The recruitment of telomerase to telomeres is a tightly regulated process which is stimulated by replication stress and mediated by the DNA damage response regulatory kinase ATR. Here, we demonstrate that nuclear filamentous actin is important for telomerase recruitment under endogenous and replication stress conditions in immortal human cells. Inhibition of nuclear actin polymerization decreases the presence of telomerase at telomeres. This process is regulated by both ATR and mTOR kinases, and employs other regulators of actin structure and function, such as WASP, ARP2/3 and myosin. Nuclear filamentous actin serves as a site for telomerase recruitment, which is mediated by telomere tethering on actin fibres in response to replication stress, allowing telomerase to localize to telomeres containing stalled replication forks. Overall, these data demonstrate that, in human cells which express telomerase, telomeric replication stress triggers the recruitment of telomerase to telomeres via a nuclear actin network, enabling telomere length maintenance.### Competing Interest StatementThe authors have declared no competing interest.

Pancreatic beta cell dedifferentiation, transdifferentiation into other islet cells and apoptosis have been implicated in beta cell failure in type 2 diabetes, although the mechanisms are poorly defined. The endoplasmic reticulum stress response factor X-box binding protein 1 (XBP1) is a major regulator of the unfolded protein response. XBP1 expression is reduced in islets of people with type 2 diabetes, but its role in adult differentiated beta cells is unclear. Here, we assessed the effects of Xbp1 deletion in adult beta cells and tested whether XBP1-mediated unfolded protein response makes a necessary contribution to beta cell compensation in insulin resistance states. Mice with inducible beta cell-specific Xbp1 deletion were studied under normal (chow diet) or metabolic stress (high-fat diet or obesity) conditions. Glucose tolerance, insulin secretion, islet gene expression, alpha cell mass, beta cell mass and apoptosis were assessed. Lineage tracing was used to determine beta cell fate. Deletion of Xbp1 in adult mouse beta cells led to beta cell dedifferentiation, beta-to-alpha cell transdifferentiation and increased alpha cell mass. Cell lineage-specific analyses revealed that Xbp1 deletion deactivated beta cell identity genes (insulin, Pdx1, Nkx6.1, Beta2, Foxo1) and derepressed beta cell dedifferentiation (Aldh1a3) and alpha cell (glucagon, Arx, Irx2) genes. Xbp1 deletion in beta cells of obese ob/ob or high-fat diet-fed mice triggered diabetes and worsened glucose intolerance by disrupting insulin secretory capacity. Furthermore, Xbp1 deletion increased beta cell apoptosis under metabolic stress conditions by attenuating the antioxidant response. These findings indicate that XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and is required for beta cell compensation and prevention of diabetes in insulin resistance states.

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

Background Breast cancer stem cells (BCSCs) are drivers of therapy-resistance, therefore are responsible for poor survival. Molecular signatures of BCSCs from primary cancers remain undefined. Here, we identify the consistent transcriptome of primary BCSCs shared across breast cancer subtypes, and we examine the clinical relevance of ITGA7, one of the genes differentially expressed in BCSCs. Methods Primary BCSCs were assessed using immunohistochemistry and fluorescently labelled using Aldefluor ( n = 17). Transcriptomes of fluorescently sorted BCSCs and matched non-stem cancer cells were determined using RNA-seq ( n = 6). ITGA7 expression was examined in breast cancers using immunohistochemistry ( n = 305), and its functional role was tested using siRNA in breast cancer cells. Results Proportions of BCSCs varied from 0 to 9.4%. 38 genes were significantly differentially expressed in BCSCs; genes were enriched for functions in vessel morphogenesis, motility, and metabolism. ITGA7 was found to be significantly downregulated in BCSCs, and low expression significantly correlated with reduced survival in patients treated with chemotherapy, and with chemoresistance in breast cancer cells in vitro. Conclusions This study is the first to define the molecular profile of BCSCs from a range of primary breast cancers. ITGA7 acts as a predictive marker for chemotherapy response, in accordance with its downregulation in BCSCs.

