RESUMO
Using multiplexed quantitative proteomics, we analyzed cell cycle-dependent changes of the human proteome. We identified >4,400 proteins, each with a six-point abundance profile across the cell cycle. Hypothesizing that proteins with similar abundance profiles are co-regulated, we clustered the proteins with abundance profiles most similar to known Anaphase-Promoting Complex/Cyclosome (APC/C) substrates to identify additional putative APC/C substrates. This protein profile similarity screening (PPSS) analysis resulted in a shortlist enriched in kinases and kinesins. Biochemical studies on the kinesins confirmed KIFC1, KIF18A, KIF2C, and KIF4A as APC/C substrates. Furthermore, we showed that the APC/C(CDH1)-dependent degradation of KIFC1 regulates the bipolar spindle formation and proper cell division. A targeted quantitative proteomics experiment showed that KIFC1 degradation is modulated by a stabilizing CDK1-dependent phosphorylation site within the degradation motif of KIFC1. The regulation of KIFC1 (de-)phosphorylation and degradation provides insights into the fidelity and proper ordering of substrate degradation by the APC/C during mitosis.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteólise , Proteômica , Sequência de Aminoácidos , Ciclo Celular , Células HeLa , Humanos , Cinesinas/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , UbiquitinaçãoRESUMO
Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal-fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse-chase LC-MS-based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine-derived fluxes: Oxidation of glucose-derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation.
Assuntos
Ciclo Celular/fisiologia , Ciclo do Ácido Cítrico/fisiologia , Glucose/metabolismo , Glutamina/metabolismo , Metaboloma/fisiologia , Metabolômica/métodos , Proliferação de Células , Cromatografia Líquida , Glicólise , Células HeLa , Humanos , Marcação por Isótopo , Espectrometria de Massas , Fosforilação Oxidativa , Consumo de Oxigênio/fisiologia , Periodicidade , Fatores de TempoRESUMO
Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.
Assuntos
Microfluídica/métodos , Proteômica/métodos , Células HEK293 , Células HeLa , Humanos , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteômica/instrumentação , Tirosina/metabolismo , UbiquitinaçãoRESUMO
The coupling of the rate of cell growth to the rate of cell division determines cell size, a defining characteristic that is central to cell function and, ultimately, to tissue architecture. The physiology of size homeostasis has fascinated generations of biologists, but the mechanism, challenged by experimental limitations, remains largely unknown. In this paper, we propose a unique optical method that can measure the dry mass of thick live cells as accurately as that for thin cells with high computational efficiency. With this technique, we quantify, with unprecedented accuracy, the asymmetry of division in lymphoblasts and epithelial cells. We can then use the Collins-Richmond model of conservation to compute the relationship between growth rate and cell mass. In attached epithelial cells, we find that due to the asymmetry in cell division and size-dependent growth rate, there is active regulation of cell size. Thus, like nonadherent cells, size homeostasis requires feedback control.
Assuntos
Crescimento Celular , Homeostase/fisiologia , Microscopia de Contraste de Fase/métodos , Animais , Adesão Celular/fisiologia , Células Cultivadas , Holografia/métodos , Lasers , Camundongos , RefratometriaRESUMO
Growth arrest-specific 2-like 3 (Gas2l3) is a newly discovered cell cycle protein and a cytoskeleton orchestrator that binds both actin filament and microtubule networks. Studies of cultured mammalian cells established Gas2l3 as a regulator of the cell division process, in particular cytokinesis and cell abscission. Thus far, the role of Gas2l3 in vivo remains entirely unknown. In order to investigate Gas2l3 in developing vertebrates, we cloned the zebrafish gene. Spatiotemporal analysis of gas2l3 expression revealed a ubiquitous maternal transcript as well as a zygotic transcript primarily restricted to brain tissues. We next conducted a series of loss-of-function experiments, and searched for developmental anomalies at the end of the segmentation period. Our analysis revealed abnormal brain morphogenesis and ventricle formation in gas2l3 knockdown embryos. This signature phenotype could be rescued by elevated levels of gas2l3 RNA. At the tissue level, gas2l3 downregulation interferes with cell proliferation, suggesting that the cell cycle activities of Gas2l3 are essential for brain tissue homeostasis. Altogether, this study provides the first insight into the function of gas2l3 in vivo, demonstrating its essential role in brain development.
