Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts

The small guanosine triphosphatase Ran is loaded with guanosine triphosphate (GTP) by the chromatin-bound guanine nucleotide exchange factor RCC1 and releases import cargoes in the nucleus during interphase. In mitosis, Ran-GTP promotes spindle assembly around chromosomes by locally discharging cargoes that regulate microtubule dynamics and organization. We used fluorescence resonance energy transfer-based biosensors to visualize gradients of Ran-GTP and liberated cargoes around chromosomes in mitotic Xenopus egg extracts. Both gradients were required to assemble and maintain spindle structure. During interphase, Ran-GTP was highly enriched in the nucleoplasm, and a steep concentration difference between nuclear and cytoplasmic Ran-GTP was established, providing evidence for a Ran-GTP gradient surrounding chromosomes throughout the cell cycle.     

Spatial coordination of spindle assembly by chromosome-mediated signaling gradients

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.     

A mitotic lamin B matrix induced by RanGTP required for spindle assembly

Mitotic spindle morphogenesis is a series of highly coordinated movements that lead to chromosome segregation and cytokinesis. We report that the intermediate filament protein lamin B, a component of the interphase nuclear lamina, functions in spindle assembly. Lamin B assembled into a matrix-like network in mitosis through a process that depended on the presence of the guanosine triphosphate-bound form of the small guanosine triphosphatase Ran. Depletion of lamin B resulted in defects in spindle assembly. Dominant negative mutant lamin B proteins that disrupt lamin B assembly in interphase nuclei also disrupted spindle assembly in mitosis. Furthermore, lamin B was essential for the formation of the mitotic matrix that tethers a number of spindle assembly factors. We propose that lamin B is a structural component of the long-sought-after spindle matrix that promotes microtubule assembly and organization in mitosis.     

Chromatin docking and exchange activity enhancement of RCC1 by histones H2A and H2B

The Ran guanosine triphosphatase (GTPase) controls nucleocytoplasmic transport, mitotic spindle formation, and nuclear envelope assembly. These functions rely on the association of the Ran-specific exchange factor, RCC1 (regulator of chromosome condensation 1), with chromatin. We find that RCC1 binds directly to mononucleosomes and to histones H2A and H2B. RCC1 utilizes these histones to bind Xenopus sperm chromatin, and the binding of RCC1 to nucleosomes or histones stimulates the catalytic activity of RCC1. We propose that the docking of RCC1 to H2A/H2B establishes the polarity of the Ran-GTP gradient that drives nuclear envelope assembly, nuclear transport, and other nuclear events.     

A kinesin-like motor inhibits microtubule dynamic instability

The motility of molecular motors and the dynamic instability of microtubules are key dynamic processes for mitotic spindle assembly and function. We report here that one of the mitotic kinesins that localizes to chromosomes, Xklp1 from Xenopus laevis, could inhibit microtubule growth and shrinkage. This effect appeared to be mediated by a structural change in the microtubule lattice. We also found that Xklp1 could act as a fast, nonprocessive, plus end-directed molecular motor. The integration of the two properties, motility and inhibition of microtubule dynamics, in one molecule emphasizes the versatile properties of kinesin family members.     

Localization of RanBP1 in Early Embryonic Development of Mice

RanBP1 is a binding protein of Ran that plays a pivotal role in nucleocytoplasmic transport.In this study,the localization and possible functions of RanBP1 were examined,during the early embryonic development of mice.Immunofluorescence results showed that RanBP1 was mainly localized in cytoplasm at mitosis interphase,and its concentration was lower in nucleus and the lowest in nucleolus.With the formation of the spindle in the early embryonic cells,RanBP1 condensed area took the shape of spindle microtubule,the concentration of RanBP1 was low in the site of chromosome.During the formation of nucleus,RanBP1 concentrated in nucleus and there were few dots of RanBP1 around the nucleolus.These dots were lost after the nucleus full growth.The results showed that RanBP1 had important roles in nucleocytoplasmic transport,spindle formation and nuclear assembly in the early embryonic development of mice.     