Obesity is caused by an imbalance between food intake and energy expenditure (EE). Here we identify a conserved pathway that links signalling through peripheral Y1 receptors (Y1R) to the control of EE. Selective antagonism of peripheral Y1R, via the non-brain penetrable antagonist BIBO3304, leads to a significant reduction in body weight gain due to enhanced EE thereby reducing fat mass. Specifically thermogenesis in brown adipose tissue (BAT) due to elevated UCP1 is enhanced accompanied by extensive browning of white adipose tissue both in mice and humans. Importantly, selective ablation of Y1R from adipocytes protects against diet-induced obesity. Furthermore, peripheral specific Y1R antagonism also improves glucose homeostasis mainly driven by dynamic changes in Akt activity in BAT. Together, these data suggest that selective peripheral only Y1R antagonism via BIBO3304, or a functional analogue, could be developed as a safer and more effective treatment option to mitigate diet-induced obesity.

It has been suggested that interleukin-6 (IL-6) produced by adipocytes in obesity leads to liver insulin resistance, although this hypothesis has never been definitively tested. Accordingly, we did so by generating adipocyte-specific IL-6-deficient (AdipoIL-6−/−) mice and studying them in the context of diet-induced and genetic obesity. Mice carrying two floxed alleles of IL-6 (C57Bl/6J) were crossed with Cre recombinase-overexpressing mice driven by the adiponectin promoter to generate AdipoIL-6−/− mice. AdipoIL-6−/− and floxed littermate controls were fed a standard chow or high-fat diet (HFD) for 16 wk and comprehensively metabolically phenotyped. In addition to a diet-induced obesity model, we also examined the role of adipocyte-derived IL-6 in a genetic model of obesity and insulin resistance by crossing the AdipoIL-6−/− mice with leptin-deficient ( ob/ob) mice. As expected, mice on HFD and ob/ob mice displayed marked weight gain and increased fat mass compared with chow-fed and ob/+ (littermate control) animals, respectively. However, deletion of IL-6 from adipocytes in either model had no effect on glucose tolerance or fasting hyperinsulinemia. We concluded that adipocyte-specific IL-6 does not contribute to whole body glucose intolerance in obese mice.

Exercise stimulates the release of molecules into the circulation, supporting the concept that inter-tissue signaling proteins are important mediators of adaptations to exercise. Recognizing that many circulating proteins are packaged in extracellular vesicles (EVs), we employed quantitative proteomic techniques to characterize the exercise-induced secretion of EV-contained proteins. Following a 1-hr bout of cycling exercise in healthy humans, we observed an increase in the circulation of over 300 proteins, with a notable enrichment of several classes of proteins that compose exosomes and small vesicles. Pulse-chase and intravital imaging experiments suggested EVs liberated by exercise have a propensity to localize in the liver and can transfer their protein cargo. Moreover, by employing arteriovenous balance studies across the contracting human limb, we identified several novel candidate myokines, released into circulation independently of classical secretion. These data identify a new paradigm by which tissue crosstalk during exercise can exert systemic biological effects.

GLUT4 is unique among specialized glucose transporters in being exclusively expressed in muscle and adipocytes. In the absence of insulin the distribution of GLUT4 is preferentially intracellular and insulin stimulation results in the movement of GLUT4 containing vesicles to the plasma membrane. This process is responsible for the insulin stimulation of glucose uptake in muscle and fat. While signalling pathways triggering the translocation of GLUT4 are well understood, the mechanisms regulating the intracellular retention of GLUT4 are less well understood. Here we report a role for β-catenin in this process. In 3T3-L1 adipocytes in which β-catenin is depleted, the levels of GLUT4 at and near the plasma membrane rise in unstimulated cells while the subsequent increase in GLUT4 at the plasma membrane upon insulin stimulation is reduced. Small molecule approaches to acutely activate or inhibit β-catenin give results that support the results obtained with siRNA and these changes are accompanied by matching changes in glucose transport into these cells. Together these results indicate that β-catenin is a previously unrecognized regulator of the mechanisms that control the insulin sensitive pool of GLUT4 transporters inside these adipocyte cells.

Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome.

Forces play diverse roles in vascular development, homeostasis and disease. VE-cadherin at endothelial cell-cell junctions links the contractile acto-myosin cytoskeletons of adjacent cells, serving as a tension-transducer. To explore tensile changes across VE-cadherin in live zebrafish, we tailored an optical biosensor approach, originally established in vitro. We validate localization and function of a VE-cadherin tension sensor (TS) in vivo. Changes in tension across VE-cadherin observed using ratio-metric or lifetime FRET measurements reflect acto-myosin contractility within endothelial cells. Furthermore, we apply the TS to reveal biologically relevant changes in VE-cadherin tension that occur as the dorsal aorta matures and upon genetic and chemical perturbations during embryonic development.

