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1.
Elife ; 92020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-33284105

RESUMO

How bulk cytoplasm generates forces to separate post-anaphase microtubule (MT) asters in Xenopus laevis and other large eggs remains unclear. Previous models proposed that dynein-based, inward organelle transport generates length-dependent pulling forces that move centrosomes and MTs outwards, while other components of cytoplasm are static. We imaged aster movement by dynein and actomyosin forces in Xenopus egg extracts and observed outward co-movement of MTs, endoplasmic reticulum (ER), mitochondria, acidic organelles, F-actin, keratin, and soluble fluorescein. Organelles exhibited a burst of dynein-dependent inward movement at the growing aster periphery, then mostly halted inside the aster, while dynein-coated beads moved to the aster center at a constant rate, suggesting organelle movement is limited by brake proteins or other sources of drag. These observations call for new models in which all components of the cytoplasm comprise a mechanically integrated aster gel that moves collectively in response to dynein and actomyosin forces.


Assuntos
Actinas/metabolismo , Actomiosina/metabolismo , Citoplasma/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Organelas/metabolismo , Animais , Citocinese , Feminino , Oócitos , Xenopus laevis
2.
Methods Mol Biol ; 1865: 175-194, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30151767

RESUMO

Xenopus oocytes and embryos are model systems optimally suited for quantitative proteomics. This is due to the availability of large amount of protein material and the ease of physical manipulation. Furthermore, facile in vitro fertilization provides superbly synchronized embryos for cell cycle and developmental stages. Here, we detail protocols developed over the last few years for sample preparation of multiplexed proteomics with TMT-tags followed by quantitative mass spectrometry analysis using the MultiNotch MS3 approach. In this approach, each condition is barcoded with an isobaric tag at the peptide level. After barcoding, samples are combined and the relative abundance of ~100,000 peptides is quantified on a mass spectrometer. High reproducibility of the sample preparation process prior to peptides being tagged and combined is of upmost importance for obtaining unbiased data. Otherwise, differences in sample handling can inadvertently appear as biological changes. We detail and exemplify the application of our sample workflow on an embryonic time-series of ten developmental stages of Xenopus laevis embryos ranging from the egg to stage 35 (just before hatching). Our accompanying paper (Chapter 14 ) details a bioinformatics pipeline to analyze the quality of the given sample preparation and strategies to convert spectra of X. laevis peptides into biologically interpretable data.


Assuntos
Embrião não Mamífero/metabolismo , Proteômica/métodos , Xenopus/embriologia , Animais , Precipitação Química , Cromatografia Líquida , Cisteína/metabolismo , Gema de Ovo/metabolismo , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Peptídeos/metabolismo , Controle de Qualidade , Extração em Fase Sólida , Proteínas de Xenopus/metabolismo
3.
Methods Mol Biol ; 1865: 195-215, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30151768

RESUMO

The oocytes, embryos, and cell-free lysates of the frog Xenopus laevis have emerged as powerful models for quantitative proteomic experiments. In the accompanying paper (Chapter 13) we describe how to prepare samples and acquire multiplexed proteomics spectra from those. As an illustrative example we use a 10-stage developmental time series from the egg to stage 35 (just before hatching). Here, we outline how to convert the ~700,000 acquired mass spectra from this time series into protein expression dynamics for ~9000 proteins. We first outline a preliminary quality-control analysis to discover any errors that occurred during sample preparation. We discuss how peptide and protein identification error rates are controlled, and how peptide and protein species are quantified. Our analysis relies on the freely available MaxQuant proteomics pipeline. Finally, we demonstrate how to start interpreting this large dataset by clustering and gene-set enrichment analysis.