Assuntos
Encéfalo/embriologia , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Microscopia de Fluorescência , Dados de Sequência Molecular , Morfogênese/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , Proteínas de Peixe-Zebra/genéticaRESUMO
We introduce a microfluidic system for simultaneously measuring single-cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 h of growth data from mouse lymphoblast and pro-B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1-S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis.
Assuntos
Crescimento Celular , Tamanho Celular , Linfócitos/citologia , Técnicas Analíticas Microfluídicas/métodos , Células Precursoras de Linfócitos B/citologia , Análise de Célula Única/métodos , Animais , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Fase G1 , Camundongos , Fase SRESUMO
Robo receptors play pivotal roles in neurodevelopment, and their deregulation is implicated in several neuropathological conditions and cancers. To date, the mechanism of Robo activation and regulation remains obscure. Here we present the crystal structure of the juxtamembrane (JM) domains of human Robo1. The structure exhibits unexpectedly high backbone similarity to the netrin and RGM binding region of neogenin and DCC, which are functionally related receptors of Robo1. Comparison of these structures reveals a conserved surface that overlaps with a cluster of oncogenic and neuropathological mutations found in all Robo isoforms. The structure also reveals the intricate folding of the JM linker, which points to its role in Robo1 activation. Further experiments with cultured cells demonstrate that exposure or relief of the folded JM linker results in enhanced shedding of the Robo1 ectodomain.
Assuntos
Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/química , Receptores Imunológicos/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/ultraestrutura , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/ultraestrutura , Estrutura Terciária de Proteína , Receptores Imunológicos/ultraestrutura , Proteínas RoundaboutRESUMO
Currently, the reliable identification of peptides and proteins is only feasible when thoroughly annotated sequence databases are available. Although sequencing capacities continue to grow, many organisms remain without reliable, fully annotated reference genomes required for proteomic analyses. Standard database search algorithms fail to identify peptides that are not exactly contained in a protein database. De novo searches are generally hindered by their restricted reliability, and current error-tolerant search strategies are limited by global, heuristic tradeoffs between database and spectral information. We propose a Bayesian information criterion-driven error-tolerant peptide search (BICEPS) and offer an open source implementation based on this statistical criterion to automatically balance the information of each single spectrum and the database, while limiting the run time. We show that BICEPS performs as well as current database search algorithms when such algorithms are applied to sequenced organisms, whereas BICEPS only uses a remotely related organism database. For instance, we use a chicken instead of a human database corresponding to an evolutionary distance of more than 300 million years (International Chicken Genome Sequencing Consortium (2004) Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695-716). We demonstrate the successful application to cross-species proteomics with a 33% increase in the number of identified proteins for a filarial nematode sample of Litomosoides sigmodontis.
Assuntos
Galinhas/genética , Filarioidea/genética , Peptídeos/química , Proteômica/métodos , Software , Algoritmos , Sequência de Aminoácidos , Animais , Teorema de Bayes , Evolução Biológica , Bases de Dados de Proteínas , Humanos , Internet , Espectrometria de Massas , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Análise de Sequência de ProteínaRESUMO
We used a suspended microchannel resonator (SMR) combined with picoliter-scale microfluidic control to measure buoyant mass and determine the 'instantaneous' growth rates of individual cells. The SMR measures mass with femtogram precision, allowing rapid determination of the growth rate in a fraction of a complete cell cycle. We found that for individual cells of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and mouse lymphoblasts, heavier cells grew faster than lighter cells.
Assuntos
Crescimento Celular , Técnicas Analíticas Microfluídicas/métodos , Animais , Bacillus subtilis/crescimento & desenvolvimento , Ciclo Celular , Escherichia coli/crescimento & desenvolvimento , Linfócitos/fisiologia , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that regulates mitosis and G1 by sequentially targeting cell-cycle regulators for ubiquitination and proteasomal degradation. The mechanism of ubiquitin chain formation by APC and the resultant chain topology remains controversial. By using a single-lysine APC substrate to dissect the topology of ubiquitinated substrates, we find that APC-catalyzed ubiquitination has an intrinsic preference for the K11 linkage of ubiquitin that is essential for substrate degradation. K11 specificity is determined by an E2 enzyme, UBE2S/E2-EPF, that elongates ubiquitin chains after the substrates are pre-ubiquitinated by UbcH10 or UbcH5. UBE2S copurifies with APC; dominant-negative Ube2S slows down APC substrate degradation in functional cell-cycle extracts. We propose that Ube2S is a critical, unique component of the APC ubiquitination pathway.