T follicular regulatory (Tfr) cells control the magnitude and specificity of the germinal centre reaction, but how regulation is contained to ensure generation of high-affinity antibody is unknown. Here we show that this balance is maintained by the reciprocal influence of interleukin (IL)-2 and IL-21. The number of IL-2-dependent FoxP3 + regulatory T cells is increased in the peripheral blood of human patients with loss-of-function mutations in the IL-21 receptor (IL-21R). In mice, IL-21:IL-21R interactions influence the phenotype of T follicular cells, reducing the expression of CXCR4 and inhibiting the expansion of Tfr cells after T-cell-dependent immunization. The negative effect of IL-21 on Tfr cells in mice is cell intrinsic and associated with decreased expression of the high affinity IL-2 receptor (CD25). Bcl-6, expressed in abundance in Tfr cells, inhibits CD25 expression and IL-21-mediated inhibition of CD25 is Bcl-6 dependent. These findings identify a mechanism by which IL-21 reinforces humoral immunity by restricting Tfr cell proliferation.

The processes regulating glucose-stimulated insulin secretion (GSIS) and its modulation by incretins in pancreatic β-cells are only partly understood. Here we investigate the involvement of β-catenin in these processes. Reducing β-catenin levels using siRNA knockdown attenuated GSIS in a range of β-cell models and blocked the ability of GLP-1 agonists and the depolarizing agent KCl to potentiate this. This could be mimicked in both β-cell models and isolated islets by short-term exposure to the β-catenin inhibitory drug pyrvinium. In addition, short-term treatment with a drug that increases β-catenin levels results in an increase in insulin secretion. The timing of these effects suggests that β-catenin is required for the processes regulating trafficking and/or release of pre-existing insulin granules rather than for those regulated by gene expression. This was supported by the finding that the overexpression of the transcriptional co-activator of β-catenin, transcription factor 7-like 2 (TCF7L2), attenuated insulin secretion, consistent with the extra TCF7L2 translocating β-catenin from the plasma membrane pool to the nucleus. We show that β-catenin depletion disrupts the intracellular actin cytoskeleton, and by using total internal reflectance fluorescence (TIRF) microscopy, we found that β-catenin is required for the glucose- and incretin-induced depletion of insulin vesicles from near the plasma membrane. In conclusion, we find that β-catenin levels modulate Ca2+-dependent insulin exocytosis under conditions of glucose, GLP-1, or KCl stimulation through a role in modulating insulin secretory vesicle localization and/or fusion via actin remodeling. These findings also provide insights as to how the overexpression of TCF7L2 may attenuate insulin secretion.

Isoforms of flavin-containing monooxygenase (FMO) are involved in xenobiotic metabolism but have also been implicated in the regulation of glucose and lipid homeostasis and in the development of atherosclerosis. However, we have recently shown that improved insulin action is associated with increased FMO expression in livers of protein kinase C-deficient mice. Here, we investigated whether FMO3 expression affected insulin signaling, glucose metabolism, and endoplasmic reticulum (ER) stress in hepatocytes. HepG2 and IHH hepatocytes were transfected with FMO3 cDNA for overexpression, or small interfering RNA for knockdown. Cells were treated with palmitate to induce insulin resistance and insulin signaling, phosphoenolpyruvate carboxykinase (PEPCK) gene expression and ER stress markers were examined by immunoblotting and RT-PCR. Glycogen synthesis was measured using [14C]glucose. Palmitate treatment reduced insulin signaling at the level of Akt phosphorylation and glycogen synthesis, which were little affected by FMO3 overexpression. However, the fatty acid also increased the levels of several ER stress markers and activation of caspase 3, which were counteracted by FMO3 overexpression and exacerbated by FMO3 knockdown. Although FMO3 expression did not reverse lipid effects on protein thiol redox in hepatocytes, it did prevent up-regulation of the gluconeogenic enzyme PEPCK by pharmacological ER stress inducers or by palmitate. ER stress and PEPCK levels were also reduced in livers of fat-fed protein kinase Cδ-deficient mice. Our data indicate that FMO3 can contribute to the regulation of glucose metabolism in the liver by reducing lipid-induced ER stress and the expression of PEPCK, independently of insulin signal transduction.

