RESUMO
The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence has suggested that microtubules also might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires γ-tubulin and augmin and is stimulated by factors previously implicated in chromatin-stimulated nucleation, guanosine triphosphate(GTP)-bound Ran and its effector, TPX2. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Meiose , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Microscopia/métodos , Óvulo/química , Óvulo/metabolismoRESUMO
BACKGROUND: Novel immunotherapies targeting cancer-associated truncated O-glycans Tn (GalNAcα-Ser/Thr) and STn (Neu5Acα2-6GalNacα-Ser/Thr) are promising strategies for cancer treatment. However, no comprehensive, antibody-based mapping of truncated O-glycans in tumours exist to guide drug development. METHODS: We used monoclonal antibodies to map the expression of truncated O-glycans in >700 tissue cores representing healthy and tumour tissues originating from breast, colon, lung, pancreas, skin, CNS and mesenchymal tissue. Patient-derived xenografts were used to evaluate Tn expression upon tumour engraftment. RESULTS: The Tn-antigen was highly expressed in breast (57%, n = 64), colorectal (51%, n = 140) and pancreatic (53%, n = 108) tumours, while STn was mainly observed in colorectal (80%, n = 140) and pancreatic (56%, n = 108) tumours. We observed no truncated O-glycans in mesenchymal tumours (n = 32) and low expression of Tn (5%, n = 87) and STn (1%, n = 75) in CNS tumours. No Tn-antigen was found in normal tissue (n = 124) while STn was occasionally observed in healthy gastrointestinal tissue. Surface expression of Tn-antigen was identified across several cancers. Tn and STn expression decreased with tumour grade, but not with cancer stage. Numerous xenografts maintained Tn expression. CONCLUSIONS: Surface expression of truncated O-glycans is limited to cancers of epithelial origin, making Tn and STn attractive immunological targets in the treatment of human carcinomas.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Neoplasias/patologia , Análise Serial de Tecidos/métodos , Animais , Anticorpos Monoclonais/imunologia , Estudos de Casos e Controles , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Gradação de Tumores , Estadiamento de Neoplasias , Transplante de Neoplasias , Neoplasias/classificação , Neoplasias/metabolismo , Regulação para CimaRESUMO
A major challenge in cell biology is to understand how nanometer-sized molecules can organize micrometer-sized cells in space and time. One solution in many animal cells is a radial array of microtubules called an aster, which is nucleated by a central organizing center and spans the entire cytoplasm. Frog (here Xenopus laevis) embryos are more than 1 mm in diameter and divide with a defined geometry every 30 min. Like smaller cells, they are organized by asters, which grow, interact, and move to precisely position the cleavage planes. It has been unclear whether asters grow to fill the enormous egg by the same mechanism used in smaller somatic cells, or whether special mechanisms are required. We addressed this question by imaging growing asters in a cell-free system derived from eggs, where asters grew to hundreds of microns in diameter. By tracking marks on the lattice, we found that microtubules could slide outward, but this was not essential for rapid aster growth. Polymer treadmilling did not occur. By measuring the number and positions of microtubule ends over time, we found that most microtubules were nucleated away from the centrosome and that interphase egg cytoplasm supported spontaneous nucleation after a time lag. We propose that aster growth is initiated by centrosomes but that asters grow by propagating a wave of microtubule nucleation stimulated by the presence of preexisting microtubules.
Assuntos
Embrião não Mamífero/citologia , Microtúbulos/fisiologia , Modelos Biológicos , Animais , Tamanho Celular , Sistema Livre de Células , Centrossomo/metabolismo , Microscopia de Fluorescência , Reologia , Xenopus laevisRESUMO
Cognitive dysfunction in aging is a major biomedical challenge. Whether treatment with klotho, a longevity factor, could enhance cognition in human-relevant models such as in nonhuman primates is unknown and represents a major knowledge gap in the path to therapeutics. We validated the rhesus form of the klotho protein in mice showing it increased synaptic plasticity and cognition. We then found that a single administration of low-dose, but not high-dose, klotho enhanced memory in aged nonhuman primates. Systemic low-dose klotho treatment may prove therapeutic in aging humans.
