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1.
Open Biol ; 14(10): 240194, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39437839

RESUMO

The FAM83 (Family with sequence similarity 83) family is highly conserved in vertebrates, but little is known of the functions of these proteins beyond their association with oncogenesis. Of the family, FAM83F is of particular interest because it is the only membrane-targeted FAM83 protein. When overexpressed, FAM83F activates the canonical Wnt signalling pathway and binds to and stabilizes p53; it therefore interacts with two pathways often dysregulated in disease. Insights into gene function can often be gained by studying the roles they play during development, and here we report the generation of fam83f knock-out (KO) zebrafish, which we have used to study the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the hatching gland of developing zebrafish embryos, and that fam83f KO embryos hatch earlier than their wild-type (WT) counterparts, despite developing at a comparable rate. We also demonstrate that fam83f KO embryos are more sensitive to ionizing radiation than WT embryos-an unexpected finding, bearing in mind the previously reported ability of FAM83F to stabilize p53. Transcriptomic analysis shows that loss of fam83f leads to downregulation of phosphatidylinositol-3-phosphate (PI(3)P) binding proteins and impairment of cellular degradation pathways, particularly autophagy, a crucial component of the DNA damage response. Finally, we show that Fam83f protein is itself targeted to the lysosome when overexpressed in HEK293T cells, and that this localization is dependent upon a C' terminal signal sequence. The zebrafish lines we have generated suggest that Fam83f plays an important role in autophagic/lysosomal processes, resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo.


Assuntos
Autofagia , Dano ao DNA , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Autofagia/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Técnicas de Inativação de Genes , Fosfatos de Fosfatidilinositol/metabolismo , Humanos , Embrião não Mamífero/metabolismo , Lisossomos/metabolismo
2.
Nat Commun ; 15(1): 4871, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38871738

RESUMO

The phenomenon of mixed/heterogenous treatment responses to cancer therapies within an individual patient presents a challenging clinical scenario. Furthermore, the molecular basis of mixed intra-patient tumor responses remains unclear. Here, we show that patients with metastatic lung adenocarcinoma harbouring co-mutations of EGFR and TP53, are more likely to have mixed intra-patient tumor responses to EGFR tyrosine kinase inhibition (TKI), compared to those with an EGFR mutation alone. The combined presence of whole genome doubling (WGD) and TP53 co-mutations leads to increased genome instability and genomic copy number aberrations in genes implicated in EGFR TKI resistance. Using mouse models and an in vitro isogenic p53-mutant model system, we provide evidence that WGD provides diverse routes to drug resistance by increasing the probability of acquiring copy-number gains or losses relative to non-WGD cells. These data provide a molecular basis for mixed tumor responses to targeted therapy, within an individual patient, with implications for therapeutic strategies.


Assuntos
Instabilidade Cromossômica , Receptores ErbB , Neoplasias Pulmonares , Mutação , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Camundongos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Terapia de Alvo Molecular/métodos , Feminino , Variações do Número de Cópias de DNA , Masculino
3.
J Microsc ; 294(3): 308-318, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38643509

RESUMO

Equipment demonstrations (demos) play an important role in the evaluation of new systems. As well as the excitement of exploring emerging technologies, a well-organised demo can help guide procurement decisions and support funding applications. However, it is easy to underestimate the substantial effort required both before and following the demo to maximise its potential impact. Here, we discuss how our approach to demos at the Crick Advanced Light Microscopy Science and Technology Platform (CALM-STP) has evolved over the last few years, emphasising the importance of a documented approach that combines quantitative with qualitative comparisons and engages with your user base in order to build up support for any potential system purchase.

4.
Life Sci Alliance ; 7(1)2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37884343

RESUMO

The zebrafish (Danio rerio) is an important biomedical model organism used in many disciplines. The phenomenon of developmental delay in zebrafish embryos has been widely reported as part of a mutant or treatment-induced phenotype. However, the detection and quantification of these delays is often achieved through manual observation, which is both time-consuming and subjective. We present KimmelNet, a deep learning model trained to predict embryo age (hours post fertilisation) from 2D brightfield images. KimmelNet's predictions agree closely with established staging methods and can detect developmental delays between populations with high confidence using as few as 100 images. Moreover, KimmelNet generalises to previously unseen data, with transfer learning enhancing its performance. With the ability to analyse tens of thousands of standard brightfield microscopy images on a timescale of minutes, we envisage that KimmelNet will be a valuable resource for the developmental biology community. Furthermore, the approach we have used could easily be adapted to generate models for other organisms.


Assuntos
Aprendizado Profundo , Peixe-Zebra , Animais , Peixe-Zebra/genética , Fenótipo
5.
Nat Cell Biol ; 25(1): 108-119, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36624187

RESUMO

Metastasis involves dissemination of cancer cells away from a primary tumour and colonization at distal sites. During this process, the mechanical properties of the nucleus must be tuned since they pose a challenge to the negotiation of physical constraints imposed by the microenvironment and tissue structure. We discovered increased expression of the inner nuclear membrane protein LAP1 in metastatic melanoma cells, at the invasive front of human primary melanoma tumours and in metastases. Human cells express two LAP1 isoforms (LAP1B and LAP1C), which differ in their amino terminus. Here, using in vitro and in vivo models that recapitulate human melanoma progression, we found that expression of the shorter isoform, LAP1C, supports nuclear envelope blebbing, constrained migration and invasion by allowing a weaker coupling between the nuclear envelope and the nuclear lamina. We propose that LAP1 renders the nucleus highly adaptable and contributes to melanoma aggressiveness.


