Nuclear pores form de novo from both sides of the nuclear envelope

Nuclear pore complexes are multiprotein channels that span the double lipid bilayer of the nuclear envelope. How new pores are inserted into the intact nuclear envelope of proliferating and differentiating eukaryotic cells is unknown. We found that the Nup107-160 complex was incorporated into assembly sites in the nuclear envelope from both the nucleoplasmic and the cytoplasmic sides. Nuclear pore insertion required the generation of Ran guanosine triphosphate in the nuclear and cytoplasmic compartments. Newly formed nuclear pore complexes did not contain structural components of preexisting pores, suggesting that they can form de novo.     

Role of ANC-1 in tethering nuclei to the actin cytoskeleton

Mutations in anc-1 (nuclear anchorage defective) disrupt the positioning of nuclei and mitochondria in Caenorhabditis elegans. ANC-1 is shown to consist of mostly coiled regions with a nuclear envelope localization domain (called the KASH domain) and an actin-binding domain; this structure was conserved with the Drosophila protein Msp-300 and the mammalian Syne proteins. Antibodies against ANC-1 localized cytoplasmically and were enriched at the nuclear periphery in an UNC-84-dependent manner. Overexpression of the KASH domain or the actin-binding domain caused a dominant negative anchorage defect. Thus, ANC-1 may connect nuclei to the cytoskeleton by interacting with UNC-84 at the nuclear envelope and with actin in the cytoplasm.     

Deep mixing of 3He: reconciling Big Bang and stellar nucleosynthesis

Low-mass stars, approximately 1 to 2 solar masses, near the Main Sequence are efficient at producing the helium isotope 3He, which they mix into the convective envelope on the giant branch and should distribute into the Galaxy by way of envelope loss. This process is so efficient that it is difficult to reconcile the low observed cosmic abundance of 3He with the predictions of both stellar and Big Bang nucleosynthesis. Here we find, by modeling a red giant with a fully three-dimensional hydrodynamic code and a full nucleosynthetic network, that mixing arises in the supposedly stable and radiative zone between the hydrogen-burning shell and the base of the convective envelope. This mixing is due to Rayleigh-Taylor instability within a zone just above the hydrogen-burning shell, where a nuclear reaction lowers the mean molecular weight slightly. Thus, we are able to remove the threat that 3He production in low-mass stars poses to the Big Bang nucleosynthesis of 3He.     

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.     

Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy

Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.     

Insulin stimulation of nucleoside triphosphatase activity in isolated nuclear envelopes

The activity of nucleoside triphosphatase, an enzyme that regulates nuclear messenger RNA transport, was measured in highly purified nuclear envelopes isolated from rat liver. Addition of picomolar concentrations of insulin to freshly prepared nuclear envelopes directly increased the enzyme activity. The major effect of insulin on this enzyme was to increase the maximum velocity of its activity; no significant effects were seen on the affinity constant. These studies raise the possibility, therefore, that the nuclear envelope is a site where insulin regulates nuclear functions.     

Nuclear mitochondria?

Recognizable mitochondria were detected in the nucleus of a leukemic cell. It is suggested that passage through enlarged nuclear pores, incorporation within a pinched off invagination, or inclusion within the nuclear envelope at telophase may have been responsible for this unusual event.     

Studies on the Programmed Cell Death in Rice During Starchy Endosperm Development

Morphological variations of the nucleus in starchy endosperm cell were observed by the electron-transmisson microscope during endosperm development in rice. Along with the development of the starchy endosperm,the nuclei of the cells showed chromatin condensation,the typical feature of programmed cell death(PCD). The nuclei also showed nucleus deformation,disruption of nuclear envelope,nucleoplasm leaking into the cytoplasm and nucleus disintegration resulting in nuclear residue formation. From the nucleus deformation to the nucleus disintegration,the morphological changes of the nucleus were orderly progressive. This indicated that the cell death of starchy endosperm in rice was programmed cell death. Evans Blue staining observation showed that the cell death was initially detected in the central part of starchy endosperm in rice,then expanded outward. The activities of superoxide dismutase(SOD)and catalase(CAT)in rice starchy endosperm both descended continuously as development progressed. The analysis of DNA of rice starchy endosperm did not show the presence of DNA laddering. The above results showed that the cell death of starchy endosperm in rice was a special form of PCD.     

