Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 48.167
Filtrar
1.
Sci Rep ; 14(1): 12998, 2024 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-38844535

RESUMO

The challenge of in-situ handling and high-resolution low-dose imaging of intact, sensitive and wet samples in their native state at nanometer scale, including live samples is met by Advanced Environmental Scanning Electron Microscopy (A-ESEM). This new generation of ESEM utilises machine learning-based optimization of thermodynamic conditions with respect to sample specifics to employ a low temperature method and an ionization secondary electron detector with an electrostatic separator. A modified electron microscope was used, equipped with temperature, humidity and gas pressure sensors for in-situ and real-time monitoring of the sample. A transparent ultra-thin film of ionic liquid is used to increase thermal and electrical conductivity of the samples and to minimize sample damage by free radicals. To validate the power of the new method, we analyze condensed mitotic metaphase chromosomes to reveal new structural features of their perichromosomal layer, and the organization of chromatin fibers, not observed before by any microscopic technique. The ability to resolve nano-structural details of chromosomes using A-ESEM is validated by measuring gold nanoparticles with achievable resolution in the lower nanometre units.


Assuntos
Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Varredura/métodos , Humanos , Ouro/química , Nanopartículas Metálicas/química , Mitose , Cromossomos/ultraestrutura
2.
PLoS Genet ; 20(6): e1011302, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38829899

RESUMO

Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1Δ or mad2Δ strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.


Assuntos
Proteínas de Ciclo Celular , Cryptococcus neoformans , Proteínas Mad2 , Fuso Acromático , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Mad2/metabolismo , Proteínas Mad2/genética , Fuso Acromático/metabolismo , Fuso Acromático/genética , Transdução de Sinais , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Mitose/genética , Cinetocoros/metabolismo , Segregação de Cromossomos/genética , Microtúbulos/metabolismo , Microtúbulos/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética
3.
Proc Natl Acad Sci U S A ; 121(25): e2323009121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38875144

RESUMO

Error correction is central to many biological systems and is critical for protein function and cell health. During mitosis, error correction is required for the faithful inheritance of genetic material. When functioning properly, the mitotic spindle segregates an equal number of chromosomes to daughter cells with high fidelity. Over the course of spindle assembly, many initially erroneous attachments between kinetochores and microtubules are fixed through the process of error correction. Despite the importance of chromosome segregation errors in cancer and other diseases, there is a lack of methods to characterize the dynamics of error correction and how it can go wrong. Here, we present an experimental method and analysis framework to quantify chromosome segregation error correction in human tissue culture cells with live cell confocal imaging, timed premature anaphase, and automated counting of kinetochores after cell division. We find that errors decrease exponentially over time during spindle assembly. A coarse-grained model, in which errors are corrected in a chromosome-autonomous manner at a constant rate, can quantitatively explain both the measured error correction dynamics and the distribution of anaphase onset times. We further validated our model using perturbations that destabilized microtubules and changed the initial configuration of chromosomal attachments. Taken together, this work provides a quantitative framework for understanding the dynamics of mitotic error correction.


Assuntos
Segregação de Cromossomos , Cinetocoros , Microtúbulos , Mitose , Fuso Acromático , Humanos , Cinetocoros/metabolismo , Fuso Acromático/metabolismo , Microtúbulos/metabolismo , Anáfase , Modelos Biológicos , Células HeLa
4.
J Exp Clin Cancer Res ; 43(1): 159, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38840237

RESUMO

BACKGROUND: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. EXPERIMENTAL DESIGN: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. RESULTS: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. CONCLUSION: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.


Assuntos
Carcinoma de Células Renais , Reparo do DNA , Neoplasias Renais , Survivina , Serina-Treonina Quinases TOR , Ensaios Antitumorais Modelo de Xenoenxerto , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/radioterapia , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Animais , Survivina/metabolismo , Humanos , Camundongos , Linhagem Celular Tumoral , Neoplasias Renais/patologia , Neoplasias Renais/radioterapia , Neoplasias Renais/tratamento farmacológico , Reparo do DNA/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Imidazóis/farmacologia , Dano ao DNA , Everolimo/farmacologia , Naftoquinonas/farmacologia , Naftoquinonas/uso terapêutico , Radiossensibilizantes/farmacologia , Radiossensibilizantes/uso terapêutico , Lipossomos/farmacologia , Inibidores de MTOR/farmacologia , Inibidores de MTOR/uso terapêutico
5.
Elife ; 132024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38864853