Assuntos
Glucuronidase , Longevidade , Camundongos , Humanos , Animais , Idoso , Glucuronidase/metabolismo , Envelhecimento , Cognição , Primatas/metabolismoRESUMO
The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Most current immunotherapeutic targets are tumor-associated antigens that are also found in healthy tissues and often do not display sufficient cancer selectivity to be used as targets for potent antibody-based immunotherapeutic treatments, such as chimeric antigen receptor (CAR) T cells. Many solid tumors, however, display aberrant glycosylation that results in expression of tumor-associated carbohydrate antigens that are distinct from healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes within existing or novel glycoprotein targets may provide the cancer selectivity needed for immunotherapy of solid tumors. However, to date only a few such glycopeptide epitopes have been targeted. Here, we used O-glycoproteomics data from multiple cell lines to identify a glycopeptide epitope in CD44v6, a cancer-associated CD44 isoform, and developed a cancer-specific mAb, 4C8, through a glycopeptide immunization strategy. 4C8 selectively binds to Tn-glycosylated CD44v6 in a site-specific manner with low nanomolar affinity. 4C8 was shown to be highly cancer specific by IHC of sections from multiple healthy and cancerous tissues. 4C8 CAR T cells demonstrated target-specific cytotoxicity in vitro and significant tumor regression and increased survival in vivo. Importantly, 4C8 CAR T cells were able to selectively kill target cells in a mixed organotypic skin cancer model having abundant CD44v6 expression without affecting healthy keratinocytes, indicating tolerability and safety.
Assuntos
Anticorpos Monoclonais , Neoplasias , Humanos , Anticorpos Monoclonais/farmacologia , Neoplasias/patologia , Glicoproteínas , Epitopos , GlicopeptídeosRESUMO
Size specification of macromolecular assemblies in the cytoplasm is poorly understood [1]. In principle, assemblies could scale with cell size or use intrinsic mechanisms. For the mitotic spindle, scaling with cell size is expected, because the function of this assembly is to physically move sister chromatids into the center of nascent daughter cells. Eggs of Xenopus laevis are among the largest cells known that cleave completely during cell division. Cell length in this organism changes by two orders of magnitude ( approximately 1200 microm to approximately 12 microm) while it develops from a fertilized egg into a tadpole [2]. We wondered whether, and how, mitotic spindle length and morphology adapt to function at these different length scales. Here, we show that spindle length increases with cell length in small cells, but in very large cells spindle length approaches an upper limit of approximately 60 microm. Further evidence for an upper limit to spindle length comes from an embryonic extract system that recapitulates mitotic spindle assembly in a test tube. We conclude that early mitotic spindle length in Xenopus laevis is uncoupled from cell length, reaching an upper bound determined by mechanisms that are intrinsic to the spindle.
Assuntos
Tamanho Celular , Fuso Acromático/fisiologia , Xenopus laevis/fisiologia , Animais , MitoseRESUMO
Anastral meiotic spindles are thought to be organized differently from astral mitotic spindles, but the field lacks the basic structural information required to describe and model them, including the location of microtubule-nucleating sites and minus ends. We measured the distributions of oriented microtubules in metaphase anastral spindles in Xenopus laevis extracts by fluorescence speckle microscopy and cross-correlation analysis. We localized plus ends by tubulin incorporation and combined this with the orientation data to infer the localization of minus ends. We found that minus ends are localized throughout the spindle, sparsely at the equator and at higher concentrations near the poles. Based on these data, we propose a model for maintenance of the metaphase steady-state that depends on continuous nucleation of microtubules near chromatin, followed by sorting and outward transport of stabilized minus ends, and, eventually, their loss near poles.