水稻淀粉胚乳细胞发育期间程序性死亡的研究

 应用透射电子显微镜观察水稻淀粉胚乳细胞核在发育期间的形态变化。结果表明,伴随淀粉胚乳发育进程,其细胞核呈现染色质凝聚这一程序性细胞死亡(PCD)的典型特征。水稻胚乳细胞还出现核变形、核膜破裂、核基质进入胞质乃至核降解形成核残体的现象,细胞核从变形到解体是以一种有序的方式进行,证实水稻淀粉胚乳的细胞死亡是程序性细胞死亡。EVANS BLUE染色结果表明,水稻淀粉胚乳细胞死亡顺序是由胚乳中部向四周扩展。随发育进程,水稻胚乳中的超氧物歧化酶(SOD)和过氧化氢酶(CAT)活性持续下降。对水稻淀粉胚乳中的DNA进行     

日本鹌鹑睾丸的电镜观察

本文是通过透射电镜观察日本鹌鹑(Coturnix coturnix japonica)雄性生殖细胞及间质细胞的超微结构。精原细胞呈椭圆形。核圆形,异染色质少,常呈小块状,多靠近核被膜,核仁明显。染色质周围颗粒及染色质间颗粒清晰可见。线粒体有时呈空泡状,嵴短而稀疏,均匀分布于胞质中。精母细胞的异染色质散在分布于核质中,由颗粒状物质聚成团块,核仁明显。线粒体数量不多,分布情况如精原细胞。这与鸡的线粒体分布位置存有差异。粗面内质网和高尔基复合体发育良好,并可见环孔板。精子细胞核大而圆,异染色质少,呈稀疏的颗粒状分布在核被膜附近及分散于核液中。线粒体电子密度较高,内质网及高尔基复合体发育中等。间质细胞形状不规則,核仁清晰,内质网及线粒体丰富,二者有极性分布现象。线粒体嵴既有小管状,又有板层状。     

Nuclear reassembly excludes large macromolecules

Interphase nucleus and cytoplasm are distinct compartments, whose soluble macromolecular contents mix when the nuclear envelope disassembles at mitosis. To determine how their interphase identities are reestablished, fibroblasts were loaded with fluorescent dextrans and then allowed to divide. Large dextrans (molecular weight of 40,000 or more) were excluded from condensed mitotic chromosomes and from newly formed, postmitotic interphase nuclei. Therefore, postmitotic reassembly of the nucleus as a compartment distinct from cytoplasm occurs by exclusion not only of organelles but also of soluble macromolecules. This might occur by exclusion of macromolecules from condensed chromatin throughout mitosis and completion of nuclear envelope assembly before nuclear expansion.     

成年型牛淋巴肉瘤淋巴样细胞的核定量超微结构研究

本文通过细胞形态的定量分析技术研究了成年型牛淋巴肉瘤(LS)淋巴样细胞与正常淋巴细胞之间核仁及核膜超微结构的定量差别。结果表明:3组LS淋巴样细胞的单位细胞核体积中核仁所占体积较正常淋巴细胞平均分别增大70.73％、48.78％及80.49％,生统处理差异显著(P<0.05)或非常显著(P<0.01),3组LS淋巴样细胞的单位核体积所拥有的核膜表面积比正常淋巴细胞分别增大15.95％、18.76％和16.52,生统处理差异显著(p<0.05)或非常显著(p<0.01)。     

A photoactivatable GFP for selective photolabeling of proteins and cells

We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometer light and remains stable for days under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics by tracking photoactivated molecules that are the only visible GFPs in the cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange.     

