RESUMEN

Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion attraction/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the "chromosome milieu" during mitosis of living human cells using an orientation-independent-differential interference contrast module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prophase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro, native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like. These results suggest that a transient rise in depletion attraction, likely triggered by the relocation of macromolecules (proteins, RNAs, and others) via nuclear envelope breakdown and by a subsequent decrease in cell volumes, contributes to mitotic chromosome condensation, shedding light on a different aspect of the condensation mechanism in living human cells.

Asunto(s)

Cromatina , Cromosomas Humanos , Mitosis , Humanos , Células HeLa , Cromatina/metabolismo , Cromosomas Humanos/metabolismo , Cromosomas Humanos/genética , Microscopía Confocal , Complejos Multiproteicos/metabolismo , Adenosina Trifosfatasas , Proteínas de Unión al ADN

RESUMEN

Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.

Asunto(s)

Adenosina Trifosfatasas , Segregación Cromosómica , Complejos Multiproteicos , Schizosaccharomyces , Proteínas de Unión al ADN/genética , Cromatina , Cromosomas , ADN , Schizosaccharomyces/genética , ARN Polimerasa II/genética , Mitosis , Proteínas de Ciclo Celular/genética

RESUMEN

Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion force/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the "chromosome milieu" during mitosis of living human cells using orientation-independent-differential interference contrast (OI-DIC) module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prometaphase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro, native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like, with further condensation. These results suggest that a transient rise in depletion force, likely triggered by the relocation of macromolecules (proteins, RNAs and others) via nuclear envelope breakdown and also by a subsequent decrease in cell-volumes, contributes to mitotic chromosome condensation, shedding light on a new aspect of the condensation mechanism in living human cells.

RESUMEN

Previous studies indicated that mitotic chromosome structure consists of many stacked layers formed by a mononucleosome sheet folded as a helicoid. This multilayer chromatin structure justifies the cylindrical shape of chromosomes and the transverse orientation of cytogenetic bands, and can explain chromosome duplication by the formation of a transient double helicoid that is split into two sister chromatids in mitosis. Here it is hypothesized that the bipolar pulling forces exerted by the mitotic spindle cause the sliding of the layers and facilitate sister chromatid resolution. This hypothesis is supported by three favorable conditions: i) There is no topological entanglement of DNA between adjacent layers; ii) The orientation (parallel to the stacked layers) of the bipolar kinetochore microtubules is adequate to produce layer sliding in opposite directions; iii) The viscous resistance to the sliding caused by the weak interactions between nucleosomes in adjacent layers can be overcome by the microtubule pulling forces.

RESUMEN

The nucleus is a complex organelle that hosts the genome and is essential for vital processes like DNA replication, DNA repair, transcription, and splicing. The genome is non-randomly organized in the three-dimensional space of the nucleus. This functional sub-compartmentalization was thought to be organized on the framework of nuclear matrix (NuMat), a non-chromatin scaffold that functions as a substratum for various molecular processes of the nucleus. More recently, nuclear bodies or membrane-less subcompartments of the nucleus are thought to arise due to phase separation of chromatin, RNA, and proteins. The nuclear architecture is an amalgamation of the relative organization of chromatin, epigenetic landscape, the nuclear bodies, and the nucleoskeleton in the three-dimensional space of the nucleus. During mitosis, the nucleus undergoes drastic changes in morphology to the degree that it ceases to exist as such; various nuclear components, including the envelope that defines the nucleus, disintegrate, and the chromatin acquires mitosis-specific epigenetic marks and condenses to form chromosome. Upon mitotic exit, chromosomes are decondensed, re-establish hierarchical genome organization, and regain epigenetic and transcriptional status similar to that of the mother cell. How this mitotic memory is inherited during cell division remains a puzzle. NuMat components that are a part of the mitotic chromosome in the form of mitotic chromosome scaffold (MiCS) could potentially be the seeds that guide the relative re-establishment of the epigenome, chromosome territories, and the nuclear bodies. Here, we synthesize the advances towards understanding cellular memory of nuclear architecture across mitosis and propose a hypothesis that a subset of NuMat proteome essential for nucleation of various nuclear bodies are retained in MiCS to serve as seeds of mitotic memory, thus ensuring the daughter cells re-establish the complex status of nuclear architecture similar to that of the mother cells, thereby maintaining the pre-mitotic transcriptional status.

