RESUMO
In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped chromosomes1. This metamorphosis is driven mainly by the combined action of condensins and topoisomerase IIα (TOP2A)2,3, and has been observed using microscopy for over a century. Nevertheless, very little is known about the structural organization of a mitotic chromosome. Here we introduce a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native metaphase chromosomes to be conducted under tightly controlled experimental conditions. We have used this method to extensively characterize chromosome mechanics and structure. Notably, we find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behaviour, distinct from that predicted by classical polymer models4. To explain this anomalous stiffening, we introduce a hierarchical worm-like chain model that describes the chromosome as a heterogeneous assembly of nonlinear worm-like chains. Moreover, through inducible degradation of TOP2A5 specifically in mitosis, we provide evidence that TOP2A has a role in the preservation of chromosome compaction. The methods described here open the door to a wide array of investigations into the structure and dynamics of both normal and disease-associated chromosomes.
Assuntos
Cromossomos Humanos , Cromossomos , Cromossomos/genética , Cromossomos/metabolismo , Cromossomos Humanos/metabolismo , DNA/química , DNA Topoisomerases Tipo II/genética , Humanos , Mitose , Óptica e FotônicaRESUMO
We demonstrate experimentally that the trident process e^{-}âe^{-}e^{+}e^{-} in a strong external field, with a spatial extension comparable to the effective radiation length, is well understood theoretically. The experiment, conducted at CERN, probes values for the strong field parameter χ up to 2.4. Experimental data and theoretical expectations using the local constant field approximation show remarkable agreement over almost 3 orders of magnitude in yield.
RESUMO
Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase timing or the initiation of chromosome condensation. Instead, it prevents chromatin condensation in prometaphase, extends the length of prometaphase, and ultimately causes cells to exit mitosis without chromosome segregation occurring. Surprisingly, we find that removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure and conclude that TOP2A is crucial for maintenance of mitotic chromosomes. Treatments with drugs used to poison/inhibit TOP2A function, such as etoposide and ICRF-193, do not phenocopy the effects on chromosome structure of TOP2A degradation by AID. Our data point to a role for TOP2A as a structural chromosome maintenance enzyme locking in condensation states once sufficient compaction is achieved.
Assuntos
Estruturas Cromossômicas/química , Cromossomos Humanos/química , DNA Topoisomerases Tipo II/metabolismo , Heterocromatina/química , Mitose , Segregação de Cromossomos , Estruturas Cromossômicas/genética , Cromossomos Humanos/genética , Citocinese , DNA Topoisomerases Tipo II/genética , Células HCT116 , Heterocromatina/genética , Humanos , MetáfaseRESUMO
A fundamental requirement in nature is for a cell to correctly package and divide its replicated genome. Condensin is a mechanical multisubunit complex critical to this process. Condensin uses ATP to power conformational changes in DNA to enable to correct DNA compaction, organization, and segregation of DNA from the simplest bacteria to humans. The highly conserved nature of the condensin complex and the structural similarities it shares with the related cohesin complex have provided important clues as to how it functions in cells. The fundamental requirement for condensin in mitosis and meiosis is well established, yet the precise mechanism of action is still an open question. Mutation or removal of condensin subunits across a range of species disrupts orderly chromosome condensation leading to errors in chromosome segregation and likely death of the cell. There are divergences in function across species for condensin. Once considered to function solely in mitosis and meiosis, an accumulating body of evidence suggests that condensin has key roles in also regulating the interphase genome. This review will examine how condensin organizes our genomes, explain where and how it binds the genome at a mechanical level, and highlight controversies and future directions as the complex continues to fascinate and baffle biologists.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Genoma/genética , Complexos Multiproteicos/fisiologia , Adenosina Trifosfatases/ultraestrutura , Animais , Segregação de Cromossomos , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Interfase , Meiose , Mitose , Complexos Multiproteicos/ultraestruturaRESUMO
Condensin, a highly conserved pentameric chromosome complex, is required for the correct organization and folding of the genome. Here, we highlight how to knock protein tags into endogenous loci to faithfully study the condensin complex in vertebrates and dissect its multiple functions. These include using the streptavidin binding peptide (SBP) to create the first genome-wide map of condensin and perform varied applications in proteomics and enzymology of the complex. The revolution in gene editing using CRISPR/Cas9 has made it possible to insert tags into endogenous loci with relative ease, allowing physiological and fully functional tagged protein to be analyzed biochemically (affinity tags), microscopically (fluorescent tags) or both purified and localized (multifunctional tags). In this chapter, we detail how to engineer vertebrate cells using CRISPR/Cas9 to provide researchers powerful tools to obtain greater precision than ever to understand how the complex interacts and behaves in cells.