RESUMO

Cohesin is a multi-subunit protein that plays a pivotal role in holding sister chromatids together during cell division. Sister chromatid cohesion 3 (SCC3), constituents of cohesin complex, is highly conserved from yeast to mammals. Since the deletion of individual cohesin subunit always causes lethality, it is difficult to dissect its biological function in both mitosis and meiosis. Here, we obtained scc3 weak mutants using CRISPR-Cas9 system to explore its function during rice mitosis and meiosis. The scc3 weak mutants displayed obvious vegetative defects and complete sterility, underscoring the essential roles of SCC3 in both mitosis and meiosis. SCC3 is localized on chromatin from interphase to prometaphase in mitosis. However, in meiosis, SCC3 acts as an axial element during early prophase I and subsequently situates onto centromeric regions following the disassembly of the synaptonemal complex. The loading of SCC3 onto meiotic chromosomes depends on REC8. scc3 shows severe defects in homologous pairing and synapsis. Consequently, SCC3 functions as an axial element that is essential for maintaining homologous chromosome pairing and synapsis during meiosis.


Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Pareamento Cromossômico , Meiose , Oryza , Meiose/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Oryza/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Coesinas , Mitose , Complexo Sinaptonêmico/metabolismo , Complexo Sinaptonêmico/genética , Sistemas CRISPR-Cas
6.
Ren Fail ; 46(2): 2365408, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38874119

RESUMO

Podocyte loss in glomeruli is a fundamental event in the pathogenesis of chronic kidney diseases. Currently, mitotic catastrophe (MC) has emerged as the main cause of podocyte loss. However, the regulation of MC in podocytes has yet to be elucidated. The current work aimed to study the role and mechanism of p53 in regulating the MC of podocytes using adriamycin (ADR)-induced nephropathy. In vitro podocyte stimulation with ADR triggered the occurrence of MC, which was accompanied by hyperactivation of p53 and cyclin-dependent kinase (CDK1)/cyclin B1. The inhibition of p53 reversed ADR-evoked MC in podocytes and protected against podocyte injury and loss. Further investigation showed that p53 mediated the activation of CDK1/cyclin B1 by regulating the expression of Wee1. Restraining Wee1 abolished the regulatory effect of p53 inhibition on CDK1/cyclin B1 and rebooted MC in ADR-stimulated podocytes via p53 inhibition. In a mouse model of ADR nephropathy, the inhibition of p53 ameliorated proteinuria and podocyte injury. Moreover, the inhibition of p53 blocked the progression of MC in podocytes in ADR nephropathy mice through the regulation of the Wee1/CDK1/cyclin B1 axis. Our findings confirm that p53 contributes to MC in podocytes through regulation of the Wee1/CDK1/Cyclin B1 axis, which may represent a novel mechanism underlying podocyte injury and loss during the progression of chronic kidney disorder.


Assuntos
Proteína Quinase CDC2 , Proteínas de Ciclo Celular , Ciclina B1 , Doxorrubicina , Mitose , Podócitos , Proteínas Tirosina Quinases , Proteína Supressora de Tumor p53 , Podócitos/metabolismo , Podócitos/patologia , Animais , Proteína Quinase CDC2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Camundongos , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Doxorrubicina/farmacologia , Ciclina B1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Humanos , Masculino
7.
Arch Dermatol Res ; 316(5): 195, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38775978