Assuntos
Melanoma , Membrana Nuclear , Humanos , Isoformas de Proteínas/metabolismo , Movimento Celular , Membrana Nuclear/metabolismo , Melanoma/genética , Melanoma/metabolismo , Microambiente Tumoral
6.
Wellcome Open Res ; 7: 275, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37614774

RESUMO

The zebrafish ( Danio rerio), is an important biomedical model organism used in many disciplines, including development, disease modeling and toxicology, to better understand vertebrate biology. The phenomenon of developmental delay in zebrafish embryos has been widely reported as part of a mutant or treatment-induced phenotype, and accurate characterization of such delays is imperative. Despite this, the only way at present to identify and quantify these delays is through manual observation, which is both time-consuming and subjective. Machine learning approaches in biology are rapidly becoming part of the toolkit used by researchers to address complex questions. In this work, we introduce a machine learning-based classifier that has been trained to detect temporal developmental differences across groups of zebrafish embryos. Our classifier is capable of rapidly analyzing thousands of images, allowing comparisons of developmental temporal rates to be assessed across and between experimental groups of embryos. Finally, as our classifier uses images obtained from a standard live-imaging widefield microscope and camera set-up, we envisage it will be readily accessible to the zebrafish community, and prove to be a valuable resource.

7.
mBio ; 12(2)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33688001

RESUMO

Malaria parasites cause disease through repeated cycles of intraerythrocytic proliferation. Within each cycle, several rounds of DNA replication produce multinucleated forms, called schizonts, that undergo segmentation to form daughter merozoites. Upon rupture of the infected cell, the merozoites egress to invade new erythrocytes and repeat the cycle. In human malarial infections, an antibody response specific for the Plasmodium falciparum protein PF3D7_1021800 was previously associated with protection against malaria, leading to an interest in PF3D7_1021800 as a candidate vaccine antigen. Antibodies to the protein were reported to inhibit egress, resulting in it being named schizont egress antigen-1 (SEA1). A separate study found that SEA1 undergoes phosphorylation in a manner dependent upon the parasite cGMP-dependent protein kinase PKG, which triggers egress. While these findings imply a role for SEA1 in merozoite egress, this protein has also been implicated in kinetochore function during schizont development. Therefore, the function of SEA1 remains unclear. Here, we show that P. falciparum SEA1 localizes in proximity to centromeres within dividing nuclei and that conditional disruption of SEA1 expression severely impacts the distribution of DNA and formation of merozoites during schizont development, with a proportion of SEA1-null merozoites completely lacking nuclei. SEA1-null schizonts rupture, albeit with low efficiency, suggesting that neither SEA1 function nor normal segmentation is a prerequisite for egress. We conclude that SEA1 does not play a direct mechanistic role in egress but instead acts upstream of egress as an essential regulator required to ensure the correct packaging of nuclei within merozoites.IMPORTANCE Malaria is a deadly infectious disease. Rationally designed novel therapeutics will be essential for its control and eradication. The Plasmodium falciparum protein PF3D7_1021800, annotated as SEA1, is under investigation as a prospective component of a malaria vaccine, based on previous indications that antibodies to SEA1 interfere with parasite egress from infected erythrocytes. However, a consensus on the function of SEA1 is lacking. Here, we demonstrate that SEA1 localizes to dividing parasite nuclei and is necessary for the correct segregation of replicated DNA into individual daughter merozoites. In the absence of SEA1, merozoites develop defectively, often completely lacking a nucleus, and, consequently, egress is impaired and/or aberrant. Our findings provide insights into the divergent mechanisms by which intraerythrocytic malaria parasites develop and divide. Our conclusions regarding the localization and function of SEA1 are not consistent with the hypothesis that antibodies against it confer protective immunity to malaria by blocking merozoite egress.


Assuntos
Antígenos de Protozoários/genética , Eritrócitos/parasitologia , Merozoítos/genética , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/genética , Esquizontes/fisiologia , Antígenos de Protozoários/metabolismo , Divisão Celular , Humanos , Merozoítos/química , Fosforilação , Plasmodium falciparum/química , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Estudos Prospectivos , Proteínas de Protozoários/metabolismo
8.
Nat Commun ; 11(1): 1746, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269212