Assuntos
Adenosina Trifosfatases/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Complexos Multiproteicos/genética , Animais , Sistemas CRISPR-Cas/genética , Mapeamento Cromossômico/métodos , Edição de Genes/métodos , Genoma/genética , Proteômica/métodos , Vertebrados/genéticaRESUMO
Ultrafine anaphase bridges (UFBs) are thin DNA threads linking the separating sister chromatids in the anaphase of mitosis. UFBs are thought to form when topological DNA entanglements between two chromatids are not resolved prior to anaphase onset. In contrast to other markers of defective chromosome segregation, UFBs cannot be detected by direct staining of the DNA, but instead can be detected using immunofluorescence-based approaches. Due to the fact that they are short-lived and fragile in nature, UFBs can be challenging to detect. In this chapter, we describe methods that have been optimized for successful detection of UFBs. We also provide guidelines for the optimization of UFBs detection depending on the antibody and the cell line to be used.
Assuntos
Anáfase/genética , Cromátides/genética , DNA , Linhagem Celular Tumoral , Centrômero/genética , Sítios Frágeis do Cromossomo , Segregação de Cromossomos , Imunofluorescência , Instabilidade Genômica , Humanos , Microscopia de Fluorescência , Telômero/genéticaRESUMO
Transcription factor-based biosensors are used to identify producer strains, a critical bottleneck in cell factory engineering. Here, we address two challenges with this methodology: transplantation of heterologous transcriptional regulators into new hosts to generate functional biosensors and biosensing of the extracellular product concentration that accurately reflects the effective cell factory production capacity. We describe the effects of different translation initiation rates on the dynamic range of a p-coumaric acid biosensor based on the Bacillus subtilis transcriptional repressor PadR by varying its ribosomal binding site. Furthermore, we demonstrate the functionality of this p-coumaric acid biosensor in Escherichia coli and Corynebacterium glutamicum. Finally, we encapsulate yeast p-coumaric acid-producing cells with E. coli-biosensing cells in picoliter droplets and, in a microfluidic device, rapidly sort droplets containing yeast cells producing high amounts of extracellular p-coumaric acid using the fluorescent E. coli biosensor signal. As additional biosensors become available, such approaches will find broad applications for screening of an extracellular product.
Assuntos
Técnicas Biossensoriais/métodos , Propionatos/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Ácidos Cumáricos , Escherichia coli/metabolismo , Fatores de Transcrição/metabolismoRESUMO
PICH is an SNF2-family DNA translocase that appears to play a role specifically in mitosis. Characterization of PICH in human cells led to the initial discovery of "ultra-fine DNA bridges" (UFBs) that connect the 2 segregating DNA masses in the anaphase of mitosis. These bridge structures, which arise from specific regions of the genome, are a normal feature of anaphase but had escaped detection previously because they do not stain with commonly used DNA dyes. Nevertheless, UFBs are important for genome maintenance because defects in UFB resolution can lead to cytokinesis failure. We reported recently that PICH stimulates the unlinking (decatenation) of entangled DNA by Topoisomerase IIα (Topo IIα), and is important for the resolution of UFBs. We also demonstrated that PICH and Topo IIα co-localize at the rDNA (rDNA). In this Extra View article, we discuss the mitotic roles of PICH and explore further the role of PICH in the timely segregation of the rDNA locus.
Assuntos
Segregação de Cromossomos/genética , DNA Helicases/metabolismo , DNA Ribossômico/genética , Mitose/genética , Não Disjunção Genética , Animais , Linhagem Celular , Galinhas , Humanos , Modelos Biológicos , Transcrição GênicaRESUMO
PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH(-/-) cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH(-/-) cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas Aviárias/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Segregação de Cromossomos/genética , DNA Helicases/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose/genética , Animais , Proteínas Aviárias/metabolismo , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Galinhas , Instabilidade Cromossômica/genética , DNA Helicases/metabolismo , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Inativação de Genes , Humanos , Linfócitos/metabolismoRESUMO
Ultra-fine DNA bridges (UFBs) are a recently identified class of mitotic DNA structures that cannot be visualized using conventional DNA staining methods (e.g. using DAPI). Their existence can currently only be revealed by immuno-fluorescent staining for proteins that bind to them, including PICH and BLM. UFBs become visible in the anaphase of mitosis, and can persist into telophase in rare cases. There are at least three different types of UFBs that can be distinguished according to the chromosomal loci from which they originate. However, it remains largely unknown how these UFBs are generated or resolved in the cell. In this article, we will review our current understanding of different types of UFBs and the potential functional role of the proteins that have been shown to be associated with them.