Assuntos
Análise de Dados , Embrião não Mamífero/metabolismo , Proteômica/métodos , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Análise por Conglomerados , Ontologia Genética , Humanos , Espectrometria de Massas , Peptídeos/metabolismo , Proteoma/metabolismo , Fatores de Tempo , Proteínas de Xenopus/metabolismo
4.
Mol Biol Cell ; 28(11): 1444-1456, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28404751

RESUMO

The chromosomal passenger complex (CPC) is a conserved, essential regulator of cell division. As such, significant anti-cancer drug development efforts have been focused on targeting it, most notably by inhibiting its AURKB kinase subunit. The CPC is activated by AURKB-catalyzed autophosphorylation on multiple subunits, but how this regulates CPC interactions with other mitotic proteins remains unclear. We investigated the hydrodynamic behavior of the CPC in Xenopus laevis egg cytosol using sucrose gradient sedimentation and in HeLa cells using fluorescence correlation spectroscopy. We found that autophosphorylation of the CPC decreases its sedimentation coefficient in egg cytosol and increases its diffusion coefficient in live cells, indicating a decrease in mass. Using immunoprecipitation coupled with mass spectrometry and immunoblots, we discovered that inactive, unphosphorylated CPC interacts with nucleophosmin/nucleoplasmin proteins, which are known to oligomerize into pentamers and decamers. Autophosphorylation of the CPC causes it to dissociate from nucleophosmin/nucleoplasmin. We propose that nucleophosmin/nucleoplasmin complexes serve as chaperones that negatively regulate the CPC and/or stabilize its inactive form, preventing CPC autophosphorylation and recruitment to chromatin and microtubules in mitosis.


Assuntos
Proteínas Nucleares/metabolismo , Nucleoplasminas/metabolismo , Animais , Divisão Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos , Células HeLa/metabolismo , Humanos , Hidrodinâmica , Microtúbulos/metabolismo , Mitose , Chaperonas Moleculares/metabolismo , Nucleofosmina , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo
5.
Angew Chem Int Ed Engl ; 56(21): 5738-5743, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28418626

RESUMO

The bromodomain-containing protein BRD9, a subunit of the human BAF (SWI/SNF) nucleosome remodeling complex, has emerged as an attractive therapeutic target in cancer. Despite the development of chemical probes targeting the BRD9 bromodomain, there is a limited understanding of BRD9 function beyond acetyl-lysine recognition. We have therefore created the first BRD9-directed chemical degraders, through iterative design and testing of heterobifunctional ligands that bridge the BRD9 bromodomain and the cereblon E3 ubiquitin ligase complex. Degraders of BRD9 exhibit markedly enhanced potency compared to parental ligands (10- to 100-fold). Parallel study of degraders with divergent BRD9-binding chemotypes in models of acute myeloid leukemia resolves bromodomain polypharmacology in this emerging drug class. Together, these findings reveal the tractability of non-BET bromodomain containing proteins to chemical degradation, and highlight lead compound dBRD9 as a tool for the study of BRD9.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Sistemas de Liberação de Medicamentos , Humanos , Ligantes , Estrutura Molecular , Pirróis/química
6.
Curr Biol ; 25(20): 2663-71, 2015 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-26441354

RESUMO

The composition of the nucleoplasm determines the behavior of key processes such as transcription, yet there is still no reliable and quantitative resource of nuclear proteins. Furthermore, it is still unclear how the distinct nuclear and cytoplasmic compositions are maintained. To describe the nuclear proteome quantitatively, we isolated the large nuclei of frog oocytes via microdissection and measured the nucleocytoplasmic partitioning of ∼9,000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus or cytoplasm; only ∼17% partition equally. A protein's native size in a complex, but not polypeptide molecular weight, is predictive of localization: partitioned proteins exhibit native sizes larger than ∼100 kDa, whereas natively smaller proteins are equidistributed. To evaluate the role of nuclear export in maintaining localization, we inhibited Exportin 1. This resulted in the expected re-localization of proteins toward the nucleus, but only 3% of the proteome was affected. Thus, complex assembly and passive retention, rather than continuous active transport, is the dominant mechanism for the maintenance of nuclear and cytoplasmic proteomes.


Assuntos
Proteínas de Anfíbios/genética , Proteínas Nucleares/genética , Proteoma/genética , Xenopus/genética , Proteínas de Anfíbios/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Proteoma/metabolismo , Xenopus/metabolismo
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