RESUMO

Chronic arsenic exposure is a global health hazard significantly associated with the development of deleterious cutaneous changes and increased keratinocyte cancer risk. Although arsenic exposure is associated with broad-scale cellular and molecular changes, gaps exist in understanding how these changes impact the skin and facilitate malignant transformation. Recently developed epigenetic "clocks" can accurately predict chronological, biological and mitotic age, as well as telomere length, on the basis of tissue DNA methylation state. Deviations of predicted from expected age (epigenetic age dysregulation) have been associated with numerous complex diseases, increased all-cause mortality and higher cancer risk. We investigated the ability of these algorithms to detect molecular changes associated with chronic arsenic exposure in the context of associated skin lesions. To accomplish this, we utilized a multi-algorithmic approach incorporating seven "clocks" (Horvath, Skin&Blood, PhenoAge, PCPhenoAge, GrimAge, DNAmTL and epiTOC2) to analyze peripheral blood of pediatric and adult cohorts of arsenic-exposed (n = 84) and arsenic-naïve (n = 33) individuals, among whom n = 18 were affected by skin lesions. Arsenic-exposed adults with skin lesions exhibited accelerated epigenetic (Skin&Blood: + 7.0 years [95% CI 3.7; 10.2], q = 6.8 × 10-4), biological (PhenoAge: + 5.8 years [95% CI 0.7; 11.0], q = 7.4 × 10-2, p = 2.8 × 10-2) and mitotic age (epiTOC2: + 19.7 annual cell divisions [95% CI 1.8; 37.7], q = 7.4 × 10-2, p = 3.2 × 10-2) compared to healthy arsenic-naïve individuals; and accelerated epigenetic age (Skin&Blood: + 2.8 years [95% CI 0.2; 5.3], q = 2.4 × 10-1, p = 3.4 × 10-2) compared to lesion-free arsenic-exposed individuals. Moreover, lesion-free exposed adults exhibited accelerated Skin&Blood age (+ 4.2 [95% CI 1.3; 7.1], q = 3.8 × 10-2) compared to their arsenic-naïve counterparts. Compared to the pediatric group, arsenic-exposed adults exhibited accelerated epigenetic (+ 3.1 to 4.4 years (95% CI 1.2; 6.4], q = 2.4 × 10-4-3.1 × 10-3), biological (+ 7.4 to 7.8 years [95% CI 3.0; 12.1] q = 1.6 × 10-3-2.8 × 10-3) and mitotic age (+ 50.0 annual cell divisions [95% CI 15.6; 84.5], q = 7.8 × 10-3), as well as shortened telomere length (- 0.23 kilobases [95% CI - 0.13; - 0.33], q = 2.4 × 10-4), across all seven algorithms. We demonstrate that lifetime arsenic exposure and presence of arsenic-associated skin lesions are associated with accelerated epigenetic, biological and mitotic age, and shortened telomere length, reflecting altered immune signaling and genomic regulation. Our findings highlight the usefulness of DNA methylation-based algorithms in identifying deleterious molecular changes associated with chronic exposure to the heavy metal, serving as potential prognosticators of arsenic-induced cutaneous malignancy.


Assuntos
Arsênio , Metilação de DNA , Epigênese Genética , Encurtamento do Telômero , Humanos , Adulto , Arsênio/efeitos adversos , Arsênio/toxicidade , Feminino , Metilação de DNA/efeitos dos fármacos , Encurtamento do Telômero/efeitos dos fármacos , Masculino , Criança , Adolescente , Adulto Jovem , Pessoa de Meia-Idade , Mitose/efeitos dos fármacos , Mitose/genética , Pele/patologia , Pele/efeitos dos fármacos , Dermatopatias/induzido quimicamente , Dermatopatias/genética , Dermatopatias/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/patologia
8.
Open Biol ; 14(5): 230460, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38806145

RESUMO

The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.


Assuntos
Aurora Quinase B , Proteínas de Ciclo Celular , Histonas , Mitose , Proteína Fosfatase 1 , Aurora Quinase B/metabolismo , Fosforilação , Humanos , Histonas/metabolismo , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Células HeLa , Fuso Acromático/metabolismo , Centrômero/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética
9.
Med Image Anal ; 95: 103204, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38761438

RESUMO

Due to the intra-class diversity of mitotic cells and the morphological overlap with similarly looking imposters, automatic mitosis detection in histopathology slides is still a challenging task. In this paper, we propose a novel mitosis detection model in a weakly supervised way, which consists of a candidate proposal network and a verification network. The candidate proposal network based on patch learning aims to separate both mitotic cells and their mimics from the background as candidate objects, which substantially reduces missed detections in the screening process of candidates. These obtained candidate results are then fed into the verification network for mitosis refinement. The verification network adopts an RBF-based subcategorization scheme to deal with the problems of high intra-class variability of mitosis and the mimics with similar appearance. We utilize the RBF centers to define subcategories containing mitotic cells with similar properties and capture representative RBF center locations through joint training of classification and clustering. Due to the lower intra-class variation within a subcategory, the localized feature space at subcategory level can better characterize a certain type of mitotic figures and can provide a better similarity measurement for distinguishing mitotic cells from nonmitotic cells. Our experiments manifest that this subcategorization scheme helps improve the performance of mitosis detection and achieves state-of-the-art results on the publicly available mitosis datasets using only weak labels.