Assuntos
Lisina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Biocatálise , Ciclo Celular , Células HeLa , Humanos , Lisina/genética , Mutação , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Securina , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Protein degradation mediated by the ubiquitin-proteasome pathway regulates signaling events in many physiological and pathological conditions. In vitro degradation assays have been instrumental in the understanding of how cell proliferation and other fundamental cellular processes are regulated. These assays are direct, time-specific and highly informative but also laborious, typically relying on low-throughput polyacrylamide gel-electrophoresis followed by autoradiography or immunoblotting. We present protein degradation on chip (pDOC), a MITOMI-based integrated microfluidic technology for discovery and analysis of proteins degradation in cell-free extracts. The platform accommodates hundreds of microchambers on which protein degradation is assayed quickly, simultaneously and using minute amounts of reagents in one or many physiochemical environments. Essentially, pDOC provides a sensitive multiplex alternative to the conventional degradation assay, with relevance to biomedical and translational research associated with regulated proteolysis.
Assuntos
Microfluídica , Microfluídica/métodos , Proteólise , Extratos Celulares , Eletroforese em Gel de Poliacrilamida , ImmunoblottingRESUMO
A balanced progression through mitosis and cell division is largely dependent on orderly phosphorylation and ubiquitin-mediated proteolysis of regulatory and structural proteins. These series of events ultimately secure genome stability and time-invariant cellular properties during cell proliferation. Two of the core enzymes regulating mitotic milestones in all eukaryotes are cyclin dependent kinase 1 (CDK1) with its coactivator cyclin B, and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Discovering mechanisms and substrates for these enzymes is vital to understanding how cells move through mitosis and segregate chromosomes with high fidelity. However, the study of these enzymes has significant challenges. Purely in vitro studies discount the contributions of yet to be described regulators and misses the physiological context of cellular environment. In vivo studies are complicated by the fact that each of these enzymes, as well as many of their regulators and downstream targets, are essential. Moreover, long-term in vivo manipulations can result in cascading, indirect effects that can distort data analysis and interpretation. Many of these challenges can be circumvented using cell-free systems, which have historically played a critical role in identifying these enzymes and their contributions under quasicellular environments. Here, we describe the preparation of a newly developed human cell-free system that recapitulates an anaphase-like state of human cells. This new toolkit complements traditional cell-free systems from human cells and frog eggs and can be easily implemented in cell biology labs for direct and quantitative studies of mitotic signaling regulated by phosphorylation, APC/C-mediated proteolysis, and beyond.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteína Quinase CDC2/metabolismo , Sistema Livre de Células/metabolismo , Ciclina B1/metabolismo , Anáfase , Ciclina B1/genética , Células HEK293 , Humanos , Mitose , Mutação , Fosforilação , Proteólise , UbiquitinaçãoRESUMO
The E2F family of transcriptional regulators sits at the center of cell cycle gene expression and plays vital roles in normal and cancer cell cycles. Whereas control of E2Fs by the retinoblastoma family of proteins is well established, much less is known about their regulation by ubiquitin pathways. Recent studies placed the Skp1-Cul1-F-box-protein (SCF) family of E3 ubiquitin ligases with the F-box protein Cyclin F at the center of E2F regulation, demonstrating temporal proteolysis of both activator and atypical repressor E2Fs. Importantly, these E2F members, in particular activator E2F1 and repressors E2F7 and E2F8, form a feedback circuit at the crossroads of cell cycle and cell death. Moreover, Cyclin F functions in a reciprocal circuit with the cell cycle E3 ligase anaphase-promoting complex/cyclosome (APC/C), which also controls E2F7 and E2F8. This review focuses on the complex contours of feedback within this circuit, highlighting the deep crosstalk between E2F, SCF-Cyclin F, and APC/C in regulating the oscillator underlying human cell cycles.