Ran调节因子RanBP1的生物学作用

RanBP1位于细胞质中,是Ran的结合蛋白,它能够抑制RCC1的活性,间接地刺激了RanGTPase活性,促进GTP的水解。RanBP1在细胞的核质运输、纺锤体的组装、细胞周期、有丝分裂控制和有丝分裂结束后的核重组中都有一定的调节作用。     

NuMA蛋白的生物学作用及其研究进展

NuMA存在于动、植物细胞中,是细胞核基质的重要组成部分,参与纺锤体极的组装与分解、核重组等过程,调节细胞周期,对细胞的有丝分裂和减数分裂具有重要作用。此外,对细胞凋亡也发挥重要作用,NuMA的降解是早期细胞调亡的标志。     

The protein kinase p90 rsk as an essential mediator of cytostatic factor activity

Persistent activation of p42 mitogen-activated protein kinase (p42 MAPK) during mitosis induces a "cytostatic factor" arrest, the arrest responsible for preventing the parthenogenetic activation of unfertilized eggs. The protein kinase p90 Rsk is a substrate of p42 MAPK; thus, the role of p90 Rsk in p42 MAPK-induced mitotic arrest was examined. Xenopus laevis egg extracts immunodepleted of Rsk lost their capacity to undergo mitotic arrest in response to activation of the Mos-MEK-1-p42 MAPK cascade of protein kinases. Replenishing Rsk-depleted extracts with catalytically competent Rsk protein restored the ability of the extracts to undergo mitotic arrest. Rsk appears to be essential for cytostatic factor arrest.     

Regulation of microtubule dynamics by reaction cascades around chromosomes

During spindle assembly, chromosomes generate gradients of microtubule stabilization through a reaction-diffusion process, but how this is achieved is not well understood. We measured the spatial distribution of microtubule aster asymmetry around chromosomes by incubating centrosomes and micropatterned chromatin patches in frog egg extracts. We then screened for microtubule stabilization gradient shapes that would generate such spatial distributions with the use of computer simulations. Only a long-range, sharply decaying microtubule stabilization gradient could generate aster asymmetries fitting the experimental data. We propose a reaction-diffusion model that combines the chromosome generated Ran-guanosine triphosphate-Importin reaction network to a secondary phosphorylation network as a potential mechanism for the generation of such gradients.     

拟南芥Ran小GTP结合蛋白在细胞有丝分裂中的定位

分别采用RT-PCR和直接荧光免疫方法分析Ran2 mRNA在拟南芥不同组织器官中的表达及Ran2的亚细胞定位。结果表明,Ran2基因在拟南芥根、茎、叶和花中均有表达;Ran2蛋白在细胞分裂间期主要定位于核膜周边,在后期定位于赤道板上和纺锤体上,末期又回到子细胞核膜周边。     

Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts

The nuclear envelope (NE) forms a controlled boundary between the cytoplasm and the nucleus of eukaryotic cells. To facilitate investigation of mechanisms controlling NE assembly, we developed a cell-free system made from Xenopus laevis eggs to study the process in the absence of chromatin. NEs incorporating nuclear pores were assembled around beads coated with the guanosine triphosphatase Ran, forming pseudo-nuclei that actively imported nuclear proteins. NE assembly required the cycling of guanine nucleotides on Ran and was promoted by RCC1, a nucleotide exchange factor recruited to beads by Ran-guanosine diphosphate (Ran-GDP). Thus, concentration of Ran-GDP followed by generation of Ran-GTP is sufficient to induce NE assembly.     

Abnormal spindle protein, Asp, and the integrity of mitotic centrosomal microtubule organizing centers

The product of the abnormal spindle (asp) gene was found to be an asymmetrically localized component of the centrosome during mitosis, required to focus the poles of the mitotic spindle in vivo. Removing Asp protein function from Drosophila melanogaster embryo extracts, either by mutation or immunodepletion, resulted in loss of their ability to restore microtubule-organizing center activity to salt-stripped centrosome preparations. This was corrected by addition of purified Asp protein. Thus, Asp appears to hold together the microtubule-nucleating gamma-tubulin ring complexes that organize the mitotic centrosome.     

Nucleated assembly of microtubules in porcine brain extracts

Disk-type structures found in extracts of porcine brain tissue appear to be required for microtubule assembly in vitro. From the morphology of the disks and the dependence of microtubule assembly on the presence of these structures, we propose that the disks are nucleation centers for the polymerization of microtubule protein.     

