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Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1–4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR–Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB–FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

Insulin controls glucose uptake into muscle and fat cells by inducing a net redistribution of glucose transporter 4 (GLUT4) from intracellular storage to the plasma membrane (PM). The TBC1D4-RAB10 signaling module is required for insulin-stimulated GLUT4 translocation to the PM, although where it intersects GLUT4 traffic was unknown. Here we demonstrate that TBC1D4-RAB10 functions to control GLUT4 mobilization from a trans-Golgi network (TGN) storage compartment, establishing that insulin, in addition to regulating the PM proximal effects of GLUT4-containing vesicles docking to and fusion with the PM, also directly regulates the behavior of GLUT4 deeper within the cell. We also show that GLUT4 is retained in an element/domain of the TGN from which newly synthesized lysosomal proteins are targeted to the late endosomes and the ATP7A copper transporter is translocated to the PM by elevated copper. Insulin does not mobilize ATP7A nor does copper mobilize GLUT4, and RAB10 is not required for copper-elicited ATP7A mobilization. Consequently, GLUT4 intracellular sequestration and mobilization by insulin is achieved, in part, through utilizing a region of the TGN devoted to specialized cargo transport in general rather than being specific for GLUT4. Our results define the GLUT4-containing region of the TGN as a sorting and storage site from which different cargo are mobilized by distinct signals through unique molecular machinery.

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR–Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A–TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A–TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers. Durocher and colleagues identify CIP2A through synthetic lethal CRISPR screens as a key regulator of adaptive feedback mechanisms controlling chromosomal stability arising from accumulated DNA lesions in BRCA-mutated tumor cells.

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin‐dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80‐ and RING‐dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding‐deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80–BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways., This study reveals that the localization of BRCA1 at DNA damage sites involves two redundant ubiquitin‐dependent pathways controlled by the RNF8 and RNF168 E3 ubiquitin ligases, with a contribution from the E3 ligase activity of BRCA1.

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of the ubiquitin-binding protein RAP80 in BRCA1 recruitment to damaged chromatin. We found that RAP80, or the phosphopeptide-binding residues in the BRCA1 BRCT domains, act redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA double-strand break sites. We show that that RNF8 E3 ubiquitin ligase acts upstream of both the RAP80- and RING-dependent activities whereas RNF168 acts uniquely upstream of the RING domain. The function of the RING domain in BRCA1 recruitment is not solely linked to its role in mediating an interaction with BARD1 since RING mutations that do not impact BARD1 interaction, such as the E2-binding deficient Ile26Ala (I26A) mutation, produce a BRCA1 protein unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine the BRCA1 I26A mutation and mutations that disable the RAP80-BRCA1 interaction are deficient in RAD51 filament formation and are hypersensitive to poly (ADP-ribose) polymerase inhibition. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for the recognition of unmethylated histone H4 Lys20 and histone H2A Lys13/Lys15 ubiquitylation by BARD1 although we cannot rule out the possibility that the RING- E2 complex itself may facilitate ubiquitylated nucleosome recognition in other ways. Finally, given that tumors expressing RING-less BRCA1 isoforms readily acquire resistance to therapy, this work suggests that targeting RAP80, or its interaction with BRCA1, could represent a novel strategy for restoring sensitivity of such tumors to DNA damaging agents.