Asunto(s)

Núcleo Celular , Cromatina , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromosomas/genética , Matriz Nuclear/metabolismo , Mitosis

RESUMEN

Condensin I is a pentameric protein complex that plays an essential role in mitotic chromosome assembly in eukaryotic cells. Although it has been shown that condensin I loading is mitosis specific, it remains poorly understood how the robust cell cycle regulation of condensin I is achieved. Here, we set up a panel of in vitro assays to demonstrate that cell cycle-specific loading of condensin I is regulated by the N-terminal tail (N-tail) of its kleisin subunit CAP-H. Deletion of the N-tail accelerates condensin I loading and chromosome assembly in Xenopus egg mitotic extracts. Phosphorylation-deficient and phosphorylation-mimetic mutations in the CAP-H N-tail decelerate and accelerate condensin I loading, respectively. Remarkably, deletion of the N-tail enables condensin I to assemble mitotic chromosome-like structures even in interphase extracts. Together with other extract-free functional assays in vitro, our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes.

Asunto(s)

Adenosina Trifosfatasas , Cromosomas , Ciclo Celular , Cromosomas/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitosis , Proteínas de Ciclo Celular/genética

RESUMEN

Mitotic chromosome assembly is an essential preparatory step for accurate transmission of the genome during cell division. During the past decades, biochemical approaches have uncovered the molecular basis of mitotic chromosomes. For example, by using cell-free assays of frog egg extracts, the condensin I complex central for the chromosome assembly process was first identified, and its functions have been intensively studied. A list of chromosome-associated proteins has been almost completed, and it is now possible to reconstitute structures resembling mitotic chromosomes with a limited number of purified factors. In this review, I introduce how far we have come in understanding the mechanism of chromosome assembly using cell-free assays and reconstitution assays, and I discuss their potential applications to solve open questions.

RESUMEN

The folding of interphase chromatin into highly compact mitotic chromosomes is one of the most recognizable changes during the cell cycle. However, the structural organization underlying this drastic compaction remains elusive. Here, we combine several super resolution methods, including structured illumination microscopy (SIM), binding-activated localization microscopy (BALM), and atomic force microscopy (AFM), to examine the structural details of the DNA within the mitotic chromosome, both in the native state and after up to 30-fold extension using single-molecule micromanipulation. Images of native chromosomes reveal widespread ∼125 nm compact granules (CGs) throughout the metaphase chromosome. However, at maximal extensions, we find exclusively ∼90 nm domains (mitotic nanodomains, MNDs) that are unexpectedly resistant to extensive forces of tens of nanonewtons. The DNA content of the MNDs is estimated to be predominantly ∼80 kb, which is comparable to the size of the inner loops predicted by a recent nested loop model of the mitotic chromosome. With this DNA content, the total volume expected of the human genome assuming closely packed MNDs is nearly identical to what is observed. Thus, altogether, these results suggest that these mechanically stable MNDs, and their higher-order assembly into CGs, are the dominant higher-level structures that underlie the compaction of chromatin from interphase to metaphase.

Asunto(s)

Cromatina , Cromosomas , Humanos , Cromosomas/metabolismo , Metafase , ADN/metabolismo , Micromanipulación

RESUMEN

We have used a combination of chemical genetics, chromatin proteomics, and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1as cells undergo synchronous mitotic entry within 15 min following release from a 1NM-PP1-induced arrest in late G2. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase, and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.

Asunto(s)

Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Cromosomas , ADN/metabolismo , Linfoma de Células B/metabolismo , Proteínas Nucleares/metabolismo , Profase , Proteoma , Proteómica , Animales , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Pollos , Cromatina/genética , ADN/genética , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/patología , Proteínas Nucleares/genética , Unión Proteica , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Factores de Tiempo

RESUMEN

Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.

