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1.
Cell ; 184(2): 384-403.e21, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33450205

RESUMEN

Many oncogenic insults deregulate RNA splicing, often leading to hypersensitivity of tumors to spliceosome-targeted therapies (STTs). However, the mechanisms by which STTs selectively kill cancers remain largely unknown. Herein, we discover that mis-spliced RNA itself is a molecular trigger for tumor killing through viral mimicry. In MYC-driven triple-negative breast cancer, STTs cause widespread cytoplasmic accumulation of mis-spliced mRNAs, many of which form double-stranded structures. Double-stranded RNA (dsRNA)-binding proteins recognize these endogenous dsRNAs, triggering antiviral signaling and extrinsic apoptosis. In immune-competent models of breast cancer, STTs cause tumor cell-intrinsic antiviral signaling, downstream adaptive immune signaling, and tumor cell death. Furthermore, RNA mis-splicing in human breast cancers correlates with innate and adaptive immune signatures, especially in MYC-amplified tumors that are typically immune cold. These findings indicate that dsRNA-sensing pathways respond to global aberrations of RNA splicing in cancer and provoke the hypothesis that STTs may provide unexplored strategies to activate anti-tumor immune pathways.


Asunto(s)
Antivirales/farmacología , Inmunidad/efectos de los fármacos , Empalmosomas/metabolismo , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Inmunidad Adaptativa/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Femenino , Amplificación de Genes/efectos de los fármacos , Humanos , Intrones/genética , Ratones , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas c-myc/metabolismo , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genética , ARN Bicatenario/metabolismo , Transducción de Señal/efectos de los fármacos , Empalmosomas/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/genética
2.
EMBO J ; 42(17): e114534, 2023 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-37469281

RESUMEN

Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.


Asunto(s)
Proteínas Cromosómicas no Histona , Nucleosomas , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Centrómero/genética , Centrómero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
4.
Development ; 148(18)2021 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-34427308

RESUMEN

Regeneration after peripheral nerve damage requires that axons re-grow to the correct target tissues in a process called target-specific regeneration. Although much is known about the mechanisms that promote axon re-growth, re-growing axons often fail to reach the correct targets, resulting in impaired nerve function. We know very little about how axons achieve target-specific regeneration, particularly in branched nerves that require distinct targeting decisions at branch points. The zebrafish vagus motor nerve is a branched nerve with a well-defined topographic organization. Here, we track regeneration of individual vagus axons after whole-nerve laser severing and find a robust capacity for target-specific, functional re-growth. We then develop a new single-cell chimera injury model for precise manipulation of axon-environment interactions and find that (1) the guidance mechanism used during regeneration is distinct from the nerve's developmental guidance mechanism, (2) target selection is specified by neurons' intrinsic memory of their position within the brain, and (3) targeting to a branch requires its pre-existing innervation. This work establishes the zebrafish vagus nerve as a tractable regeneration model and reveals the mechanistic basis of target-specific regeneration.


Asunto(s)
Axones/fisiología , Regeneración Nerviosa/fisiología , Nervio Vago/fisiología , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Neuronas/fisiología , Traumatismos de los Nervios Periféricos/fisiopatología
5.
Nature ; 561(7723): 331-337, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30185905

RESUMEN

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.

6.
Nucleic Acids Res ; 48(5): 2621-2642, 2020 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-31863590

RESUMEN

Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a cancer-associated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 localization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely 'hijacked' by cytoplasmic Malat1 to the cytoplasm, resulting in the depletion of nuclear TDP-43 and redistribution of TDP-43 binding to repetitive element transcripts and mRNAs encoding mitotic and nuclear-cytoplasmic regulators. The SINE promotes Malat1 nuclear retention by facilitating Malat1 binding to HNRNPK, a protein that drives RNA nuclear retention, potentially through direct interactions of the SINE with KHDRBS1 and TRA2A, which bind to HNRNPK. Losing these RNA-protein interactions due to the SINE deletion likely creates more available TDP-43 binding sites on Malat1 and subsequent TDP-43 aggregation. These results highlight the significance of lncRNA TEs in TDP-43 proteostasis with potential implications in both cancer and neurodegenerative diseases.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteostasis/genética , ARN Largo no Codificante/genética , Elementos de Nucleótido Esparcido Corto/genética , Apoptosis , Línea Celular , Citoplasma/metabolismo , Daño del ADN , Estrés del Retículo Endoplásmico , Activación Enzimática , Dosificación de Gen , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Humanos , Mitosis , Modelos Biológicos , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de Secuencia/genética , eIF-2 Quinasa
7.
J Cell Sci ; 131(19)2018 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-30185525