Assuntos
Ciclinas/metabolismo , Fatores de Transcrição E2F/metabolismo , Ubiquitina/metabolismo , Animais , Ciclo Celular/genética , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Humanos , ProteóliseRESUMO
E2F8 is a transcriptional repressor that antagonizes E2F1 at the crossroads of the cell cycle, apoptosis, and cancer. Previously, we discovered that E2F8 is a direct target of the APC/C ubiquitin ligase. Nevertheless, it remains unknown how E2F8 is dynamically controlled throughout the entirety of the cell cycle. Here, using newly developed human cell-free systems that recapitulate distinct inter-mitotic and G1 phases and a continuous transition from prometaphase to G1, we reveal an interlocking dephosphorylation switch coordinating E2F8 degradation with mitotic exit and the activation of APC/CCdh1. Further, we uncover differential proteolysis rates for E2F8 at different points within G1 phase, accounting for its accumulation in late G1 while APC/CCdh1 is still active. Finally, we demonstrate that the F-box protein Cyclin F regulates E2F8 in G2-phase. Altogether, our data define E2F8 regulation throughout the cell cycle, illuminating an extensive coordination between phosphorylation, ubiquitination and transcription in mammalian cell cycle.
Assuntos
Ciclo Celular/fisiologia , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Sistema Livre de Células , Ciclinas/metabolismo , Fator de Transcrição E2F1/metabolismo , Fase G1/fisiologia , Fase G2/fisiologia , Células HeLa , Humanos , Mitose/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteólise , Proteínas Recombinantes/metabolismo , UbiquitinaçãoRESUMO
Autophosphorylation of receptor and non-receptor tyrosine kinases is a common molecular switch with broad implications for pathogeneses and therapy of cancer and other human diseases. Technologies for large-scale discovery and analysis of autophosphorylation are limited by the inherent difficulty to distinguish between phosphorylation and autophosphorylation in vivo and by the complexity associated with functional assays of receptors kinases in vitro. Here, we report a method for the direct detection and analysis of tyrosine autophosphorylation using integrated microfluidics and freshly synthesized protein arrays. We demonstrate the efficacy of our platform in detecting autophosphorylation activity of soluble and transmembrane tyrosine kinases, and the dependency of in vitro autophosphorylation assays on membranes. Our method, Integrated Microfluidics for Autophosphorylation Discovery (IMAD), is high-throughput, requires low reaction volumes and can be applied in basic and translational research settings. To our knowledge, it is the first demonstration of posttranslational modification analysis of membrane protein arrays.
Assuntos
Ensaios de Triagem em Larga Escala , Técnicas Analíticas Microfluídicas/instrumentação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Membrana Celular/metabolismo , Biblioteca Gênica , Células HEK293 , Humanos , Fosforilação , Análise Serial de Proteínas , Proteínas Tirosina Quinases/genéticaRESUMO
Cancer is the second leading cause of death globally. Matching proper treatment and dosage is crucial for a positive outcome. Any given drug may affect patients with similar tumors differently. Personalized medicine aims to address this issue. Unfortunately, most cancer samples cannot be expanded in culture, limiting conventional cell-based testing. Herein, presented is a microfluidic device that combines a drug microarray with cell microscopy. The device can perform 512 experiments to test chemosensitivity and resistance to a drug array. MCF7 and 293T cells are cultured inside the device and their chemosensitivity and resistance to docetaxel, applied at various concentrations, are determined. Cell mortality is determined as a function of drug concentration and exposure time. It is found that both cell types form cluster morphology within the device, not evident in conventional tissue culture under similar conditions. Cells inside the clusters are less sensitive to drugs than dispersed cells. These findings support a heterogenous response of cancer cells to drugs. Then demonstrated is the principle of drug microarrays by testing cell response to four different drugs at four different concentrations. This approach may enable the personalization of treatment to the particular tumor and patient and may eventually improve final patient outcome.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Neoplasias , Medicina de Precisão , Humanos , Células MCF-7 , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismoRESUMO
Cytokinesis is an intensively studied process by which the cell cytoplasm divides to produce two daughter cells. Like any other aspect of cell cycle research, the study of cytokinesis relies heavily on cell synchronization. However, the synchronization of cells during cytokinesis is challenging due to the rapid nature of this process and the shortage of cell cycle blocking agents specifically targeting this phase. Here, we demonstrate the use of standard flow cytometry for directly isolating cytokinetic cells from an asynchronous population of normally proliferating cells. This approach is based on a cell cycle marker whose temporal proteolysis, in combination with DNA quantification or cell size approximation, distinguishes cells undergoing cytokinesis. Furthermore, by avoiding doublet discrimination, typically used in flow cytometry analyses, we were able to further increase selectivity, specifically purifying cells at late cytokinesis. Our method circumvents checkpoint activation, cell cycle arrest, and any other means of pre-synchronization. These qualities, as demonstrated for both unattached and adherent cells, enable high selectivity for cytokinetic cells despite their overall low abundance in an asynchronous population. The sorted cells can then be readily used for cell biological, biochemical, and genomic applications to facilitate cytokinesis and cell cycle research.