RESUMEN

Transcription factors play a key role in maintaining cell identity. One mechanism of such cell memory after multiple rounds of cell division cycles is through persistent mitotic chromosome binding, although how individual transcription factors achieve mitotic chromosome retention is not completely understood. Here we show that PAX6, a lineage-determining transcription factor, coats mitotic chromosomes. Using deletion and point mutants associated with human ocular diseases in live-cell imaging analysis, we identified two regions, MCR-D1 and MCR-D2, that were responsible for mitotic chromosome retention of PAX6. We also identified three nuclear localization signals (NLSs) that contributed to mitotic chromosome retention independent of their nuclear import functions. Full mitotic chromosome retention required the presence of DNA-binding domains as well as NLSs within MCR-Ds. Furthermore, disease-associated mutations and NLS mutations changed the distribution of intrinsically disordered regions (IDRs) in PAX6. Our findings not only identify PAX6 as a novel mitotic chromosome retention factor but also demonstrate that the mechanism of mitotic chromosome retention involves sequence-specific DNA binding, NLSs, and molecular conformation determined by IDRs. These findings link mitotic chromosome retention with PAX6-related pathogenesis and imply similar mechanisms for other lineage-determining factors in the PAX family.

Asunto(s)

Cromosomas Humanos/metabolismo , Oftalmopatías/genética , Mitosis/genética , Factor de Transcripción PAX6/metabolismo , Células HEK293 , Humanos , Microscopía Intravital , Mutación , Factor de Transcripción PAX6/genética

RESUMEN

Ki-67 is one of the most famous marker proteins used by histologists to identify proliferating cells. Indeed, over 30 000 articles referring to Ki-67 are listed on PubMed. Here, we review some of the current literature regarding the protein. Despite its clinical importance, our knowledge of the molecular biology and biochemistry of Ki-67 is far from complete, and its exact molecular function(s) remain enigmatic. Furthermore, reports describing Ki-67 function are often contradictory, and it has only recently become clear that this proliferation marker is itself dispensable for cell proliferation. We discuss the unusual organization of the protein and its mRNA and how they relate to various models for its function. In particular, we focus on ways in which the intrinsically disordered structure of Ki-67 might aid in the assembly of the still-mysterious mitotic chromosome periphery compartment by controlling liquid-liquid phase separation of nucleolar proteins and RNAs.

Asunto(s)

Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Antígeno Ki-67/química , Antígeno Ki-67/metabolismo , Mitosis , Animales , Proliferación Celular , Humanos

RESUMEN

Tethering of viral genomes to host chromosomes has been recognized in a variety of DNA and RNA viruses. It can occur during both the productive cycle and latent infection and may impact viral genomes in manifold ways including their protection, localization, transcription, replication, integration, and segregation. Tethering is typically accomplished by dedicated viral proteins that simultaneously associate with both the viral genome and cellular chromatin via nucleic acid, histone and/or non-histone protein interactions. Some of the most prominent tethering proteins have been identified in DNA viruses establishing sustained latent infections, including members of the papillomaviruses and herpesviruses. Herpesvirus particles have linear genomes that circularize in infected cell nuclei and usually persist as extrachromosomal episomes. In several γ-herpesviruses, tethering facilitates the nuclear retention and faithful segregation of viral episomes during cell division, thus contributing to persistence of these viruses in the absence of infectious particle production. However, it has not been studied whether the genomes of human Cytomegalovirus (hCMV), the prototypical ß-herpesvirus, are tethered to host chromosomes. Here we provide evidence by fluorescence in situ hybridization that hCMV genomes associate with the surface of human mitotic chromosomes following infection of both non-permissive myeloid and permissive fibroblast cells. This chromosome association occurs at lower frequency in the absence of the immediate-early 1 (IE1) proteins, which bind to histones and have been implicated in the maintenance of hCMV episomes. Our findings point to a mechanism of hCMV genome maintenance through mitosis and suggest a supporting but non-essential role of IE1 in this process.