Ability of the c-mos product to associate with and phosphorylate tubulin.

The mos proto-oncogene product, pp39mos, is a protein kinase and has been equated with cytostatic factor (CSF), an activity in unfertilized eggs that is thought to be responsible for the arrest of meiosis at metaphase II. The biochemical properties and potential substrates of pp39mos were examined in unfertilized eggs and in transformed cells in order to study how the protein functions both as CSF and in transformation. The pp39mos protein associated with polymers under conditions that favor tubulin oligomerization and was present in an approximately 500-kilodalton "core" complex under conditions that favor depolymerization. beta-Tubulin was preferentially coprecipitated in pp39mos immunoprecipitates and was the major phosphorylated product in a pp39mos-dependent immune complex kinase assay. Immunofluorescence analysis of NIH 3T3 cells transformed with Xenopus c-mos showed that pp39mos colocalizes with tubulin in the spindle during metaphase and in the midbody and asters during telophase. Disruption of microtubules with nocodazole affected tubulin and pp39mos organization in the same way. It therefore appears that pp39mos is a tubulin-associated protein kinase and may thus participate in the modification of microtubules and contribute to the formation of the spindle. This activity expressed during interphase in somatic cells may be responsible for the transforming activity of pp39mos.     

猪颗粒细胞中Ran的定位及作用

试验成功地培养了猪颗粒细胞,并采用激光共聚焦显微技术研究细胞有丝分裂过程中Ran的定位变化。结果表明,猪间期颗粒细胞中,Ran主要分布于细胞核内,核仁内没有Ran分布,细胞质中Ran浓度极低。核膜破裂,细胞进入有丝分裂,Ran分布到整个细胞,但在染色体周围浓集;在有丝分裂中期,浓集区形态和纺锤体的形态一致;在有丝分裂后期和末期,Ran的浓集区随纺锤体的拉长而分布于被分开的两组染色体之间;有丝分裂结束后,随着细胞核的形成,Ran在染色体周围浓集,核膜形成后又浓集于细胞核内,在整个有丝分裂中,Ran在细胞分裂区的细胞膜下未见特殊分布。这些分布与Ran在细胞周期中的作用是一致的。     

Spindle multipolarity is prevented by centrosomal clustering

Most tumor cells are characterized by increased genomic instability and chromosome segregational defects, often associated with hyperamplification of the centrosome and the formation of multipolar spindles. However, extra centrosomes do not always lead to multipolarity. Here, we describe a process of centrosomal clustering that prevented the formation of multipolar spindles in noncancer cells. Noncancer cells needed to overcome this clustering mechanism to allow multipolar spindles to form at a high frequency. The microtubule motor cytoplasmic dynein was a critical part of this coalescing machinery, and in some tumor cells overexpression of the spindle protein NuMA interfered with dynein localization, promoting multipolarity.     

The centromeric protein Sgo1 is required to sense lack of tension on mitotic chromosomes

Chromosome alignment on the mitotic spindle is monitored by the spindle checkpoint. We identify Sgo1, a protein involved in meiotic chromosome cohesion, as a spindle checkpoint component. Budding yeast cells with mutations in SGO1 respond normally to microtubule depolymerization but not to lack of tension at the kinetochore, and they have difficulty attaching sister chromatids to opposite poles of the spindle. Sgo1 is thus required for sensing tension between sister chromatids during mitosis, and its degradation when they separate may prevent cell cycle arrest and chromosome loss in anaphase, a time when sister chromatids are no longer under tension.     

The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A

Initiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.     

植物GTP结合蛋白的研究进展

GTP结合蛋白(G蛋白)广泛存在于生物界,在细胞信息传导中起着极为重要的开关作用,它可以调控真核生物中高度保守的跨膜信号传导通路。G蛋白可以划分为异源三聚体G蛋白和低分子量G蛋白(Ran)两种类型。Ran是一种大量分布于细胞核内的小分子的GTP酶,在核质运输以及微管蛋白成核过程中具有十分重要的作用。本文主要对G蛋白以及低分子量G蛋白Ran的研究进展做一综述。