Intranuclear injection of anti-actin antibodies into Xenopus oocytes blocks chromosome condensation

The role of contractile proteins in the structural organisation of the interphase nucleus and of metaphase chromosomes is largely unknown. Actin has been found in interphase nuclei of different species, especially in association with condensed chromatin. In the germinal vesicle (nucleus) of Xenopus oocytes, actin has been localised in the nuclear gel supporting the chromosomes and the extrachromosomal nucleoli. It has been reported that the premeiotic lampbrush chromosomes in these germinal vesicles are positively stained for actin and tubulin by the immunoperoxidase technique. Moreover, the longitudinal contraction of these chromosomes is ATP dependent. Therefore it has been suggested that actin participates in the structural organisation of the highly specialised lampbrush chromosomes. However, actin is not a major component of the metaphase chromosome scaffold. The results reported here suggest that actin is involved in the condensation of Xenopus chromosomes.     

Identification of specific binding proteins for a nuclear location sequence

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex and are directed by specific nuclear location sequences (NLS) in the proteins. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells and the nuclear accumulation of chimaeric proteins or of non-nuclear proteins conjugated to synthetic peptides. The best-characterized NLS is the simian virus 40 large T-antigen sequence. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus.     

Steroid-induced meiotic division in Xenopus laevis oocytes: surface and calcium.

Progesterone reinitiates meiotic maturation in Xenopus oocytes. Evidence is reported which indicates that the steroid acts at the level of the cell surface and suggests that an induced change of Ca2+ distribution triggers in turn a cascade of cytoplasmic events including protein synthesis and germinal vesicle (nucleus) breakdown. These novel features of steroid hormone action in amphibian oocytes are discussed in relation to presently accepted views of the mechanism of action of steroid hormones in somatic cells.     

核内肌动蛋白的研究进展

肌动蛋白是细胞质中的一种重要的胞质蛋白,承担多种细胞质功能,近年的研究工作表明在细胞核中存在肌动蛋白,而且,肌动蛋白还参与了核内多种生理活动,如DNA的转录、RNA的转运等,综述了近年来有关核内肌动蛋白的研究进展。     

Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2

Calcium is believed to control a variety of cellular processes, often with a high degree of spatial and temporal precision. For a cell to use Ca2+ in this manner, mechanisms must exist for controlling the ion in a localized fashion. We have now gained insight into such mechanisms from studies which measured Ca2+ in single living cells with high resolution using a digital imaging microscope and the highly fluorescent Ca2+-sensitive dye, Fura-2. Levels of Ca2+ in the cytoplasm, nucleus and sarcoplasmic reticulum (SR) are clearly different. Free [Ca2+] in the nucleus and SR was greater than in the cytoplasm and these gradients were abolished by Ca2+ ionophores. When external Ca2+ was raised above normal in the absence of ionophores, free cytoplasmic Ca2+ increased but nuclear Ca2+ did not. Thus, nuclear [Ca2+] appears to be regulated independently of cytoplasmic [Ca2+] by gating mechanisms in the nuclear envelope. The observed regulation of intranuclear Ca2+ in these contractile cells may thus be seen as a way to prevent fluctuation in Ca2+-linked nuclear processes during the rise in cytoplasmic [Ca2+] which triggers contraction. The approach described here offers the opportunity of following changes in Ca2+ in cellular compartments in response to a wide range of stimuli, allowing new insights into the role of local changes in Ca2+ in the regulation of cell function.     

Calcium-dependent phosphorylation of histone H3 in butyrate-treated HeLa cells

Ca2+ is prominant in the control of cell proliferation and function. However, the biochemical mechanism(s) mediating its effects on nuclear events is unknown. We report here that Ca2+, at physiological concentrations, stimulates the phosphorylation of histone H3 by an endogenous protein kinase in HeLa cell nuclei. Also, pretreatment of cells with Na butyrate, which increases histone acetylation, selectively increases the susceptability of histone H3 to phosphorylation by the protein kinase. Our results reveal a potential link between histone H3 acetylation and phosphorylation, modifications which are thought to have important effects on chromatin structure and function and suggest a possible mechanism whereby stimuli at the cell surface (such as hormones, mitogens and drugs) may influence biochemical events at the nuclear level; changes in the intracellular Ca2+ concentration may influence the phosphorylation of chromosomal proteins, mediated by Ca2+ -dependent kinases in th nucleus.     

Intracellular transport of microinjected 5S and small nuclear RNAs

The mechanism by which some RNAs are segregated in the cell nucleus was analysed by microinjecting 32 P-labelled total RNA from HeLa cells into the cytoplasm of Xenopus oocytes. Small nuclear RNAs (u1, U2, U4, U5 and U6) migrated into the cell nucleus, where they became 30-60 fold more concentrated than in the cytoplasm. Other RNAs, such as tRNA and 7S RNA, remained in the cytoplasm, while 5S RNA became concentrated in the nucleolus. Studies with lupus erythematosus antibodies showed that the migrating RNAs become associated with oocyte RNA-binding proteins.     