Assuntos
Meiose , Metáfase , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Densitometria , Técnicas In Vitro , Modelos Biológicos , Tubulina (Proteína)/metabolismo , Xenopus laevisRESUMO
Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This "poleward flux" of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose-responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.
Assuntos
Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Cinesinas/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/fisiologia , Proteínas de Xenopus/metabolismo , Animais , Extratos Celulares/química , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/fisiologia , Citocinese/fisiologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Citometria por Imagem , Cinesinas/antagonistas & inibidores , Metáfase/efeitos dos fármacos , Metáfase/fisiologia , Microscopia de Fluorescência , Proteínas Motores Moleculares , Oócitos/química , Pirimidinas/farmacologia , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Tionas/farmacologia , Proteínas de Xenopus/antagonistas & inibidores , Xenopus laevisRESUMO
We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Endocitose/fisiologia , Proteínas Fúngicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Aurora Quinases , Proteínas Fúngicas/genética , Proteína Quinase C , Proteínas Serina-Treonina Quinases/genética , Pirazóis/química , Pirimidinas/química , Receptores Proteína Tirosina Quinases/genética , Receptores de Superfície Celular/metabolismo , Leveduras/genética , Leveduras/metabolismo , Leveduras/ultraestruturaRESUMO
Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) alpha, Impbeta, and Imp7. While Impalpha and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impbeta specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.
Assuntos
Microtúbulos/fisiologia , Oócitos/fisiologia , Xenopus laevis/fisiologia , Proteína ran de Ligação ao GTP/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Meiose , Microtúbulos/ultraestrutura , Oócitos/citologia , Transporte Proteico , Interferência de RNA , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/metabolismo , Proteína ran de Ligação ao GTP/genéticaRESUMO
In this study we find that the function of BRCA1 inhibits the microtubule nucleation function of centrosomes. In particular, cells in early S phase have quiescent centrosomes due to BRCA1 activity, which inhibits the association of gamma-tubulin with centrosomes. We find that modification of either of two specific lysine residues (Lys-48 and Lys-344) of gamma-tubulin, a known substrate for BRCA1-dependent ubiquitination activity, led to centrosome hyperactivity. Interestingly, mutation of gamma-tubulin lysine 344 had a minimal effect on centrosome number but a profound effect on microtubule nucleation function, indicating that the processes regulating centrosome duplication and microtubule nucleation are distinct. Using an in vitro aster formation assay, we found that BRCA1-dependent ubiquitination activity directly inhibits microtubule nucleation by centrosomes. Mutant BRCA1 protein that was inactive as a ubiquitin ligase did not inhibit aster formation by the centrosome. Further, a BRCA1 carboxy-terminal truncation mutant that was an active ubiquitin ligase lacked domains critical for the inhibition of centrosome function. These experiments reveal an important new functional assay regulated by the BRCA1-dependent ubiquitin ligase, and the results suggest that the loss of this BRCA1 activity could cause the centrosome hypertrophy and subsequent aneuploidy typically found in breast cancers.