RESUMO

In mammalian cell lines, the endosomal sorting complex required for transport (ESCRT)-III mediates abscission, the process that physically separates daughter cells and completes cell division. Cep55 protein is regarded as the master regulator of abscission, because it recruits ESCRT-III to the midbody (MB), the site of abscission. However, the importance of this mechanism in a mammalian organism has never been tested. Here we show that Cep55 is dispensable for mouse embryonic development and adult tissue homeostasis. Cep55-knockout offspring show microcephaly and primary neural progenitors require Cep55 and ESCRT for survival and abscission. However, Cep55 is dispensable for cell division in embryonic or adult tissues. In vitro, division of primary fibroblasts occurs without Cep55 and ESCRT-III at the midbody and is not affected by ESCRT depletion. Our work defines Cep55 as an abscission regulator only in specific tissue contexts and necessitates the re-evaluation of an alternative ESCRT-independent cell division mechanism.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Células Cultivadas , Córtex Cerebral/anormalidades , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fibroblastos/metabolismo , Deleção de Genes , Genótipo , Rim/anormalidades , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcefalia/patologia , Mitose
9.
J Biol Chem ; 294(49): 18639-18649, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31653703

RESUMO

Rigorous spatiotemporal regulation of cell division is required to maintain genome stability. The final stage in cell division, when the cells physically separate (abscission), is tightly regulated to ensure that it occurs after cytokinetic events such as chromosome segregation. A key regulator of abscission timing is Aurora B kinase activity, which inhibits abscission and forms the major activity of the abscission checkpoint. This checkpoint prevents abscission until chromosomes have been cleared from the cytokinetic machinery. Here we demonstrate that the mitosis-specific CDK11p58 kinase specifically forms a complex with cyclin L1ß that, in late cytokinesis, localizes to the stem body, a structure in the middle of the intercellular bridge that forms between two dividing cells. Depletion of CDK11 inhibits abscission, and rescue of this phenotype requires CDK11p58 kinase activity or inhibition of Aurora B kinase activity. Furthermore, CDK11p58 kinase activity is required for formation of endosomal sorting complex required for transport III filaments at the site of abscission. Combined, these data suggest that CDK11p58 kinase activity opposes Aurora B activity to enable abscission to proceed and result in successful completion of cytokinesis.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Western Blotting , Divisão Celular/genética , Divisão Celular/fisiologia , Segregação de Cromossomos/genética , Segregação de Cromossomos/fisiologia , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Citocinese/genética , Citocinese/fisiologia , Imunofluorescência , Células HeLa , Humanos , Mitose/genética , Mitose/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Imagem com Lapso de Tempo
10.
Open Biol ; 4: 130190, 2014 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-24451548

RESUMO

The final step of cytokinesis is abscission when the intercellular bridge (ICB) linking the two new daughter cells is broken. Correct construction of the ICB is crucial for the assembly of factors involved in abscission, a failure in which results in aneuploidy. Using live imaging and subdiffraction microscopy, we identify new anillin-septin cytoskeleton-dependent stages in ICB formation and maturation. We show that after the formation of an initial ICB, septin filaments drive ICB elongation during which tubules containing anillin-septin rings are extruded from the ICB. Septins then generate sites of further constriction within the mature ICB from which they are subsequently removed. The action of the anillin-septin complex during ICB maturation also primes the ICB for the future assembly of the ESCRT III component Chmp4B at the abscission site. These studies suggest that the sequential action of distinct contractile machineries coordinates the formation of the abscission site and the successful completion of cytokinesis.


Assuntos
Proteínas Contráteis/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Septinas/metabolismo , Segregação de Cromossomos , Proteínas Contráteis/antagonistas & inibidores , Proteínas Contráteis/genética , Citocinese , Células HeLa , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Septinas/antagonistas & inibidores , Septinas/genética
11.
EMBO J ; 30(10): 1919-27, 2011 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-21505420

RESUMO

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites.


Assuntos
Adenosina Trifosfatases/metabolismo , Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Sítios de Ligação , Segregação de Cromossomos , DNA Fúngico/genética , DNA Fúngico/metabolismo , Deleção de Genes , Ligação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
12.
Dev Cell ; 19(2): 232-44, 2010 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-20708586

RESUMO

Sister chromatid separation is initiated at anaphase onset by the activation of separase, which removes cohesins from chromosomes. However, it remains elusive how sister chromatid separation is completed along the entire chromosome length. Here we found that, during early anaphase in Saccharomyces cerevisiae, sister chromatids separate gradually from centromeres to telomeres, accompanied by regional chromosome stretching and subsequent recoiling. The stretching results from residual cohesion between sister chromatids, which prevents their immediate separation. This residual cohesion is at least partly dependent on cohesins that have escaped removal by separase at anaphase onset. Meanwhile, recoiling of a stretched chromosome region requires condensins and generates forces to remove residual cohesion. We provide evidence that condensins promote chromosome recoiling directly in vivo, which is distinct from their known function in resolving sister chromatids. Our work identifies residual sister chromatid cohesion during early anaphase and reveals condensins' roles in chromosome recoiling, which eliminates residual cohesion to complete sister chromatid separation.


Assuntos
Adenosina Trifosfatases/metabolismo , Anáfase , Cromátides/metabolismo , Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Adenosina Trifosfatases/genética , Cromossomos/química , Proteínas de Ligação a DNA/genética , Complexos Multiproteicos/genética , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
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