Asunto(s)

Citomegalovirus , Proteínas Inmediatas-Precoces , Cromosomas , Citomegalovirus/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Hibridación Fluorescente in Situ , Proteínas Virales

RESUMEN

Heterochromatin is identified as a potential factor driving diversification of species. To understand the magnitude of heterochromatin variation within the Anopheles gambiae complex of malaria mosquitoes, we analyzed metaphase chromosomes in An. arabiensis, An. coluzzii, An. gambiae, An. merus, and An. quadriannulatus. Using fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA), a highly repetitive fraction of DNA, and heterochromatic Bacterial Artificial Chromosome (BAC) clones, we established the correspondence of pericentric heterochromatin between the metaphase and polytene X chromosomes of An. gambiae. We then developed chromosome idiograms and demonstrated that the X chromosomes exhibit qualitative differences in their pattern of heterochromatic bands and position of satellite DNA (satDNA) repeats among the sibling species with postzygotic isolation, An. arabiensis, An. merus, An. quadriannulatus, and An. coluzzii or An. gambiae. The identified differences in the size and structure of the X chromosome heterochromatin point to a possible role of repetitive DNA in speciation of mosquitoes. We found that An. coluzzii and An. gambiae, incipient species with prezygotic isolation, share variations in the relative positions of the satDNA repeats and the proximal heterochromatin band on the X chromosomes. This previously unknown genetic polymorphism in malaria mosquitoes may be caused by a differential amplification of DNA repeats or an inversion in the sex chromosome heterochromatin.

Asunto(s)

Anopheles/genética , Variación Estructural del Genoma , Heterocromatina/genética , Cromosomas Politénicos/genética , Cromosoma X/genética , Animales , ADN Satélite/genética

RESUMEN

High mobility group box 1 (HMGB1) is an abundant non-histone nuclear protein that functions as a structural protein of chromatin, regulating genome replication and recombination, mRNA transcription and DNA repair. HMGB1 has been implicated in the tumorigenesis of various cancer types, and the upregulation of HMGB1 has been demonstrated in glioma cells. However, the association between HMGB1 and the mitotic chromosomes in glioma remains uncharacterized. In the present study, the sub-cellular localization of HMGB1 in glioma tissues and cells was investigated. In addition, enhanced green fluorescent protein (EGFP)-tagging of the human HMGB1 protein and chromosome spreading were used to investigate the combination of HMGB1 with mitotic chromosomes. The results of the current study indicated that HMGB1 was localized to the nucleus and the cytoplasm, and it was determined to combine with the condensed chromosomes of proliferating cells in paraformaldehyde (PFA)-fixed glioma tissues. However, HMGB1 was also associated with interphase (but not mitotic chromosomes) when fixed with chilled methanol and 5% (v/v) acetic acid or PFA in vitro. Data from live cell imaging and chromosome spreading indicated the association of HMGB1 with mitotic chromosomes in glioma cells. The present results suggest that HMGB1 combines with mitotic chromosomes in glioma cells, and that the use of fixatives may result in the dissociation of the HMGB1-DNA interaction. Therefore, in live specimens and chromosome spreads, EGFP fusion proteins may represent an accurate indicator for the determination of the correct localization and interaction of HMGB1 in glioma cells.

RESUMEN

The condensin protein complex plays a key role in the structural organization of genomes. How the ATPase activity of its SMC subunits drives large-scale changes in chromosome topology has remained unknown. Here we reconstruct, at near-atomic resolution, the sequence of events that take place during the condensin ATPase cycle. We show that ATP binding induces a conformational switch in the Smc4 head domain that releases its hitherto undescribed interaction with the Ycs4 HEAT-repeat subunit and promotes its engagement with the Smc2 head into an asymmetric heterodimer. SMC head dimerization subsequently enables nucleotide binding at the second active site and disengages the Brn1 kleisin subunit from the Smc2 coiled coil to open the condensin ring. These large-scale transitions in the condensin architecture lay out a mechanistic path for its ability to extrude DNA helices into large loop structures.

Asunto(s)

Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Proteínas Portadoras/química , Chaetomium/genética , Proteínas Cromosómicas no Histona/química , Proteínas de Unión al ADN/química , ADN/química , Complejos Multiproteicos/química , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Chaetomium/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Cromosomas/ultraestructura , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Células HeLa , Humanos , Modelos Moleculares , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Molecular dynamics simulation is a powerful tool used in modern molecular modeling, which enables a deeper comprehension of the physical behavior of atoms and molecules at a micro level. In this study, we simulated mitotic chromosome assembly mediated by condensins, a class of large protein complexes containing a pair of structural maintenance of chromosomes (SMC) subunits that are central to this process. In this chapter, we present the construction of a coarse-grained physical model of chromosomal DNA fibers and condensin molecules, and monitoring of the function of condensins in mitotic chromosome assembly, using computer-based molecular dynamics simulation. We explain how our model of chromosomes and condensins may be simulated using a package of molecular dynamics simulation. Procedures involved in calculating the observables of dynamics are described, together with an example of the simulation results.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Cromosomas/fisiología , Proteínas de Unión al ADN/metabolismo , Mitosis/fisiología , Complejos Multiproteicos/metabolismo , Simulación de Dinámica Molecular , Subunidades de Proteína/metabolismo