Assuntos
Proteína BRCA1/metabolismo , Centrossomo/ultraestrutura , Ubiquitina/metabolismo , Aneuploidia , Proteína BRCA1/fisiologia , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Centrossomo/metabolismo , Relação Dose-Resposta a Droga , Humanos , Lisina/química , Microscopia de Fluorescência , Microtúbulos/ultraestrutura , Mutação , Peptídeos/química , Fenótipo , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Fase S , Fatores de Tempo , Transfecção , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The Nercc1 protein kinase autoactivates in vitro and is activated in vivo during mitosis. Autoactivation in vitro requires phosphorylation of the activation loop at threonine 210. Mitotic activation of Nercc1 in mammalian cells is accompanied by Thr210 phosphorylation and involves a small fraction of total Nercc1. Mammalian Nercc1 coimmunoprecipitates gamma-tubulin and the activated Nercc1 polypeptides localize to the centrosomes and spindle poles during early mitosis, suggesting that active Nercc has important functions at the microtubular organizing center during cell division. To test this hypothesis, we characterized the Xenopus Nercc1 orthologue (XNercc). XNercc endogenous to meiotic egg extracts coprecipitates a multiprotein complex that contains gamma-tubulin and several components of the gamma-tubulin ring complex and localizes to the poles of spindles formed in vitro. Reciprocally, immunoprecipitates of the gamma-tubulin ring complex polypeptide Xgrip109 contain XNercc. Immunodepletion of XNercc from egg extracts results in delayed spindle assembly, fewer bipolar spindles, and the appearance of aberrant microtubule structures, aberrations corrected by addition of purified recombinant XNercc. XNercc immunodepletion also slows aster assembly induced by Ran-GTP, producing Ran-asters of abnormal size and morphology. Thus, Nercc1 contributes to both the centrosomal and the chromatin/Ran pathways that collaborate in the organization of a bipolar spindle.
Assuntos
Centrossomo/enzimologia , Mitose/fisiologia , Proteínas Quinases/metabolismo , Fuso Acromático/fisiologia , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Oócitos/enzimologia , Ligação Proteica , Proteínas Quinases/genética , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis , Proteína ran de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. RESULTS: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with gamma-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. CONCLUSIONS: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and gamma-TuRC.
Assuntos
Centrossomo/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Cromatografia Líquida , Primers do DNA , Imunofluorescência , Immunoblotting , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Plasmídeos/genética , Análise de Sequência de DNA , Xenopus , Proteínas de Xenopus/metabolismo , Proteína ran de Ligação ao GTP/metabolismoRESUMO
For many years, microtubule research has depended on tubulin purified from cow and pig brains, which may not be ideal for experiments using proteins or extracts from non-brain tissues and cold-blooded organisms. Here, we describe a method to purify functional tubulin from the eggs of the frog, Xenopus laevis. This tubulin has many benefits for the study of microtubules and microtubule based structures assembled in vitro at room temperature. Frog tubulin lacks many of the highly stabilizing posttranslational modifications present in pig brain-derived tubulin, and polymerizes efficiently at room temperature. In addition, fluorescently labeled frog egg tubulin incorporates into meiotic spindles assembled in egg extract more efficiently than brain tubulin, and is thus superior as a probe for Xenopus egg extract experiments. Frog egg tubulin will provide excellent opportunities to identify active nucleation complexes and revisit microtubule polymerization dynamics in vitro.
Assuntos
Oócitos/metabolismo , Tubulina (Proteína)/isolamento & purificação , Tubulina (Proteína)/metabolismo , Xenopus laevis/metabolismo , Animais , Sistema Livre de Células , Microscopia de Fluorescência , Microtúbulos/metabolismo , Espectrometria de Fluorescência , Fuso Acromático/metabolismo , Coloração e Rotulagem , Suínos , Tubulina (Proteína)/químicaRESUMO
Mitotic spindles specify cleavage planes in early embryos by communicating their position and orientation to the cell cortex using microtubule asters that grow out from the spindle poles during anaphase. Chromatin also plays a poorly understood role. Polyspermic fertilization provides a natural experiment in which aster pairs from the same spindle (sister asters) have chromatin between them, whereas asters pairs from different spindles (nonsisters) do not. In frogs, only sister aster pairs induce furrows. We found that only sister asters recruited two conserved furrow-inducing signaling complexes, chromosome passenger complex (CPC) and Centralspindlin, to a plane between them. This explains why only sister pairs induce furrows. We then investigated factors that influenced CPC recruitment to microtubule bundles in intact eggs and a cytokinesis extract system. We found that microtubule stabilization, optimal starting distance between asters, and proximity to chromatin all favored CPC recruitment. We propose a model in which proximity to chromatin biases initial CPC recruitment to microtubule bundles between asters from the same spindle. Next a positive feedback between CPC recruitment and microtubule stabilization promotes lateral growth of a plane of CPC-positive microtubule bundles out to the cortex to position the furrow.