RESUMEN

Stress granules (SGs) assemble under stress-induced conditions that inhibit protein synthesis, including phosphorylation of eIF2α, inhibition of the RNA helicase eIF4a proteins or inactivation of mTORC1. Classically defined SGs are composed of translation initiation factors, 40S ribosomes, RNA-binding proteins and poly(A)+ mRNAs. As such, they represent an important compartment for storage of mRNAs and regulation of their translation. Emerging work on SGs indicates that these structures might promote cellular survival in diverse disease states. Yet, much work on SG formation and function employs acute stress conditions, which might not accurately reflect the chronic stresses that manifest in human disease. Here, we used prolonged nutrient starvation to model and investigate SG formation and function during chronic stress in a human cell line and mouse embryonic fibroblasts. Surprisingly, we found that SGs that form under chronic nutrient starvation lack 40S ribosomes, do not actively exchange their constituent components with cytoplasmic pools and promote cell death. We named these SGs starvation-induced SGs (stSGs). Our results on stSGs imply that SG assembly and function in the context of prolonged nutrient starvation stress differ significantly from what has been described for acute stress conditions.


Asunto(s)
Apoptosis , Gránulos Citoplasmáticos/metabolismo , Animales , Línea Celular Tumoral , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Cinética , Ratones , Biosíntesis de Proteínas , Ribosomas/metabolismo
8.
Development ; 142(15): 2704-18, 2015 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-26160902

RESUMEN

Dachsous (Dchs), an atypical cadherin, is an evolutionarily conserved regulator of planar cell polarity, tissue size and cell adhesion. In humans, DCHS1 mutations cause pleiotropic Van Maldergem syndrome. Here, we report that mutations in zebrafish dchs1b and dchs2 disrupt several aspects of embryogenesis, including gastrulation. Unexpectedly, maternal zygotic (MZ) dchs1b mutants show defects in the earliest developmental stage, egg activation, including abnormal cortical granule exocytosis (CGE), cytoplasmic segregation, cleavages and maternal mRNA translocation, in transcriptionally quiescent embryos. Later, MZdchs1b mutants exhibit altered dorsal organizer and mesendodermal gene expression, due to impaired dorsal determinant transport and Nodal signaling. Mechanistically, MZdchs1b phenotypes can be explained in part by defective actin or microtubule networks, which appear bundled in mutants. Accordingly, disruption of actin cytoskeleton in wild-type embryos phenocopied MZdchs1b mutant defects in cytoplasmic segregation and CGE, whereas interfering with microtubules in wild-type embryos impaired dorsal organizer and mesodermal gene expression without perceptible earlier phenotypes. Moreover, the bundled microtubule phenotype was partially rescued by expressing either full-length Dchs1b or its intracellular domain, suggesting that Dchs1b affects microtubules and some developmental processes independent of its known ligand Fat. Our results indicate novel roles for vertebrate Dchs in actin and microtubule cytoskeleton regulation in the unanticipated context of the single-celled embryo.


Asunto(s)
Actinas/metabolismo , Cadherinas/metabolismo , Citoesqueleto/fisiología , Microtúbulos/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Cadherinas/genética , Cartilla de ADN/genética , Exocitosis/fisiología , Femenino , Inmunohistoquímica , Hibridación in Situ , Microscopía Confocal , Imagen Óptica , Ovario/anatomía & histología , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas de Pez Cebra/genética
10.
Sci Rep ; 14(1): 6013, 2024 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-38472343

RESUMEN

Nuclear membrane rupture is a physiological response to multiple in vivo processes, such as cell migration, that can cause extensive genome instability and upregulate invasive and inflammatory pathways. However, the underlying molecular mechanisms of rupture are unclear and few regulators have been identified. In this study, we developed a reporter that is size excluded from re-compartmentalization following nuclear rupture events. This allows for robust detection of factors influencing nuclear integrity in fixed cells. We combined this with an automated image analysis pipeline in a high-content siRNA screen to identify new proteins that both increase and decrease nuclear rupture frequency in cancer cells. Pathway analysis identified an enrichment of nuclear membrane and ER factors in our hits and we demonstrate that one of these, the protein phosphatase CTDNEP1, is required for nuclear stability. Analysis of known rupture determinants, including an automated quantitative analysis of nuclear lamina gaps, are consistent with CTDNEP1 acting independently of actin and nuclear lamina organization. Our findings provide new insights into the molecular mechanism of nuclear rupture and define a highly adaptable program for rupture analysis that removes a substantial barrier to new discoveries in the field.


