A cell-free system for light-dependent nuclear import of phytochrome

Translocation from the cytosol to the nucleus is an essential step in phytochrome (phy) signal transduction. In the case of phytochrome A (phyA), this step occurs with the help of FHY1 (far-red-elongated hypocotyl 1), a specific transport protein. To investigate the components involved in phyA transport, we used a cell-free system that facilitates the controlled addition of transport factors. For this purpose, we isolated nuclei from the unicellular green algae Acetabularia acetabulum . These nuclei are up to 100 μm in diameter and allow easy detection of imported proteins. Experiments with isolated nuclei of Acetabularia showed that FHY1 is sufficient for phyA transport. The reconstituted system demonstrates all the characteristics of phytochrome transport in Arabidopsis thaliana . In addition, FHY1 was also actively exported from the nucleus, consistent with its role as a shuttle protein in plants. Therefore, we believe that isolated Acetabularia nuclei may be used as a general tool to study nuclear transport of plant proteins.     

Evaluation of mammalian cell-free systems of nuclear disassembly and assembly.

Mammalian cell-free systems are very useful for the biochemical and structural study of nuclear disassembly and assembly. Through experimental manipulations, the role of specific proteins in these processes can be studied. Recently, we intended to examine the involvement of integral and peripheral inner nuclear membrane proteins in nuclear disassembly and assembly. However, we could not achieve proper disassembly when isolated interphase HeLa nuclei were exposed to mitotic soluble extracts obtained from the same cell line and containing cyclin B1. Homogenates of synchronized mitotic HeLa cells left to reassemble their nuclei generated incomplete nuclear envelopes on chromatin masses. Digitonin-permeabilized mitotic cells also assembled incomplete nuclei, generating a lot of cytoplasmic inclusions of inner nuclear membrane proteins as an intermediate. These results were therefore used as a basis for a critical evaluation of mammalian cell-free systems. We present here evidence that cell synchronization itself can interfere with the progress of nuclear assembly, possibly by causing aberrant nuclear disassembly and/or by inducing the formation of an abnormal number of mitotic spindles.     

Lamin disassembly kinetics: A cell-free system with extracts from mitotic HeLa cells

We describe a cell-free system in which a postribosomal supernatant from metaphase HeLa cells induces prophase-like changes in permeabilized HeLa cell populations as evidenced by the nuclear lamin disassembly and chromatin condensation. We have attempted to characterize the cell-free system with permeabilized HeLa cells. First, by extracting lamins with agents known to disrupt the noncovalent interactions in the supramolecular lamin aggregate in interphase using polyclonal and a newly established monoclonal anti-lamin Ab 2E3, uniform extraction of lamins was achieved with urea and deoxycholate whereas the cation Mg2+ and 2-mercaptoethanol had little effect on the disassembly of interphase lamins. Second, cytoplasmic extract from mitotic HeLa cells, synchronized by a nitrous oxide metaphase arrest, was tested. It had a differential effect on interphase lamin depolymerization. Nuclei in G1 phase of the cell cycle were more resistant against the mitotic extracts than cells in S and G2 phase. The results are discussed in terms of a possible inactivation of mitotic extracts by factors present in nuclei in early interphase.     

Cotranslational disassembly of flock house virus in a cell-free system.

Intact, purified particles of the nodaviruses flock house virus and nodamura virus that were either transfected into cells that were resistant to infection or introduced into in vitro translation systems directed the synthesis of viral proteins. We infer that direct interaction of these nodavirus particles with cytoplasmic components mediated virion disassembly that resulted in release of the viral RNA.     

A role for DNA in the formation of nuclear estradiol-receptor complex in a cell-free system

Destruction of the DNA component of the nuclear pellet fraction of rat uterus homogenates with DNase prevented the formation of “nuclear” estradiol-receptor complex upon subsequent incubation with supernatant fraction containing 8S cytoplasmic estrogen-receptor complex. Nuclear pellet pretreatment with RNase had no effect on the reaction. DNase did not appear to release any component capable of binding estradiol or the 8S estradiol-receptor complex.     