BACKGROUND: Lower extremity amputation is a serious complication of diabetes mellitus and occurs most commonly in persons who have a foot ulcer. PURPOSE: To examine variables that affect the rate of lower extremity amputation in patients with diabetes and infected foot ulcers. METHODS: A prospective observational study was performed including all consecutive patients who were 18 to 65 years, had a diagnosis of diabetes, and a foot ulcer showing clinical signs of infection. Patients were followed for 6 months or until ulcer healing, minor, or major amputation. A total of 81 persons were enrolled. Demographic variables were obtained, and clinical assessments, blood tests, and radiological investigations were performed. Ulcers were categorized using the Perfusion, Extent, Depth, Infection and Sensation classification system. Differences between variables and outcomes were assessed using the Wilcoxon test, Fisher’s exact test, Chi-square test, and t-test. RESULTS: Mean patient age was 54.58 ± 9.04 years, and the majority (61, 75%) were male. After 6 months, 33 (41%) were healed, 2 patients died, and 17 (21%) underwent major and 24 (30%) minor amputations. Major amputation rates were significantly higher in patients with a high Perfusion, Extent, Depth, Infection and Sensation score (6.92 ± 1.36; P = .005), elevated HbA1c (%) (9.43 ± 2.19; P = .049), presence of growth on wound culture (41 [64.1%]; P = .016), culture sensitivity to beta lactam (20 [31.2%]; P = .012), and presence of peripheral arterial disease seen on arterial Doppler ultrasound (P < .001). Minor amputation rates were higher in men (P = .02) and in the presence of peripheral arterial disease (P = .01). CONCLUSION: The presence of the above factors in persons with diabetes and foot ulcer with clinical signs of infection should alert the clinician to the need for focused and individualized treatment to attempt to prevent amputation.

Amplification of the gene encoding cyclin E (CCNE1) is an oncogenic driver in several malignancies and is associated with chemoresistance and poor prognosis. To uncover therapeutic targets forCCNE1-amplified tumors, we undertook genome-scale CRISPR/Cas9-based synthetic lethality screens in cellular models ofCCNE1amplification. Here, we report that increasingCCNE1dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumor regressions when combined with gemcitabine in models ofCCNE1-amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively inCCNE1overexpressing-cells, promoting early mitosis in cells undergoing DNA synthesis.CCNE1overexpression disrupts CDK1 homeostasis at least in part through an early activation of the FOXM1/MYBL2/MuvB-dependent mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy forCCNE1-amplified cancers.

BRCA1/2-mutated cancer cells must adapt to the genome instability caused by their deficiency in homologous recombination. Identifying and targeting these adaptive mechanisms may provide new therapeutic strategies. Here we present the results of genome-scale CRISPR/Cas9-based synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells that identified the gene encoding CIP2A as essential in a wide range of BRCA1- and BRCA2-mutated cells. Unlike PARP inhibition, CIP2A-deficiency does not cause accumulation of replication-associated DNA lesions that require homologous recombination for their repair. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient cells and tumors, indicating that targeting this mitotic chromosome stability process represents an attractive synthetic-lethal therapeutic strategy for BRCA1- and BRCA2-mutated cancers.

This model enables an individual to input an image and output a description for the same. The research paper makes use of the functionalities of Deep Learning and NLP (Natural Language Processing). Image Caption Generation is an important task as it allows us automate the task of generating captions for any image. This functionality enables us to easily organize files without paying heed to the task of captioning. It is also important for making dynamic web pages. This paper is for people who are visually impaired or suffer from short sightedness. So, rather than looking at an image with trouble they can easily read the caption generated by this model in a larger format. It can also be used to give description of a video in real time on later implementation for a video.

Insulin controls glucose uptake into muscle and fat cells by inducing a net redistribution of GLUT4 from intracellular storage to the plasma membrane (PM). The TBC1D4-RAB10 signaling module is required for insulin-stimulated GLUT4 translocation to the PM, although where it intersects GLUT4 traffic was unknown. Here we demonstrate that TBC1D4-RAB10 functions to control GLUT4 mobilization from a Trans Golgi Network (TGN) storage compartment, establishing that insulin, in addition to regulating the PM proximal effects of GLUT4-containing vesicles docking to and fusion with the PM, also directly regulates the behavior of GLUT4 deeper within the cell. We also show that GLUT4 is retained in an element/domain of the TGN from which newly synthesized lysosomal proteins are targeted to the late endosomes and the ATP7A copper transporter is translocated to the PM by elevated copper. Insulin does not mobilize ATP7A nor does copper mobilize GLUT4. Consequently, GLUT4 intracellular sequestration and mobilization by insulin is achieved, in part, through utilizing a region of the TGN devoted to specialized cargo transport in general rather than being specific for GLUT4. Our results define GLUT4-containing region of the TGN as a sorting and storage site from which different cargo are mobilized by distinct signals.