Asunto(s)
Actinas , Membrana Nuclear , Membrana Nuclear/metabolismo , Actinas/metabolismo , Movimiento Celular , Lámina Nuclear/metabolismo , Núcleo Celular/metabolismo
11.
Curr Biol ; 34(15): 3327-3341.e9, 2024 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-38955177

RESUMEN

Organisms experience constant nutritional flux. Mechanisms at the interface of opposing nutritional states-scarcity and surplus-enable organismal energy homeostasis. Contingent on nutritional stores, adipocytes secrete adipokines, such as the fat hormone leptin, to signal nutrient status to the central brain. Increased leptin secretion underlies metabolic dysregulation during common obesity, but the molecular mechanisms regulating leptin secretion from human adipocytes are poorly understood. Here, we report that Atg8/LC3 family proteins, best known for their role in autophagy during nutrient scarcity, play an evolutionarily conserved role during nutrient surplus by promoting adipokine secretion. We show that in a well-fed state, Atg8/LC3 promotes the secretion of the Drosophila functional leptin ortholog unpaired 2 (Upd2) and leptin from human adipocytes. Proteomic analyses reveal that LC3 directs leptin to a secretory pathway in human cells. We identified LC3-dependent extracellular vesicle (EV) loading and secretion (LDELS) as a required step for leptin release, highlighting a unique secretory route adopted by leptin in human adipocytes. In Drosophila, mutations to Upd2's Atg8 interaction motif (AIM) result in constitutive adipokine retention. Atg8-mediated Upd2 retention alters lipid storage and hunger response and rewires the bulk organismal transcriptome in a manner conducive to starvation survival. Thus, Atg8/LC3's bidirectional role in nutrient sensing-conveying nutrient surplus and responding to nutrient deprivation-enables organisms to manage nutrient flux effectively. We posit that decoding how bidirectional molecular switches-such as Atg8/LC3-operate at the nexus of nutritional scarcity and surplus will inform therapeutic strategies to tackle chronic metabolic disorders.


Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Transducción de Señal , Animales , Humanos , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Adipocitos/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Leptina/metabolismo , Leptina/genética , Nutrientes/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Autofagia
12.
bioRxiv ; 2024 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-39005354

RESUMEN

Bacterial vaginosis (BV) is a dysbiosis of the vaginal microbiome that is prevalent in reproductive-age women worldwide. Adverse outcomes associated with BV include an increased risk of sexually acquired Human Immunodeficiency Virus (HIV), yet the immunological mechanisms underlying this association are not well understood. To investigate BV driven changes to cervicovaginal tract (CVT) and circulating T cell phenotypes, participants with or without BV provided vaginal tract (VT) and ectocervical (CX) tissue biopsies and peripheral blood mononuclear cells (PBMC). Immunofluorescence analysis of genital mucosal tissues revealed a reduced density of CD3+CD4+CCR5+ cells in the VT lamina propria of individuals with compared to those without BV (median 243.8 cells/mm2 BV- vs 106.9 cells/mm2 BV+, p=0.043). High-parameter flow cytometry of VT biopsies revealed an increased frequency in individuals with compared to those without BV of dysfunctional CD39+ conventional CD4+ T cells (Tconv) (median frequency 15% BV- vs 30% BV+, padj=0.0331) and tissue-resident CD69+CD103+ Tconv (median frequency 24% BV- vs 38% BV+, padj=0.0061), previously reported to be implicated in HIV acquisition and replication. Our data suggests that BV elicits diverse and complex VT T cell alterations and expands on potential immunological mechanisms that may promote adverse outcomes including HIV susceptibility.

13.
Development ; 137(7): 1127-35, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20215349

RESUMEN

During early zebrafish development the nodal signalling pathway patterns the embryo into three germ layers, in part by inducing the expression of no tail (ntl), which is essential for correct mesoderm formation. When nodal signalling is inhibited ntl fails to be expressed in the dorsal margin, but ventral ntl expression is unaffected. These observations indicate that ntl transcription is under both nodal-dependent and nodal-independent regulation. Consistent with these observations and with a role for ntl in mesoderm formation, some somites form within the tail region of embryos lacking nodal signalling. In an effort to understand how ntl is regulated and thus how mesoderm forms, we have mapped the elements responsible for nodal-dependent and nodal-independent expression of ntl in the margin of the embryo. Our work demonstrates that expression of ntl in the margin is the consequence of two separate enhancers, which act to mediate different mechanisms of mesoderm formation. One of these enhancers responds to nodal signalling, and the other to Wnt and BMP signalling. We demonstrate that the nodal-independent regulation of ntl is essential for tail formation. Misexpression of Wnt and BMP ligands can induce the formation of an ectopic tail, which contains somites, in embryos devoid of nodal signalling, and this tail formation is dependent on ntl function. Similarly, nodal-independent tail somite formation requires ntl. At later stages in development ntl is required for notochord formation, and our analysis has also led to the identification of the enhancer required for ntl expression in the developing notochord.