大麦淀粉胚乳发育期间程序性细胞死亡的研究

 【目的】大麦胚乳发育过程中,淀粉胚乳细胞变成死细胞,本研究阐明这种死亡方式是一种特殊形式的程序性细胞死亡（PCD）。【方法】利用透射电镜和荧光显微镜观察淀粉胚乳细胞核的结构变化,利用琼脂糖凝胶电泳和TUNEL检测DNA断裂,并对抗氧化酶活性动态和籽粒灌浆速率进行测量。【结果】伴随淀粉胚乳发育进程,大麦淀粉胚乳细胞核呈现出核变形、染色质凝集、核膜破裂和核瓦解形成核残体等一系列PCD过程中核的变化特征。琼脂糖凝胶电泳和TUNEL结果都表明,核DNA发生了有规律的断裂,但DAPI染色结果却表明,核残体并没有从死亡的淀粉胚乳细胞中消失,而是分散存在于淀粉粒之间。Evan’s blue染色表明,死亡的淀粉胚乳细胞在胚乳组织中是随机发生的。在淀粉胚乳细胞核衰退解体和无核的胞质发育过程中,胚乳内抗氧化酶都存在一定的活性,籽粒的粒重也在不断增加。【结论】大麦淀粉胚乳在核衰退解体和无核化的胞质发育时期,胚乳细胞维持较高的代谢活性,没有表现衰退解体特征,直到被贮藏物质所充实才死亡,淀粉胚乳细胞的发育是一种特殊形式的PCD过程。     

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.     

感染叶锈菌的小麦细胞间隙液对小麦悬浮细胞程序性死亡的诱导

以小麦悬浮细胞为材料,分别用感染叶锈菌小种165的小麦细胞间隙液(IWF165)和未接菌的小麦细胞间隙液(IWFck)对其进行处理,处理后不同时间点取样进行形态学观察和DNA电泳。结果表明,用IWF165处理后的悬浮细胞可观察到明显的染色质凝聚化、边缘化和细胞核解体等现象;同时DNA电泳结果显示IWF165处理后的细胞基因组DNA发生降解并形成明显的DNA梯状条带。而用IWFck处理的悬浮细胞,其细胞核结构完整,染色质分布均匀;DNA电泳没有出现DNA梯状条带。在感染叶锈菌的小麦细胞间隙液中存在着激发子,它能够诱导小麦悬浮细胞发生程序性死亡。     

Expression of the art/trs protein of HIV and study of its role in viral envelope synthesis

The art/trs transactivator protein of human immunodeficiency virus (HIV) was expressed in mammalian cells as a 19-kilodalton protein that was immunoreactive with sera from HIV-infected patients. Separate plasmids encoding the art/trs protein, the tat protein, or the envelope glycoprotein gp120 were used to demonstrate that both art/trs and tat are absolutely required for the synthesis of gp120 from its cognate messenger RNA. In addition, both the tat and art/trs proteins influence the level of envelope RNA. The results suggest that art/trs and tat may be ideal targets for potential anti-HIV agents in AIDS therapy.     

Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators

Rhizobial bacteria enter a symbiotic interaction with legumes, activating diverse responses in roots through the lipochito oligosaccharide signaling molecule Nod factor. Here, we show that NSP2 from Medicago truncatula encodes a GRAS protein essential for Nod-factor signaling. NSP2 functions downstream of Nod-factor-induced calcium spiking and a calcium/calmodulin-dependent protein kinase. We show that NSP2-GFP expressed from a constitutive promoter is localized to the endoplasmic reticulum/nuclear envelope and relocalizes to the nucleus after Nod-factor elicitation. This work provides evidence that a GRAS protein transduces calcium signals in plants and provides a possible regulator of Nod-factor-inducible gene expression.