Distribution of two distinct Ca2+-ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells

Many cellular functions are regulated by activation of cell-surface receptors that mobilize calcium from internal stores sensitive to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The nature of these internal calcium stores and their localization in cells is not clear and has been a subject of debate. It was originally suggested that the Ins(1,4,5)P3-sensitive store is the endoplasmic reticulum, but a new organelle, the calciosome, identified by its possession of the calcium-binding protein, calsequestrin, and a Ca2+-ATPase-like protein of relative molecular mass 100,000 (100K), has been described as a potential Ins(1,4,5)P3-sensitive calcium store. Direct evidence on whether the calciosome is the Ins(1,4,5)P3-sensitive store is lacking. Using monoclonal antibodies raised against the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, we show that bovine adrenal chromaffin cells contain two Ca2+-ATPase-like proteins with distinct subcellular distributions. A 100K Ca2+-ATPase-like protein is diffusely distributed, whereas a 140K Ca2+-ATPase-like protein is restricted to a region in close proximity to the nucleus. In addition, Ins(1,4,5)P3-generating agonists result in a highly localized rise in cytosolic calcium concentration ([Ca2+]i) initiated in a region close to the nucleus, whereas caffeine results in a rise in [Ca2+]i throughout the cytoplasm. Our results indicate that chromaffin cells possess two calcium stores with distinct Ca2+-ATPases and that the organelle with the 100K Ca2+-ATPase is not the Ins(1,4,5)P3-sensitive store.     

Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is a widespread receptor-coupled signalling system at the plasma membrane of most eukaryotic cells. The existence of an entirely separate nuclear phosphoinositide signalling system is suggested from evidence that purified nuclei synthesize PtdInsP2 and phosphatidylinositol 4-phosphate (PtdInsP) in vitro and that a transient decrease in the mass of these lipids occurs when Swiss 3T3 cells are cultured in the presence of insulin-like growth factor-1 (IGF-1). These IGF-1-dependent changes in inositol lipids coincide with an increase in nuclear diacyglycerol and precede translocation to the nucleus and activation of protein kinase C (refs 5, 6). Circumstantial evidence that links these changes with mitosis comes from the isolation of a 3T3 clone that expresses the type-1 IGF receptor and binds IGF-1 peptide but does not respond mitogenically or show transient mass changes in nuclear inositol lipids. A key question is how IGF-1 initiates the rapid breakdown of PtdInsP and PtdInsP2 in the nucleus. Here we present evidence that nuclei of 3T3 cells contain the beta-isozyme of phosphoinositidase C, whereas the gamma-isozyme is confined to the cytoplasm and that IGF-1 treatment stimulates exclusively the activity of nuclear phosphoinositidase C.     

RanGAP mediates GTP hydrolysis without an arginine finger

GTPase-activating proteins (GAPs) increase the rate of GTP hydrolysis on guanine nucleotide-binding proteins by many orders of magnitude. Studies with Ras and Rho have elucidated the mechanism of GAP action by showing that their catalytic machinery is both stabilized by GAP binding and complemented by the insertion of a so-called 'arginine finger' into the phosphate-binding pocket. This has been proposed as a universal mechanism for GAP-mediated GTP hydrolysis. Ran is a nuclear Ras-related protein that regulates both transport between the nucleus and cytoplasm during interphase, and formation of the mitotic spindle and/or nuclear envelope in dividing cells. Ran-GTP is hydrolysed by the combined action of Ran-binding proteins (RanBPs) and RanGAP. Here we present the three-dimensional structure of a Ran-RanBP1-RanGAP ternary complex in the ground state and in a transition-state mimic. The structure and biochemical experiments show that RanGAP does not act through an arginine finger, that the basic machinery for fast GTP hydrolysis is provided exclusively by Ran and that correct positioning of the catalytic glutamine is essential for catalysis.     

Site-directed mutagenesis of the regulatory light-chain Ca2+/Mg2+ binding site and its role in hybrid myosins

The regulatory light chains, small polypeptides located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation. The demonstration that the regulatory light chains on scallop myosin can be replaced by light chains from other myosins has allowed us to compare the functional capabilities of different light chains, but has not enabled us to probe the role of features, such as the Ca2+/Mg2+ binding site, that are common to all of them. Here, we describe the use of site-directed mutagenesis to study the function of that site. We synthesized the chicken skeletal myosin light chain in Escherichia coli and constructed mutants with substitutions within the Ca2+/Mg2+ binding site. When the aspartate residues at the first and sixth Ca2+ coordination positions are replaced by uncharged alanines, the light chains have a reduced Ca2+ binding capacity but still bind to scallop myosin with high affinity. Unlike the wild-type skeletal light chain which inhibits myosin interaction with actin, the mutants activate it. Thus, an intact Ca2+/Mg2+ binding site in the N-terminal region of the light chain is essential for regulating the interaction of myosin with actin.     