Assuntos
Neoplasias da Mama , Mitose , Mitose/fisiologia , Humanos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Feminino , Interpretação de Imagem Assistida por Computador/métodos , Algoritmos , Aprendizado Profundo
10.
Cell Cycle ; 23(5): 613-627, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38752903

RESUMO

Ubiquitin like with PHD and ring finger domains 2 (UHRF2) regulates the cell cycle and epigenetics as a multi-domain protein sharing homology with UHRF1. UHRF1 functions with DNMT1 to coordinate daughter strand methylation during DNA replication, but UHRF2 can't perform this function, and its roles during cell cycle progression are not well defined. UHRF2 role as an oncogene vs. tumor suppressor differs in distinct cell types. UHRF2 interacts with E2F1 to control Cyclin E1 (CCNE1) transcription. UHRF2 also functions in a reciprocal loop with Cyclin E/CDK2 during G1, first as a direct target of CDK2 phosphorylation, but also as an E3-ligase with direct activity toward both Cyclin E and Cyclin D. In this study, we demonstrate that UHRF2 is expressed in early G1 following either serum stimulation out of quiescence or in cells transiting directly out of M-phase, where UHRF2 protein is lost. Further, UHRF2 depletion in G2/M is reversed with a CDK1 specific inhibitor. UHRF2 controls expression levels of cyclins and CDK inhibitors and controls its own transcription in a negative-feedback loop. Deletion of UHRF2 using CRISPR/Cas9 caused a delay in passage through each cell cycle phase. UHRF2 loss culminated in elevated levels of cyclins but also the CDK inhibitor p27KIP1, which regulates G1 passage, to reduce retinoblastoma phosphorylation and increase the amount of time required to reach G1/S passage. Our data indicate that UHRF2 is a central regulator of cell-cycle pacing through its complex regulation of cell cycle gene expression and protein stability.


Assuntos
Ciclina E , Fase G1 , Mitose , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Humanos , Ciclina E/metabolismo , Ciclina E/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Ciclo Celular/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 2 Dependente de Ciclina/genética , Fosforilação , Proteínas Oncogênicas
11.
Nature ; 630(8015): 214-221, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38811726

RESUMO

The canonical mitotic cell cycle coordinates DNA replication, centriole duplication and cytokinesis to generate two cells from one1. Some cells, such as mammalian trophoblast giant cells, use cell cycle variants like the endocycle to bypass mitosis2. Differentiating multiciliated cells, found in the mammalian airway, brain ventricles and reproductive tract, are post-mitotic but generate hundreds of centrioles, each of which matures into a basal body and nucleates a motile cilium3,4. Several cell cycle regulators have previously been implicated in specific steps of multiciliated cell differentiation5,6. Here we show that differentiating multiciliated cells integrate cell cycle regulators into a new alternative cell cycle, which we refer to as the multiciliation cycle. The multiciliation cycle redeploys many canonical cell cycle regulators, including cyclin-dependent kinases (CDKs) and their cognate cyclins. For example, cyclin D1, CDK4 and CDK6, which are regulators of mitotic G1-to-S progression, are required to initiate multiciliated cell differentiation. The multiciliation cycle amplifies some aspects of the canonical cell cycle, such as centriole synthesis, and blocks others, such as DNA replication. E2F7, a transcriptional regulator of canonical S-to-G2 progression, is expressed at high levels during the multiciliation cycle. In the multiciliation cycle, E2F7 directly dampens the expression of genes encoding DNA replication machinery and terminates the S phase-like gene expression program. Loss of E2F7 causes aberrant acquisition of DNA synthesis in multiciliated cells and dysregulation of multiciliation cycle progression, which disrupts centriole maturation and ciliogenesis. We conclude that multiciliated cells use an alternative cell cycle that orchestrates differentiation instead of controlling proliferation.