The newly identified shieldin complex, composed of SHLD1, SHLD2, SHLD3, and REV7, lies downstream of 53BP1 and acts to inhibit DNA resection and promote NHEJ. Here, we show that Shld2 (−/−) mice have defective class switch recombination (CSR) and that loss of SHLD2 can suppress the embryonic lethality of a Brca1 (Δ11) mutation, highlighting its role as a key effector of 53BP1. Lymphocyte development and RAG1/2‐mediated recombination were unaffected by SHLD2 deficiency. Interestingly, a significant fraction of Shld2 (−/−) primary B‐cells and 53BP1‐ and shieldin‐deficient CH12F3‐2 B‐cells permanently lose expression of immunoglobulin upon induction of CSR; this population of Ig‐negative cells is also seen in other NHEJ‐deficient cells and to a much lesser extent in WT cells. This loss of Ig is due to recombination coupled with overactive resection and loss of coding exons in the downstream acceptor constant region. Collectively, these data show that SHLD2 is the key effector of 53BP1 and critical for CSR in vivo by suppressing large deletions within the Igh locus.

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat., Caveolae are spherical nanodomains of the plasma membrane generated by assembly of caveolin and cavin proteins. Here, the authors show that fuzzy electrostatic interactions between caveolin-1 and Cavin1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

Sec16A is known to be required for COPII vesicle formation from the ER. Here, Bruno et al. show that, independent of its role at the ER, Sec16A is a RAB10 effector involved in the insulin-stimulated formation of specialized transport vesicles that ferry the GLUT4 glucose transporter to the plasma membrane of adipocytes., RAB10 is a regulator of insulin-stimulated translocation of the GLUT4 glucose transporter to the plasma membrane (PM) of adipocytes, which is essential for whole-body glucose homeostasis. We establish SEC16A as a novel RAB10 effector in this process. Colocalization of SEC16A with RAB10 is augmented by insulin stimulation, and SEC16A knockdown attenuates insulin-induced GLUT4 translocation, phenocopying RAB10 knockdown. We show that SEC16A and RAB10 promote insulin-stimulated mobilization of GLUT4 from a perinuclear recycling endosome/TGN compartment. We propose RAB10–SEC16A functions to accelerate formation of the vesicles that ferry GLUT4 to the PM during insulin stimulation. Because GLUT4 continually cycles between the PM and intracellular compartments, the maintenance of elevated cell-surface GLUT4 in the presence of insulin requires accelerated biogenesis of the specialized GLUT4 transport vesicles. The function of SEC16A in GLUT4 trafficking is independent of its previously characterized activity in ER exit site formation and therefore independent of canonical COPII-coated vesicle function. However, our data support a role for SEC23A, but not the other COPII components SEC13, SEC23B, and SEC31, in the insulin stimulation of GLUT4 trafficking, suggesting that vesicles derived from subcomplexes of COPII coat proteins have a role in the specialized trafficking of GLUT4.

Plasma membrane tension is an important factor that regulates many key cellular processes. Membrane trafficking is tightly coupled to membrane tension and can modulate the latter by addition or removal of the membrane. However, the cellular pathway(s) involved in these processes are poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin and clathrin-independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon reduction of tension. On the other hand, inhibition of the CG pathway results in lower membrane tension, while up regulation significantly enhances membrane tension. We find that vinculin, a well-studied mechanotransducer, mediates the tension-dependent regulation of the CG pathway. Vinculin negatively regulates a key CG pathway regulator, GBF1, at the plasma membrane in a tension dependent manner. Thus, the CG pathway operates in a negative feedback loop with membrane tension which leads to a homeostatic regulation of membrane tension.