Actin has an ill‐defined role in the trafficking of GLUT4 glucose transporter vesicles to the plasma membrane (PM). We have identified novel actin filaments defined by the tropomyosin Tpm3.1 at glucose uptake sites in white adipose tissue (WAT) and skeletal muscle. In Tpm 3.1‐overexpressing mice, insulin‐stimulated glucose uptake was increased; while Tpm3.1‐null mice they were more sensitive to the impact of high‐fat diet on glucose uptake. Inhibition of Tpm3.1 function in 3T3‐L1 adipocytes abrogates insulin‐stimulated GLUT4 translocation and glucose uptake. In WAT, the amount of filamentous actin is determined by Tpm3.1 levels and is paralleled by changes in exocyst component (sec8) and Myo1c levels. In adipocytes, Tpm3.1 localizes with MyoIIA, but not Myo1c, and it inhibits Myo1c binding to actin. We propose that Tpm3.1 determines the amount of cortical actin that can engage MyoIIA and generate contractile force, and in parallel limits the interaction of Myo1c with actin filaments. The balance between these actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the PM.

Basic in vitro systems can be used to model and assess complex diseases, such as cancer. Recent advances in this field include the incorporation of multiple cell types and extracellular matrix proteins into three-dimensional (3D) models to recapitulate the structure, organization and functionality of live tissue in situ. Cells within such a 3D environment behave very differently from cells on two-dimensional (2D) substrates, as cell-matrix interactions trigger signalling pathways and cellular responses in 3D, which may not be observed in 2D. Thus, the use of 3D systems can be advantageous for the assessment of disease progression over 2D set-ups alone. Here, we highlight the current advantages and challenges of employing 3D systems in the study of cancer and provide an overview to guide the appropriate use of distinct models in cancer research.

Glycogen synthase kinase 3 (GSK3) is essential for normal development and function of the central nervous system. It is especially important for regulating neurotransmission, although the downstream substrates mediating this function are not yet clear. In the present paper, we report the lipid kinase phosphatidylinositol 4-kinase II α (PI4KIIα) is a novel substrate of GSK3 that regulates trafficking and cell-surface expression of neurotransmitter receptors in neurons. GSK3 phosphorylates two distinct sites in the N-terminus of PI4KIIα (Ser5 and Ser47), promoting binding to the adaptor protein 3 (AP-3) complex for trafficking to the lysosome to be degraded. Blocking phosphorylation reduces trafficking to the lysosome, stabilizing PI4KIIα and its cargo proteins for redistribution throughout the cell. Importantly, a reduction in PI4KIIα expression or phosphorylation increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression at the surface of hippocampal neurons. These studies implicate signalling between GSK3 and PI4KIIα as a novel regulator of vesicular trafficking and neurotransmission in the brain.

Quantitative analysis of the dynamics of tiny cellular and subcellular structures in time-lapse cell microscopy sequences requires the development of a reliable multi-target tracking method capable of tracking numerous similar targets in the presence of high levels of noise, high target density, maneuvering motion patterns and intricate interactions. The linear Gaussian jump Markov system probability hypothesis density (LGJMS-PHD) filter is a recent Bayesian tracking filter that is well-suited for this task. However, the existing recursion equations for this filter do not consider a state-dependent transition probability matrix. As required in many biological applications, we propose a new closed-form recursion that incorporates this assumption and introduce a general framework for particle tracking using the proposed filter. We apply our scheme to multi-target tracking in total internal reflection fluorescence microscopy (TIRFM) sequences and evaluate the performance of our filter against the existing LGJMS-PHD and IMM-JPDA filters.

Regulated GLUT4 trafficking is a key action of insulin. Quantitative stepwise analysis of this process provides a powerful tool for pinpointing regulatory nodes that contribute to insulin regulation and insulin resistance. We describe a novel GLUT4 construct and workflow for the streamlined dissection of GLUT4 trafficking; from simple high throughput screens to high resolution analyses of individual vesicles. We reveal single cell heterogeneity in insulin action highlighting the utility of this approach each cell displayed a unique and highly reproducible insulin response, implying that each cell is hard-wired to produce a specific output in response to a given stimulus. These data highlight that the response of a cell population to insulin is underpinned by extensive heterogeneity at the single cell level. This heterogeneity is pre-programmed within each cell and is not the result of intracellular stochastic events.