Assuntos
Ciclo Celular , Diferenciação Celular , Centríolos , Cílios , Cílios/metabolismo , Animais , Centríolos/metabolismo , Camundongos , Humanos , Replicação do DNA , Mitose , Quinases Ciclina-Dependentes/metabolismo , Feminino , Ciclinas/metabolismo , Masculino
12.
mBio ; 15(6): e0067624, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38722185

RESUMO

An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK + HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting-dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting-dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild-type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. E2-TopBP1 interaction promotes mitotic acetylation of CHK2, promoting phosphorylation and activation of the DNA damage response (DDR). The results present a new model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis, and activates the DDR. This is a novel mechanism of HPV16 activation of the DDR, a requirement for the viral life cycle. IMPORTANCE: Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here, we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. We also demonstrate that the E2-TopBP1 interaction activates the DDR. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.


Assuntos
Proteínas de Transporte , Dano ao DNA , Proteínas de Ligação a DNA , Papillomavirus Humano 16 , Mitose , Proteínas Oncogênicas Virais , Sirtuína 1 , Humanos , Acetilação , Sirtuína 1/metabolismo , Sirtuína 1/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas Oncogênicas Virais/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Interações Hospedeiro-Patógeno , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteína p300 Associada a E1A/metabolismo , Proteína p300 Associada a E1A/genética , Linhagem Celular
13.
Cancer Lett ; 593: 216970, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38763475

RESUMO

Venetoclax is a BCL2-targeted drug employed in treating various cancers, particularly hematologic malignancies. Venetoclax combination therapies are increasingly recognized as promising treatment strategies for acute myeloid leukemia (AML). In this study, we conducted an unbiased drug screen and identified anlotinib, a promising multi-targeted receptor tyrosine kinase inhibitor with oral activity currently utilized in the treatment of solid tumor, as a potent enhancer of venetoclax's anticancer activity in AML. Our investigation encompassed AML cell lines, primary cells, and mouse models, demonstrating effective low-dose combination therapy of anlotinib and venetoclax with minimal cytopenia or organ damage. Proteomic analysis revealed abnormal mitotic signals induced by this combination in AML cells. Mechanistically, anlotinib synergized with venetoclax by suppressing ARPP19 protein, leading to sustained activation of PP2A-B55δ. This inhibited AML cells from entering the mitotic phase, culminating in mitotic catastrophe and apoptosis. Additionally, we identified a specific synthetic lethal vulnerability in AML involving an ARPP19 mutation at S62 phosphorylation. These findings underscore the therapeutic potential of anlotinib and venetoclax combination therapy in AML, warranting further clinical investigation.


Assuntos
Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes , Sinergismo Farmacológico , Indóis , Leucemia Mieloide Aguda , Mitose , Quinolinas , Sulfonamidas , Ensaios Antitumorais Modelo de Xenoenxerto , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Humanos , Sulfonamidas/farmacologia , Sulfonamidas/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Animais , Quinolinas/farmacologia , Quinolinas/administração & dosagem , Mitose/efeitos dos fármacos , Camundongos , Indóis/farmacologia , Indóis/administração & dosagem , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico
14.
J Cell Sci ; 137(11)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38770570

RESUMO

Dinoflagellates are marine organisms that undergo seasonal proliferation events known as algal blooms. Vegetative cell proliferation is a main contributing factor in these events. However, mechanistical understanding of mitosis and cytokinesis in dinoflagellates remains rudimentary. Using an optimized immunofluorescence protocol, we analysed changes in microtubule organization occurring during the mitotic cycle of the toxic dinoflagellate Ostreopsis cf. ovata. We find that the flagella and the cortical microtubule array persist throughout the mitotic cycle. Two cytoplasmic microtubule bundles originate from the ventral area, where the basal bodies are located - a cortical bundle and a cytoplasmic bundle. The latter associates with the nucleus in the cell centre before mitosis and with the acentrosomal extranuclear spindle during mitosis. Analysis of tubulin post-translational modifications identifies two populations of spindle microtubules - polar acetylated microtubules, whose length is constant, and central tyrosinated microtubules, which elongate during chromosome segregation. During cell division a microtubule-rich structure forms along the dorsal-ventral axis, associated with the site of cytokinesis, consistent with a cytokinetic mechanism that is independent of the actomyosin ring typical of animal and yeast cells.