Assuntos
Separação Celular/métodos , Animais , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/metabolismo , Citocinese , Citometria de Fluxo , Células HeLa , Humanos , Camundongos , Coloração e RotulagemRESUMO
Animal cell division ends with the cutting of the microtubule and membrane intercellular bridge connecting the 2 daughter cells. This process, known as cytokinetic abscission (abscission), is widely regarded as the last step of cytokinesis, i.e., the last step of the cell cycle. Major breakthroughs have been recently achieved, illuminating mechanistic aspects of abscission; however, the timing of abscission with respect to the mammalian cell cycle remains unclear. In this study, we carefully measured the onset and progression of abscission in dividing cells expressing a G1 reporter. We conclude that abscission commences long after cells enter the G1 phase. Affiliating abscission with G1 is beyond semantics since it essentially postulates that the last step of the cell cycle is regulated in, and probably by, the following cycle.
Assuntos
Citocinese , Células 3T3 , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Fase G1 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Vídeo , Imagem com Lapso de Tempo , Proteína Vermelha FluorescenteRESUMO
Cell cycle research greatly relies on synchronization of proliferating cells. However, effective synchronization of mammalian cells is commonly achieved by long exposure to one or more cell cycle blocking agents. These chemicals are, by definition, hazardous (some more than others), pose uneven cell cycle arrest, thus introducing unwanted variables. The challenge of synchronizing proliferating cells in G1 is even greater; this process typically involves the release of drug-arrested cells into the cycle that follows, a heterogeneous process that can truly limit synchronization. Moreover, drug-based synchronization decouples the cell cycle from cell growth in ways that are understudied and intolerable for those who investigate the relationship between these two processes. In this study we showed that cell size, as approximated by a single light-scatter parameter available in all standard sorters, can be used for synchronizing proliferating mammalian cells in G1 with minimal or no risk to either the cell cycle or cell growth. The power and selectivity of our method are demonstrated for human HEK293 cells that, despite their many advantages, are suboptimal for synchronization, let alone in G1. Our approach is readily available, simple, fast, and inexpensive; it is independent of any drugs or dyes, and nonhazardous. These properties are relevant for the study of the mammalian cell cycle, specifically in the context of G1 and cell growth.
Assuntos
Proliferação de Células , Separação Celular/métodos , Citometria de Fluxo/métodos , Fase G1/fisiologia , Western Blotting , Técnicas de Cultura de Células , Tamanho Celular , DNA/análise , DNA/metabolismo , Células HEK293 , HumanosRESUMO
Different types of mature B-cell lymphocytes are overall highly similar. Nevertheless, some B cells proliferate intensively, while others rarely do. Here, we demonstrate that a simple binary classification of gene expression in proliferating vs. resting B cells can identify, with remarkable selectivity, global in vivo regulators of the mammalian cell cycle, many of which are also post-translationally regulated by the APC/C E3 ligase. Consequently, we discover a novel regulatory network between the APC/C and the E2F transcription factors and discuss its potential impact on the G1-S transition of the cell cycle. In addition, by focusing on genes whose expression inversely correlates with proliferation, we demonstrate the inherent ability of our approach to also identify in vivo regulators of cell differentiation, cell survival, and other antiproliferative processes. Relying on data sets of wt, non-transgenic animals, our approach can be applied to other cell lineages and human data sets.