An in vitro nuclear disassembly system reveals a role for the RanGTPase system and microtubule-dependent steps in nuclear envelope breakdown

During prophase, vertebrate cells disassemble their nuclear envelope (NE) in the process of NE breakdown (NEBD). We have established an in vitro assay that uses mitotic Xenopus laevis egg extracts and semipermeabilized somatic cells bearing a green fluorescent protein-tagged NE marker to study the molecular requirements underlying the dynamic changes of the NE during NEBD by live microscopy. We applied our in vitro system to analyze the role of the Ran guanosine triphosphatase (GTPase) system in NEBD. Our study shows that high levels of RanGTP affect the dynamics of late steps of NEBD in vitro. Also, inhibition of RanGTP production by RanT24N blocks the dynamic rupture of nuclei, suggesting that the local generation of RanGTP around chromatin may serve as a spatial cue in NEBD. Furthermore, the microtubule-depolymerizing drug nocodazole interferes with late steps of nuclear disassembly in vitro. High resolution live cell imaging reveals that microtubules are involved in the completion of NEBD in vivo by facilitating the efficient removal of membranes from chromatin.     

Unusual nuclear structures in meiotic prophase of fission yeast: a cytological analysis

Earlier results from sectioned nuclei indicating that Schizosaccharomyces pombe does not develop a classical tripartite synaptonemal complex (SC) during meiotic prophase are confirmed by spreading of whole nuclei. The linear elements appearing during prophase I resemble the axial cores (SC precursors) of other organisms. The number of linear elements in haploid, diploid, and tetraploid strains is always higher than the chromosome number, implying that they are not formed continuously along the chromosomes. Time course experiments reveal that the elements appear after DNA replication and form networks and bundles. Later they separate and approximately 24 individual elements with a total length of 34 microns are observed before degradation and meiotic divisions. Parallel staining of DNA reveals changes in nuclear shape during meiotic prophase. Strains with a mei4 mutation are blocked at a late prophase stage. In serial sections we additionally observed a constant arrangement of the spindle pole body, the nucleolus, and the presumptive centromere cluster. Thus, S. pombe manages to recombine and segregate its chromosomes without SC. This might correlate with the absence of crossover interference. We propose a mechanism for chromosome pairing with initial recognition of the homologs at the centromeres and suggest functions of the linear elements in preparation of the chromosomes for meiosis I disjunction. With the spreading technique combined genetic, molecular, and cytological approaches become feasible in S. pombe. This provides an opportunity to study essential meiotic functions in the absence of SCs which may help to clarify the significance of the SC and its components for meiotic chromosome structure and function.     

A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters

We describe here a novel proteoliposome reconstitution system for functional analysis of plant membrane transporters that is based on a modified wheat germ cell-free translation system. We established optimized conditions for the reconstitution system with Arabidopsis thaliana phosphoenolpyruvate/phosphate translocator 1 (AtPPT1) as a model transporter. A high activity of AtPPT1 was achieved by synthesis of the protein in the presence of both a detergent such as Brij35 and liposomes. We also determined the substrate specificities of three putative rice PPT homologs with this system. The cell-free proteoliposome reconstitution system provides a valuable tool for functional analysis of transporter proteins.     

Disassembly of the nuclear envelope of spisula oocytes in a cell-free system

Nuclei isolated from oocytes of the surf clam Spisula solidissima are disassembled when exposed to extracts from maturing oocytes. In the course of this process the nuclear lamina undergoes a marked reduction in size and the nuclear membrane appears to be fragmented into vesicles. These events are accompanied by extensive phosphorylation of the oocyte 67-kDa lamin and its solubilization. The changes observed are similar to those which occur in vivo in activated Spisula oocytes. Nuclear envelope breakdown in vitro requires ATP and Mg2+, but not Ca2+. It is not affected by protease inhibitors and is inhibited by alkaline phosphatase.     