Assuntos
Dinoflagellida , Microtúbulos , Mitose , Microtúbulos/metabolismo , Dinoflagellida/metabolismo , Dinoflagellida/citologia , Citocinese , Fuso Acromático/metabolismo , Divisão Celular , Tubulina (Proteína)/metabolismo
15.
DNA Repair (Amst) ; 139: 103692, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38759435

RESUMO

Over the past few decades, unbiased approaches such as genetic screening and protein affinity purification have unveiled numerous proteins involved in DNA double-strand break (DSB) repair and maintaining genome stability. However, despite our knowledge of these protein factors, the underlying molecular mechanisms governing key cellular events during DSB repair remain elusive. Recent evidence has shed light on the role of non-protein factors, such as RNA, in several pivotal steps of DSB repair. In this review, we provide a comprehensive summary of these recent findings, highlighting the significance of ribosomal RNA (rRNA) as a critical mediator of DNA damage response, meiosis, and mitosis. Moreover, we discuss potential mechanisms through which rRNA may influence genome integrity.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Instabilidade Genômica , RNA Ribossômico , Humanos , RNA Ribossômico/metabolismo , RNA Ribossômico/genética , Animais , Mitose , Meiose
16.
J Inorg Biochem ; 257: 112610, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38761580

RESUMO

Drug resistance has been a major problem for cancer chemotherapy, especially for glioblastoma multiforme that is aggressive, heterogeneous and recurrent with <3% of a five-year survival and limited methods of clinical treatment. To overcome the problem, great efforts have recently been put in searching for agents inducing death of tumor cells via various non-apoptotic pathways. In the present work, we report for the first time that vanadyl complexes, i.e. bis(acetylacetonato)oxidovanadium (IV) (VO(acac)2), can cause mitotic catastrophe and methuotic death featured by catastrophic macropinocytic vacuole accumulation particularly in glioblastoma cells (GCs). Hence, VO(acac)2 strongly suppressed growth of GCs with both in vitro (IC50 = 4-6 µM) and in vivo models, and is much more potent than the current standard-of-care drug Temozolomide. The selective index is as high as ∼10 or more on GCs over normal neural cells. Importantly, GCs respond well to vanadium treatment regardless whether they are carrying IDH1 wild type gene that causes drug resistance. VO(acac)2 may induce methuosis via the Rac-Mitogen-activated protein kinase kinase 4 (MKK4)-c-Jun N-terminal kinase (JNK) signaling pathway. Furthermore, VO(acac)2-induced methuosis is not through a immunogenicity mechanism, making vanadyl complexes safe for interventional therapy. Overall, our results may encourage development of novel vanadium complexes promising for treatment of neural malignant tumor cells.


Assuntos
Complexos de Coordenação , Glioblastoma , Mitose , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Humanos , Mitose/efeitos dos fármacos , Animais , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Camundongos , Vanadatos/farmacologia , Vanadatos/química , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Camundongos Nus
17.
Nat Commun ; 15(1): 4211, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38760334

RESUMO

The cumulative number of stem cell divisions in a tissue, known as mitotic age, is thought to be a major determinant of cancer-risk. Somatic mutational and DNA methylation (DNAm) clocks are promising tools to molecularly track mitotic age, yet their relationship is underexplored and their potential for cancer risk prediction in normal tissues remains to be demonstrated. Here we build and validate an improved pan-tissue DNAm counter of total mitotic age called stemTOC. We demonstrate that stemTOC's mitotic age proxy increases with the tumor cell-of-origin fraction in each of 15 cancer-types, in precancerous lesions, and in normal tissues exposed to major cancer risk factors. Extensive benchmarking against 6 other mitotic counters shows that stemTOC compares favorably, specially in the preinvasive and normal-tissue contexts. By cross-correlating stemTOC to two clock-like somatic mutational signatures, we confirm the mitotic-like nature of only one of these. Our data points towards DNAm as a promising molecular substrate for detecting mitotic-age increases in normal tissues and precancerous lesions, and hence for developing cancer-risk prediction strategies.