RESUMEN

Chromatin condenses several folds to form mitotic chromosomes during cell division and decondenses post-mitotically to reoccupy their nuclear territory and regain their specific transcriptional profile in a precisely lineage specific manner. This necessitates that the features of nuclear architecture and DNA topology persist through mitosis. We compared the proteome of nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an equivalent biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS). Our study elucidates that as much as 67% of the NuMat proteins are retained in the MiCS indicating that the features of nuclear architecture in interphase nucleus are retained on the mitotic chromosomes. Proteins of the NuMat/MiCS have large dynamic range of MS signal and were detected in sub-femtomolar amounts. Chromatin/RNA binding proteins with hydrolase and helicase activity are highly enriched in NuMat as well as MiCS. Although several transcription factors involved in functioning of interphase nucleus are present exclusively in NuMat, protein components responsible for assembly of membrane-less nuclear bodies are uniquely retained in MiCS. Our study clearly indicates that the features of nuclear architecture, in the structural context of NuMat, are retained in MiCS and possibly play an important role in maintenance of cell lineage specific transcriptional status during cell division and thereby, serve as components of cellular memory.

Asunto(s)

Cromosomas/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Mitosis , Matriz Nuclear/metabolismo , Animales , Electroforesis en Gel Bidimensional , Proteoma/metabolismo , Control de Calidad , Espectrometría de Masas en Tándem

RESUMEN

The mitotic chromosome, which is composed of a pair of sister chromatids, is a large macromolecular assembly that ensures faithful transmission of genetic information into daughter cells. Despite its fundamental importance, how a nucleosome fiber is folded and assembled into a large-scale chromatid structure remains poorly understood. To address this question, we have established a biochemically tractable system in which mitotic chromatids can be reconstituted in vitro by mixing a simple substrate (sperm nucleus) and a limited number of purified factors. The minimum set of required factors includes core histones, three histone chaperones, topoisomerase II, and condensin I. In this article, we describe a set of protocols for the preparation of key reagents and the setup of reconstitution reactions. We believe that this classical approach of biochemical reconstitution will be of great help to dissect the mechanisms of action of individual factors during mitotic chromatid assembly and to assess the contribution of nucleosome dynamics to this process from a fresh angle. © 2018 by John Wiley & Sons, Inc.

Asunto(s)

Cromátides/metabolismo , Técnicas Citológicas/métodos , Mitosis , Adenosina Trifosfatasas/metabolismo , Animales , Extractos Celulares , Núcleo Celular/metabolismo , Cromatografía de Afinidad , Proteínas de Unión al ADN/metabolismo , Femenino , Histonas/metabolismo , Masculino , Complejos Multiproteicos/metabolismo , Óvulo/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Espermatozoides/metabolismo , Xenopus

RESUMEN

Chromosomes consist of enormously long DNA molecules plus the proteins that package and regulate the transcription and replication of this DNA. In order to understand both the composition of the bulk chromatin that packages the DNA and the specialized structures that direct its segregation (e.g., centromeres and kinetochores), one requirement is to have a list of the component proteins of mitotic chromosomes. Identification and quantitation of these proteins and their modifications require the ability to isolate chromosomes and analyze their proteome by mass spectrometry. Here, we describe a step-by-step protocol to isolate mitotic chromosomes from vertebrate cells. The chromosome proteins may be labeled in vivo with heavy stable isotope for quantitative proteomics. We then go through the proteomics workflow from preparation of samples to their analysis in the mass spectrometer. Finally, we describe some of the software used in processing of output data for statistical and bioinformatic analysis.

Asunto(s)

Cromosomas/metabolismo , Espectrometría de Masas/métodos , Mitosis , Proteoma/metabolismo , Vertebrados/metabolismo , Animales , Fraccionamiento Químico , Pollos , Cromatografía por Intercambio Iónico