Asunto(s)
Tipificación del Cuerpo/fisiología , Inducción Embrionaria/fisiología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/fisiología , Proteínas de Dominio T Box/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra , Animales , Animales Modificados Genéticamente , Proteínas Fetales , Proteína Nodal/genética , Proteína Nodal/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal/fisiología , Proteínas de Dominio T Box/genética , Pez Cebra/anatomía & histología , Pez Cebra/embriología , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética
14.
Mol Biol Cell ; 34(3): ar15, 2023 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-36598808

RESUMEN

Cells are subjected to a barrage of daily insults that often lead to their cortices being ripped open and requiring immediate repair. An important component of the cell's repair response is the formation of an actomyosin ring at the wound periphery to mediate its closure. Here we show that inhibition of myosin or the linear actin nucleation factors Diaphanous and/or dishevelled associated activator of morphogenesis results in a disrupted contractile apparatus and delayed wound closure. We also show that the branched actin nucleators WASp and SCAR function nonredundantly as scaffolds to assemble and maintain this contractile actomyosin cable. Removing branched actin leads to the formation of smaller circular actin-myosin structures at the cell cortex and to slow wound closure. Removing linear and branched actin simultaneously results in failed wound closure. Surprisingly, removal of branched actin and myosin results in the formation of parallel linear F-actin filaments that undergo a chiral swirling movement to close the wound, uncovering a new mechanism of cell wound closure. Taken together, we demonstrate the roles of different actin substructures that are required for optimal actomyosin ring formation and the extraordinary resilience of the cell to undergo wound repair when it is unable to form different subsets of these substructures.


Asunto(s)
Citoesqueleto de Actina , Actinas , Actomiosina , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Citocinesis , Miosinas/metabolismo
15.
Elife ; 122023 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-37489578

RESUMEN

Integrin-mediated cell attachment rapidly induces tyrosine kinase signaling. Despite years of research, the role of this signaling in integrin activation and focal adhesion assembly is unclear. We provide evidence that the Src-family kinase (SFK) substrate Cas (Crk-associated substrate, p130Cas, BCAR1) is phosphorylated and associated with its Crk/CrkL effectors in clusters that are precursors of focal adhesions. The initial phospho-Cas clusters contain integrin ß1 in its inactive, bent closed, conformation. Later, phospho-Cas and total Cas levels decrease as integrin ß1 is activated and core focal adhesion proteins including vinculin, talin, kindlin, and paxillin are recruited. Cas is required for cell spreading and focal adhesion assembly in epithelial and fibroblast cells on collagen and fibronectin. Cas cluster formation requires Cas, Crk/CrkL, SFKs, and Rac1 but not vinculin. Rac1 provides positive feedback onto Cas through reactive oxygen, opposed by negative feedback from the ubiquitin proteasome system. The results suggest a two-step model for focal adhesion assembly in which clusters of phospho-Cas, effectors and inactive integrin ß1 grow through positive feedback prior to integrin activation and recruitment of core focal adhesion proteins.


Asunto(s)
Adhesiones Focales , Fosfoproteínas , Fosforilación , Adhesiones Focales/metabolismo , Fosfoproteínas/metabolismo , Integrina beta1/metabolismo , Proteína Sustrato Asociada a CrK/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Integrinas/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo
16.
bioRxiv ; 2023 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-36711558

RESUMEN

Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.

17.
bioRxiv ; 2023 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-37398267

RESUMEN

Nuclear membrane rupture is a physiological response to multiple in vivo processes, such as cell migration, that can cause extensive genome instability and upregulate invasive and inflammatory pathways. However, the underlying molecular mechanisms of rupture are unclear and few regulators have been identified. In this study, we developed a reporter that is size excluded from re-compartmentalization following nuclear rupture events. This allows for robust detection of factors influencing nuclear integrity in fixed cells. We combined this with an automated image analysis pipeline in a high-content siRNA screen to identify new proteins that both increase and decrease nuclear rupture frequency in cancer cells. Pathway analysis identified an enrichment of nuclear membrane and ER factors in our hits and we demonstrate that one of these, the protein phosphatase CTDNEP1, is required for nuclear stability. Further analysis of known rupture contributors, including a newly developed automated quantitative analysis of nuclear lamina gaps, strongly suggests that CTDNEP1 acts in a new pathway. Our findings provide new insights into the molecular mechanism of nuclear rupture and define a highly adaptable program for rupture analysis that removes a substantial barrier to new discoveries in the field.