Assuntos
Metilação de DNA , Epigênese Genética , Mitose , Mutação , Neoplasias , Lesões Pré-Cancerosas , Humanos , Mitose/genética , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Neoplasias/genética , Neoplasias/patologia , Células-Tronco/metabolismo
18.
Cell Death Dis ; 15(5): 342, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38760378

RESUMO

U3 snoRNA is essential for ribosome biogenesis during interphase. Upon mitotic onset, the nucleolus disassembles and U3 snoRNA relocates to the perichromosomal region (PR) to be considered as a chromosome passenger. Whether U3 controls mitosis remains unknown. Here, we demonstrate that U3 snoRNA is required for mitotic progression. We identified DDX21 as the predominant U3-binding protein during mitosis and confirmed that U3 snoRNA colocalizes with DDX21 in the PR. DDX21 knockdown induces mitotic catastrophe and similar mitotic defects caused by U3 snoRNA depletion. Interestingly, the uniform PR distribution of U3 snoRNA and DDX21 is interdependent. DDX21 functions in mitosis depending on its PR localization. Mechanistically, U3 snoRNA regulates DDX21 PR localization through maintaining its mobility. Moreover, Cy5-U3 snoRNA downsizes the fibrous condensates of His-DDX21 at proper molecular ratios in vitro. This work highlights the importance of the equilibrium between U3 snoRNA and DDX21 in PR formation and reveals the potential relationship between the PR assembly and mitotic regulation.


Assuntos
RNA Helicases DEAD-box , Mitose , RNA Nucleolar Pequeno , Humanos , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , RNA Nucleolar Pequeno/metabolismo , RNA Nucleolar Pequeno/genética , Células HeLa
19.
Nature ; 630(8015): 116-122, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38778110

RESUMO

Eukaryotes have evolved towards one of two extremes along a spectrum of strategies for remodelling the nuclear envelope during cell division: disassembling the nuclear envelope in an open mitosis or constructing an intranuclear spindle in a closed mitosis1,2. Both classes of mitotic remodelling involve key differences in the core division machinery but the evolutionary reasons for adopting a specific mechanism are unclear. Here we use an integrated comparative genomics and ultrastructural imaging approach to investigate mitotic strategies in Ichthyosporea, close relatives of animals and fungi. We show that species in this clade have diverged towards either a fungal-like closed mitosis or an animal-like open mitosis, probably to support distinct multinucleated or uninucleated states. Our results indicate that multinucleated life cycles favour the evolution of closed mitosis.


Assuntos
Evolução Biológica , Estágios do Ciclo de Vida , Mesomycetozoea , Mitose , Filogenia , Animais , Genômica , Mesomycetozoea/genética , Mesomycetozoea/fisiologia , Mesomycetozoea/citologia , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Fuso Acromático/metabolismo , Fungos/classificação
20.
J Cell Biol ; 223(9)2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-38781028

RESUMO

Maintenance of ploidy depends on the mitotic kinase Aurora B, the catalytic subunit of the chromosomal passenger complex (CPC) whose proficient activity is supported by HP1 enriched at inner centromeres. HP1 is known to associate with INCENP of the CPC in a manner that depends on the PVI motif conserved across HP1 interactors. Here, we found that the interaction of INCENP with HP1 requires not only the PVI motif but also its C-terminally juxtaposed domain. Remarkably, these domains conditionally fold the ß-strand (PVI motif) and the α-helix from a disordered sequence upon HP1 binding and render INCENP with high affinity to HP1. This bipartite binding domain termed SSH domain (Structure composed of Strand and Helix) is necessary and sufficient to attain a predominant interaction of HP1 with INCENP. These results identify a unique HP1-binding module in INCENP that ensures enrichment of HP1 at inner centromeres, Aurora B activity, and thereby mitotic fidelity.


Assuntos
Aurora Quinase B , Centrômero , Homólogo 5 da Proteína Cromobox , Ligação Proteica , Humanos , Aurora Quinase B/metabolismo , Aurora Quinase B/genética , Sítios de Ligação , Centrômero/metabolismo , Homólogo 5 da Proteína Cromobox/genética , Homólogo 5 da Proteína Cromobox/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Mitose
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...