肌动蛋白在子代杆状病毒胞内运输过程中的作用

重组病毒AcMNPV-GFP-actin(ph-)能在晚期和极晚期成功表达外源肌动蛋白,其生长特性与野生型AcMNPV没有明显的差别.在重组病毒同步感染的Sf9细胞中,肌动蛋白从细胞质转运到细胞核内,然后又向细胞质转运并且发生聚合;到感染极晚期从细胞核全部转运到细胞质,此时受染细胞核中进行正常的病毒粒子的组装.用CD处理重组病毒感染的细胞并不能阻止肌动蛋白在细胞内转运的变化规律,只是推迟了变化出现的时间,并使肌动蛋白发生聚合.     

Synthetic peptides as nuclear localization signals

The nuclear envelope defines a compartment boundary which is penetrated by pores that mediate a remarkable transport process. Precursor RNAs are retained in the nucleus, while processed messenger RNA, transfer RNA and ribosomal subunits are transported to the cytoplasm. Proteins destined for the nucleus become localized soon after synthesis and again following mitosis, while cytoplasmic proteins are excluded. The process is highly specific: a single base change in vertebrate initiator tRNAMet (tRNAiMet) reduces the rate of export 20-fold; a point mutation within the simian virus 40 (SV40) large-T antigen, converting Lys 128 to Thr or Asn, prevents import. Lys 128 lies within a short 'signal' sequence which, when fused to large non-nuclear proteins, causes their accumulation in nuclei. Regions of other eukaryotic proteins also seem to contain nuclear localization signals, although a single consensus sequence has not emerged. We report here that a synthetic peptide containing 10 residues of large-T antigen sequence serves as a nuclear localization signal when cross-linked to bovine serum albumin (BSA) or immunoglobulin G (IgG) and microinjected in Xenopus oocytes. Substitution of Thr at the position of Lys 128 in this peptide renders it six- to sevenfold less effective. The uptake of peptide-linked BSA is saturable, and the rate is diminished by co-injection of free peptide. These findings are indicative of a receptor-mediated uptake process. With the use of anti-peptide antibodies, a family of proteins is revealed in nuclear but not cytoplasmic extracts of human lymphocytes which contain large-T antigen-like sequences.     

野生大豆根尖中柱鞘细胞脱分化的超微结构初探

野生大豆根尖成熟区的中柱鞘细胞在自然条件下即会发生脱分化。通过研究此类脱分化细胞的超微结构，揭示出：在中柱鞘细胞发化过程中，细胞由相对静止到活跃活动。细胞核膜向核基质深度凹入，形成数量不等充满细胞质的凹陷，胞基质和细胞器如线粒体、内质网和内质网形成的小囊泡深入凹陷中。脱分化的细胞核膨大，核膜上核孔密集分布，核周围聚集线粒体、粗面内质网和大量的核糖体，细胞核和细胞质之间进行着活跃的物质和能量交换活动     

中华蟾蜍卵巢性类固醇激素受体的免疫细胞化学研究

为探讨两栖动物卵巢内不同性类固醇激素受体在卵子发育中的调控作用.采用免疫细胞化学方法,对中华蟾蜍不同发育时期卵泡中的雌激素受体、孕激素受体和雄激素受体进行了定位检测.结果发现,三种受体在不同发育时期的卵泡中均有阳性反应:雌激素受体在卵黄合成期和卵黄合成后期的滤泡细胞表达为强阳性,在卵母细胞胞质和核膜中呈弱阳性反应;孕激素受体在卵黄合成早期的滤泡细胞和卵母细胞质、核膜中表达均较弱,在卵黄合成后期表达均增强;雄激素受体在卵黄合成早期的滤泡细胞和卵母细胞胞质、核膜中表达也均较弱,在卵黄合成后期卵母细胞胞质和核膜中表达略有增强.     

Modulation of spectrin-actin assembly by erythrocyte adducin

The spectrin-based membrane skeleton, an assembly of proteins tightly associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Spectrin, the most abundant component of this assembly, is an elongated and flexible molecule that, with potentiation by protein 4.1, is cross-linked at its ends by short actin filaments to form a lattice beneath the membrane. These and other proteins stabilize the plasma membrane, organize integral membrane proteins and maintain specialized regions of the cell surface. A membrane-skeleton-associated calmodulin-binding protein of erythrocytes is a major substrate for Ca2+- and phospholipid-dependent protein kinase C (ref. 5), and thus is a target for Ca2+ by two regulatory pathways. Here we demonstrate that this protein, called adducin: (1) binds tightly in vitro to spectrin-actin complexes but with much less affinity either to spectrin or to actin alone; (2) promotes assembly of additional spectrin molecules onto actin filaments; and (3) is inhibited in its ability to induce the binding of additional spectrin molecules to actin by micromolar concentrations of calmodulin and Ca2+. Adducin may be involved in the action of Ca2+ on erythrocyte membrane skeleton and in the assembly of spectrin-actin complexes.