A Highlights from MBoC Selection: akirin is required for diakinesis bivalent structure and synaptonemal complex disassembly at meiotic prophase I

During meiosis, evolutionarily conserved mechanisms regulate chromosome remodeling, leading to the formation of a tight bivalent structure. This bivalent, a linked pair of homologous chromosomes, is essential for proper chromosome segregation in meiosis. The formation of a tight bivalent involves chromosome condensation and restructuring around the crossover. The synaptonemal complex (SC), which mediates homologous chromosome association before crossover formation, disassembles concurrently with increased condensation during bivalent remodeling. Both chromosome condensation and SC disassembly are likely critical steps in acquiring functional bivalent structure. The mechanisms controlling SC disassembly, however, remain unclear. Here we identify akir-1 as a gene involved in key events of meiotic prophase I in Caenorhabditis elegans. AKIR-1 is a protein conserved among metazoans that lacks any previously known function in meiosis. We show that akir-1 mutants exhibit severe meiotic defects in late prophase I, including improper disassembly of the SC and aberrant chromosome condensation, independently of the condensin complexes. These late-prophase defects then lead to aberrant reconfiguring of the bivalent. The meiotic divisions are delayed in akir-1 mutants and are accompanied by lagging chromosomes. Our analysis therefore provides evidence for an important role of proper SC disassembly in configuring a functional bivalent structure.     

Improved methods for purification and assay of eukaryotic messenger ribonucleic acids and ribosomes. Quantitative analysis of their interaction in a fractionated reticulocyte cell-free system.

The polyadenylic acid-containing messenger ribonucleic acids of eukaryotic cells are rapidly isolated and deproteinized in a simple and gentle one-step procedure. The polyribosome fraction, dissolved in 0.5 M NaCl/0.5 percent sodium dodecyl sulfate, is passed through an oligo(dT)-cellulose column which is then washed with the solvent until proteins and contaminating ribonucleic acids are fully removed. Deproteinized messenger ribonucleic acid is then eluted by lowering the ionic strength. This method gives highly purified and active messenger ribonucleic acids from all tissues tested. The yield is approximately 1.5 to 2 percent of the polyribosomal ribonucleic acid. Messenger ribonucleic acids are assayed in a rabbit reticulocyte-derived, messenger-dependent, cell-free protein-synthesizing system modified from Crystal et al. (Crystal, R. G., Nienhuis, A. W., Elson, N. A., and Anderson, W.F. (1972) J. Biol. Chem. 247, 5357-5368). This system synthesizes proteins at an almost linear rate for at least 2 hours. During this period, each globin messenger ribonucleic acid directs the synthesis of several globin molecules. Each active ribosome synthesizes a globin molecule every 6 to 7 min, but only a small fraction of the ribosomes or messengers are active at any instant. Translation occurs mainly on di- and monoribosomes although larger sized polysomes also occur. Several lines of evidence suggest that globin messenger ribonucleic acid requires "activation" before it can be utilized and that a messenger activation step of protein synthesis initiation is rate-limiting in this cell-free system.     

A novel cell-free protein synthesis system

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30–45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.     

The nuclear pores of early meiotic prophase nuclei of plants

Summary Pollen mother cells at early meiotic prophase fromFritillaria lanceolata, F. mutica, Tulbaghia violacea, the lily “Formobel”,Triticum aegilopoides, T. dicoccoides, T. aestivum and synaptic and asynaptic forms ofT. durum were studied in thin sections with the electron microscope (a) in relation to distribution of nuclear pores (b) in respect of fine structure of the pore complex in those of the first four. The pores were distributed in random clusters during leptotene to pachytene in all plants, except in the two forms ofT. durum where there were either no pores or so few that they were not detectable. Probably correlated with this, the two membranes of the nuclear envelope were often widely separated and frequently sacculated. No pores were seen at leptotene in the part of the envelope to which, in theFritillarias and lily, the nucleolus was adpressed at this time. Evidence supporting a recent model which proposes that annuli are composed of three rings of eight granular subunits was obtained. These subunits as well as a dense central element, observed in most pores, were composed of filaments about 3 nm in diameter and evidently protein in character. There was evidence of a continuity between filaments in the central element and those in the rings of subunits which encircle the pore aperture at both the nuclear and cytoplasmic sides of the pore. In profiles of pores knobbed filaments were sometimes seen extending laterally from the pore wall into the perinuclear space at two sides. Questions concerning the role of the annulus are discussed. The author wish to thank Mr. R. F. Scott for construction to the model.     

A cell-free replication system for human polyomavirus JC DNA.

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase alpha-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origin-containing plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.