18.
iScience ; 26(2): 105965, 2023 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-36824274

RESUMEN

Despite the knowledge that protein translation and various metabolic reactions that create and sustain cellular life occur in the cytoplasm, the structural organization within the cytoplasm remains unclear. Recent models indicate that cytoplasm contains viscous fluid and elastic solid phases. We separated these viscous fluid and solid elastic compartments, which we call the cytosol and cytomatrix, respectively. The distinctive composition of the cytomatrix included structural proteins, ribosomes, and metabolome enzymes. High-throughput analysis revealed unique biosynthetic pathways within the cytomatrix. Enrichment of biosynthetic pathways in the cytomatrix indicated the presence of immobilized biocatalysis. Enzymatic immobilization and segregation can surmount spatial impediments, and the local pathway segregation may form cytoplasmic organelles. Protein translation was reprogrammed within the cytomatrix under the restriction of protein synthesis by drug treatment. The cytosol and cytomatrix are an elaborately interconnected network that promotes operational flexibility in healthy cells and the survival of malignant cells.

19.
Cancer Res Commun ; 3(9): 1756-1769, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37674528

RESUMEN

Mechanisms for Helicobacter pylori (Hp)-driven stomach cancer are not fully understood. In a transgenic mouse model of gastric preneoplasia, concomitant Hp infection and induction of constitutively active KRAS (Hp+KRAS+) alters metaplasia phenotypes and elicits greater inflammation than either perturbation alone. Gastric single-cell RNA sequencing showed that Hp+KRAS+ mice had a large population of metaplastic pit cells that expressed the intestinal mucin Muc4 and the growth factor amphiregulin. Flow cytometry and IHC-based immune profiling revealed that metaplastic pit cells were associated with macrophage and T-cell inflammation. Accordingly, expansion of metaplastic pit cells was prevented by gastric immunosuppression and reversed by antibiotic eradication of Hp. Finally, MUC4 expression was significantly associated with proliferation in human gastric cancer samples. These studies identify an Hp-associated metaplastic pit cell lineage, also found in human gastric cancer tissues, whose expansion is driven by Hp-dependent inflammation. Significance: Using a mouse model, we have delineated metaplastic pit cells as a precancerous cell type whose expansion requires Hp-driven inflammation. In humans, metaplastic pit cells show enhanced proliferation as well as enrichment in precancer and early cancer tissues, highlighting an early step in the gastric metaplasia to cancer cascade.


Asunto(s)
Helicobacter pylori , Neoplasias Gástricas , Humanos , Animales , Ratones , Proteínas Proto-Oncogénicas p21(ras) , Modelos Animales de Enfermedad , Inflamación
20.
Elife ; 112022 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-36201241

RESUMEN

Diet-induced obesity leads to dysfunctional feeding behavior. However, the precise molecular nodes underlying diet-induced feeding motivation dysregulation are poorly understood. The fruit fly is a simple genetic model system yet displays significant evolutionary conservation to mammalian nutrient sensing and energy balance. Using a longitudinal high-sugar regime in Drosophila, we sought to address how diet-induced changes in adipocyte lipid composition regulate feeding behavior. We observed that subjecting adult Drosophila to a prolonged high-sugar diet degrades the hunger-driven feeding response. Lipidomics analysis reveals that longitudinal exposure to high-sugar diets significantly alters whole-body phospholipid profiles. By performing a systematic genetic screen for phospholipid enzymes in adult fly adipocytes, we identify Pect as a critical regulator of hunger-driven feeding. Pect is a rate-limiting enzyme in the phosphatidylethanolamine (PE) biosynthesis pathway and the fly ortholog of human PCYT2. We show that disrupting Pect activity only in the Drosophila fat cells causes insulin resistance, dysregulated lipoprotein delivery to the brain, and a loss of hunger-driven feeding. Previously human studies have noted a correlation between PCYT2/Pect levels and clinical obesity. Now, our unbiased studies in Drosophila provide causative evidence for adipocyte Pect function in metabolic homeostasis. Altogether, we have uncovered that PE phospholipid homeostasis regulates hunger response.


Asunto(s)
Drosophila melanogaster , Hambre , Animales , Drosophila , Drosophila melanogaster/genética , Cuerpo Adiposo/metabolismo , Conducta Alimentaria/fisiología , Humanos , Hambre/fisiología , Mamíferos , Obesidad/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolípidos/metabolismo , Azúcares/metabolismo
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