RESUMO
Recent evidence points at a role of protein interaction gradients around chromatin in mitotic spindle morphogenesis in large eukaryotic cells. Here, we explain how gradients can arise over distances of tens of microns around supramolecular structures from mixtures of soluble molecules. We discuss how coupled sets of such reaction diffusion processes generate the spatial information that determines the local dynamics of microtubules required to form a bipolar spindle. We argue that such reaction diffusion processes are involved in the self-organization of supramolecular structures in the cell.
Assuntos
Cromossomos/metabolismo , Citoplasma/fisiologia , Fuso Acromático/metabolismo , Animais , Extratos Celulares , Centrossomo/metabolismo , Cromossomos/fisiologia , GTP Fosfo-Hidrolases/química , Modelos Biológicos , Fosforilação , Transdução de Sinais , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Xenopus laevis/metabolismo , Proteína ran de Ligação ao GTP/metabolismoRESUMO
We present Monte Carlo (MC) simulations of the stretching of a single chromatin fiber. The model approximates the DNA by a flexible polymer chain with Debye-Hückel electrostatics and uses a two-angle zigzag model for the geometry of the linker DNA connecting the nucleosomes. The latter are represented by flat disks interacting via an attractive Gay-Berne potential. Our results show that the stiffness of the chromatin fiber strongly depends on the linker DNA length. Furthermore, changing the twisting angle between nucleosomes from 90 degrees to 130 degrees increases the stiffness significantly. An increase in the opening angle from 22 degrees to 34 degrees leads to softer fibers for small linker lengths. We observe that fibers containing a linker histone at each nucleosome are stiffer compared to those without the linker histone. The simulated persistence lengths and elastic moduli agree with experimental data. Finally, we show that the chromatin fiber does not behave as an isotropic elastic rod, but its rigidity depends on the direction of deformation: Chromatin is much more resistant to stretching than to bending.
Assuntos
Cromatina/química , Cromatina/ultraestrutura , DNA/química , DNA/ultraestrutura , Modelos Químicos , Modelos Moleculares , Força Compressiva , Simulação por Computador , Elasticidade , Modelos Estatísticos , Método de Monte Carlo , Conformação de Ácido Nucleico , Estresse Mecânico , Resistência à TraçãoRESUMO
Production of Ran-guanosine triphosphate (GTP) around chromosomes induces local nucleation and plus end stabilization of microtubules (MTs). The nuclear protein TPX2 is required for RanGTP-dependent MT nucleation. To find the MT stabilizer, we affinity purify nuclear localization signal (NLS)-containing proteins from Xenopus laevis egg extracts. This NLS protein fraction contains the MT stabilization activity. After further purification, we used mass spectrometry to identify proteins in active fractions, including cyclin-dependent kinase 11 (Cdk11). Cdk11 localizes on spindle poles and MTs in Xenopus culture cells and egg extracts. Recombinant Cdk11 demonstrates RanGTP-dependent MT stabilization activity, whereas a kinase-dead mutant does not. Inactivation of Cdk11 in egg extracts blocks RanGTP-dependent MT stabilization and dramatically decreases the spindle assembly rate. Simultaneous depletion of TPX2 completely inhibits centrosome-dependent spindle assembly. Our results indicate that Cdk11 is responsible for RanGTP-dependent MT stabilization around chromosomes and that this local stabilization is essential for normal rates of spindle assembly and spindle function.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Extratos Celulares , Linhagem Celular , Cromossomos/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/isolamento & purificação , Insetos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Microtúbulos/ultraestrutura , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/isolamento & purificação , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oócitos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fuso Acromático/genética , Fuso Acromático/ultraestrutura , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Xenopus laevis , Proteína ran de Ligação ao GTP/genéticaRESUMO
During cell division, chromosomes are distributed to daughter cells by the mitotic spindle. This system requires spatial cues to reproducibly self-organize. We report that such cues are provided by chromosome-mediated interaction gradients between the small guanosine triphosphatase (GTPase) Ran and importin-beta. This produces activity gradients that determine the spatial distribution of microtubule nucleation and stabilization around chromosomes and that are essential for the self-organization of microtubules into a bipolar spindle.
Assuntos
Cromossomos/metabolismo , Microtúbulos/metabolismo , Mitose , Transdução de Sinais , Fuso Acromático/metabolismo , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Cromatina/metabolismo , Sinais (Psicologia) , Difusão , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Masculino , Matemática , Metáfase , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Oócitos , Proteínas Recombinantes/metabolismo , Espermatozoides , Xenopus laevisRESUMO
The conformation of mononucleosome complexes reconstituted with recombinant core histones on a 614-basepair-long DNA fragment containing the Xenopus borealis 5S rRNA nucleosome positioning sequence was studied by scanning/atomic force microscopy in the absence or presence of linker histone H1. Imaging without prior fixation was conducted with air-dried samples and with mononucleosomes that were injected directly into the scanning force microscopy fluid cell and visualized in buffer. From a quantitative analysis of approximately 1,700 complexes, the following results were obtained: i), In the absence of H1, a preferred location of the nucleosome at the X. borealis 5S rRNA sequence in the center of the DNA was detected. From the distribution of nucleosome positions, an energy difference of binding to the 5S rRNA sequence of DeltaDeltaG approximately 3 kcal mol(-1) as compared to a random sequence was estimated. Upon addition of H1, a significantly reduced preference of nucleosome binding to this sequence was observed. ii), The measured entry-exit angles of the DNA at the nucleosome in the absence of H1 showed two maxima at 81 +/- 29 degrees and 136 +/- 18 degrees (air-dried samples), and 78 +/- 25 degrees and 137 +/- 25 degrees (samples imaged in buffer solution). In the presence of H1, the species with the smaller entry-exit angle was stabilized, yielding average values of 88 +/- 34 degrees for complexes in air and 85 +/- 10 degrees in buffer solution. iii), The apparent contour length of the nucleosome complexes was shortened by 34 +/- 13 nm as compared to the free DNA due to wrapping of the DNA around the histone octamer complex. Considering an 11 nm diameter of the nucleosome core complex, this corresponds to a total of 145 +/- 34 basepairs that are wound around the nucleosome.