microfluidic chamber we determine how oxygenation, ventilation, healthy pulmonary endothelium and the microvascular structure support thrombopoiesis. We also show a critical role for the actin regulator Tropomyosin 4 in the final steps of platelet formation in lung vasculature. This work reveals the mechanisms of thrombopoiesis in lung vasculature and informs approaches to large-scale generation of platelets.

Representative images of spindle phenotypes.

Apical extrusion is a tissue-intrinsic process that allows epithelia to eliminate unfit or surplus cells. This is exemplified by the early extrusion of apoptotic cells, which is critical to maintain the epithelial barrier and prevent inflammation. Apoptotic extrusion is an active mechanical process, which involves mechanotransduction between apoptotic cells and their neighbors, as well as local changes in tissue mechanics. Here we report that the preexisting mechanical tension at adherens junctions (AJs) conditions the efficacy of apoptotic extrusion. Specifically, increasing baseline mechanical tension by overexpression of a phosphomimetic Myosin II regulatory light chain (MRLC) compromises apoptotic extrusion. This occurs when tension is increased in either the apoptotic cell or its surrounding epithelium. Further, we find that the proinflammatory cytokine, TNFα, stimulates Myosin II and increases baseline AJ tension to disrupt apical extrusion, causing apoptotic cells to be retained in monolayers. Importantly, reversal of mechanical tension with an inhibitory MRLC mutant or tropomyosin inhibitors is sufficient to restore apoptotic extrusion in TNFα-treated monolayers. Together, these findings demonstrate that baseline levels of tissue tension are important determinants of apoptotic extrusion, which can potentially be coopted by pathogenetic factors to disrupt the homeostatic response of epithelia to apoptosis.

Aurora-A inhibitor MLN8237 (MLN) shows no or low synergy with VCR or ATM.