SummaryCaveolae are cell-surface membrane invaginations that play critical roles in cellular processes including signaling and membrane homeostasis. The cavin proteins, in cooperation with caveolins, are essential for caveola formation. Here we show that a minimal N-terminal domain of the cavins, termed HR1, is required and sufficient for their homo- and hetero-oligomerization. Crystal structures of the mouse cavin1 and zebrafish cavin4a HR1 domains reveal highly conserved trimeric coiled-coil architectures, with intersubunit interactions that determine the specificity of cavin-cavin interactions. The HR1 domain contains a basic surface patch that interacts with polyphosphoinositides and coordinates with additional membrane-binding sites within the cavin C terminus to facilitate membrane association and remodeling. Electron microscopy of purified cavins reveals the existence of large assemblies, composed of a repeating rod-like structural element, and we propose that these structures polymerize through membrane-coupled interactions to form the unique striations observed on the surface of caveolae in vivo.

In this protocol we describe a quantitative biochemical assay to assess the efficiency of endoplasmic reticulum (ER) to Golgi protein transport in adipocytes (Bruno et al., 2016). The assay takes advantage of the fact that adipocytes secrete various bioactive proteins, known as adipokines. As a measure of ER to Golgi flux we determine the rate of bulk secretion of the adipokine adipsin post washout of Brefeldin A (BFA) treatment using immunoblotting. Because BFA treatment results in an accumulation of adipsin in the ER, the exit of adipsin from the ER upon BFA washout is synchronized across cells and experimental conditions. Thus, using this simple assay one can robustly determine if perturbations, such as knocking down a protein, have an effect on ER to Golgi protein transport.

Insulin activation of phosphatidylinositol 3-kinase (PI3K) regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but not regulation of Glut4. This uncoupling of PI3K effects occurs despite E4-ORF1 activating PI3K and downstream signaling to levels achieved by insulin. Although E4-ORF1 does not fully recapitulate insulin's effects on Glut4, it enhances insulin-stimulated insertion of Glut4-containing vesicles to the plasma membrane independent of Rab10, a key regulator of Glut4 trafficking. E4-ORF1 also stimulates plasma membrane translocation of ubiquitously expressed Glut1 glucose transporter, an effect that is likely essential for E4-ORF1 to promote an anabolic metabolism in a broad range of cell types.

The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions. We show that a subset of NPM and nucleolin localizes to the inner leaflet of plasma membrane and forms specific complexes with K-Ras but not other Ras isoforms. Active GTP-loaded and inactive GDP-loaded K-Ras both interact with NPM, although NPM-K-Ras binding is increased by growth factor receptor activation. NPM and nucleolin both stabilize K-Ras levels on the plasma membrane, but NPM concurrently increases the clustered fraction of GTP-K-Ras. The increase in nanoclustered GTP-K-Ras in turn enhances signal gain in the MAPK pathway. In summary these results reveal novel extranucleolar functions for NPM and nucleolin as regulators of K-Ras nanocluster formation and activation of the MAPK pathway. The study also identifies a new class of K-Ras nanocluster regulator that operates independently of the structural scaffold galectin-3.

The release of extracellular vesicles (EVs) is important for both normal physiology and disease. However, a basic understanding of the targeting of EV cargoes, composition and mechanism of release is lacking. Here we present evidence that the divalent metal ion transporter (DMT1) is unexpectedly regulated through release in EVs. This process involves the Nedd4-2 ubiquitin ligase, and the adaptor proteins Arrdc1 and Arrdc4 via different budding mechanisms. We show that mouse gut explants release endogenous DMT1 in EVs. Although we observed no change in the relative amount of DMT1 released in EVs from gut explants in Arrdc1 or Arrdc4 deficient mice, the extent of EVs released was significantly reduced indicating an adaptor role in biogenesis. Furthermore, using Arrdc1 or Arrdc4 knockout mouse embryonic fibroblasts, we show that both Arrdc1 and Arrdc4 are non-redundant positive regulators of EV release. Our results suggest that DMT1 release from the plasma membrane into EVs may represent a novel mechanism for the maintenance of iron homeostasis, which may also be important for the regulation of other membrane proteins.