Assuntos
Comunicação Celular/fisiologia , Fertilização/fisiologia , Óvulo/fisiologia , Fuso Acromático/fisiologia , Xenopus/fisiologia , Anáfase/fisiologia , Animais , Cromatina/metabolismo , Citocinese/fisiologia , Feminino , Microtúbulos/fisiologia , Fuso Acromático/metabolismo , Zigoto/fisiologiaRESUMO
Cell division in prokaryotes and eukaryotes is commonly initiated by the well-controlled binding of proteins to the cytoplasmic side of the cell membrane. However, a precise characterization of the spatiotemporal dynamics of membrane-bound proteins is often difficult to achieve in vivo. Here, we present protocols for the use of supported lipid bilayers to rebuild the cytokinetic machineries of cells with greatly different dimensions: the bacterium Escherichia coli and eggs of the vertebrate Xenopus laevis. Combined with total internal reflection fluorescence microscopy, these experimental setups allow for precise quantitative analyses of membrane-bound proteins. The protocols described to obtain glass-supported membranes from bacterial and vertebrate lipids can be used as starting points for other reconstitution experiments. We believe that similar biochemical assays will be instrumental to study the biochemistry and biophysics underlying a variety of complex cellular tasks, such as signaling, vesicle trafficking, and cell motility.
Assuntos
Membrana Celular/metabolismo , Escherichia coli/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Xenopus laevis/metabolismo , Animais , Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Microscopia de Fluorescência/métodos , Óvulo/metabolismo , Ligação Proteica/fisiologia , Extratos de Tecidos/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
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 â¼9,000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus or cytoplasm; only â¼17% 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 â¼100 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/metabolismoRESUMO
Cell-free cytoplasm isolated from meiotic Xenopus egg extracts reconstitutes microtubule phenomena in vitro. These crude extracts assemble bipolar meiotic spindles and are readily fractionated for biochemical assays, providing a good tool to dissect molecular mechanism. We developed techniques for immunoelectron microscopy of microtubule structures assembled in perfusion chambers and in solution.
Assuntos
Citoesqueleto/ultraestrutura , Microscopia Eletrônica/métodos , Microtúbulos/ultraestrutura , Animais , Citoesqueleto/metabolismo , Técnicas de Preparação Histocitológica , Microtúbulos/metabolismo , Óvulo , XenopusRESUMO
The large cells in early vertebrate development face an extreme physical challenge in organizing their cytoplasm. For example, amphibian embryos have to divide cytoplasm that spans hundreds of micrometres every 30 min according to a precise geometry, a remarkable accomplishment given the extreme difference between molecular and cellular scales in this system. How do the biochemical reactions occurring at the molecular scale lead to this emergent behaviour of the cell as a whole? Based on recent findings, we propose that the centrosome plays a crucial role by initiating two autocatalytic reactions that travel across the large cytoplasm as chemical waves. Waves of mitotic entry and exit propagate out from centrosomes using the Cdk1 oscillator to coordinate the timing of cell division. Waves of microtubule-stimulated microtubule nucleation propagate out to assemble large asters that position spindles for the following mitosis and establish cleavage plane geometry. By initiating these chemical waves, the centrosome rapidly organizes the large cytoplasm during the short embryonic cell cycle, which would be impossible using more conventional mechanisms such as diffusion or nucleation by structural templating. Large embryo cells provide valuable insights to how cells control chemical waves, which may be a general principle for cytoplasmic organization.
Assuntos
Anuros/embriologia , Padronização Corporal/fisiologia , Ciclo Celular/fisiologia , Centrossomo/metabolismo , Embrião não Mamífero/fisiologia , Modelos Biológicos , Transdução de Sinais/fisiologia , Animais , Proteína Quinase CDC2/metabolismo , Microtúbulos/fisiologiaRESUMO
We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.