Nuclear Reassembly Research Articles

Diacylglycerols as putative second messengers that regulate phototransductive membrane turnover by arthropods

1. Arthropod phototransduction cascades release diacylglycerols (DAGs). We have suggested that DAGs may activate protein kinase Cs (PKCs) to induce phosphorylations of proteins that regulate nuclear transcriptions of rhabdomeral membrane precursors during circadian phototransductive membrane turnover. 2. When retinas of a crab, Leptograpsus, held in vitro after illumination at dawn were treated with an inhibitor of diacylglycerol lipases, U-57908, endomembranes proliferated from nuclear envelopes and differentiated to renew rhabdomeral microvilli, following the pathways described by Stowe (1980). 3. When retinas were treated with exogenous, 1,2-dioctanoyl-sn-glycerol alone rhabdomeres were rapidly stripped of microvilli. 4. A combination of 1,2-dioctanoyl-sn-glycerol and U-57908 induced an initial endocytosis of rhabdomeral membrane, followed by proliferation of endomembranes from nuclear envelopes and reassembly of rhabdoms. 5. Two hypothetical processes are implied: (i) PKCs activated peripherally by DAGs released by phototransduction are translocated to photoreceptor nuclei where they induce the phosphorylation of proteins that regulate transcriptions, (ii) Peripherally, activated PKCs may determine phosphorylation of the cytoplasmic domains of integral rhabdomeral membrane proteins to create signals that initiate endocytosis. The latter process, however, may not occur in vivo. 6. The model for the regulation of phototransductive membrane turnover rests upon assumptions about the intracellular compartmentalisation of arthropod photo-receptors. They have not been critically examined. Crab R1-7 nuclear envelopes seem to possess a high population of nuclear pores. It may be mandatory for the transfer of peripheral signals to intra-nuclear sites.

NuMA is required for the proper completion of mitosis.

NuMA is a 236-kD intranuclear protein that during mitosis is distributed into each daughter cell by association with the pericentrosomal domain of the spindle apparatus. The NuMA polypeptide consists of globular head and tail domains separated by a discontinuous 1500 amino acid coiled-coil spacer. Expression of human NuMA lacking its globular head domain results in cells that fail to undergo cytokinesis and assemble multiple small nuclei (micronuclei) in the subsequent interphase despite the appropriate localization of the truncated NuMA to both the nucleus and spindle poles. This dominant phenotype is morphologically identical to that of the tsBN2 cell line that carries a temperature-sensitive mutation in the chromatin-binding protein RCC1. At the restrictive temperature, these cells end mitosis without completing cytokinesis followed by micronucleation in the subsequent interphase. We demonstrate that the wild-type NuMA is degraded in the latest mitotic stages in these mutant cells and that NuMA is excluded from the micronuclei that assemble post-mitotically. Elevation of NuMA levels in these mutant cells by forcing the expression of wild-type NuMA is sufficient to restore post-mitotic assembly of a single normal-sized nucleus. Expression of human NuMA lacking its globular tail domain results in NuMA that fails both to target to interphase nuclei and to bind to the mitotic spindle. In the presence of this mutant, cells transit through mitosis normally, but assemble micronuclei in each daughter cell. The sum of these findings demonstrate that NuMA function is required during mitosis for the terminal phases of chromosome separation and/or nuclear reassembly.

Chapter 17 Systems for the Study of Nuclear Assembly, DNA Replication, and Nuclear Breakdown in Xenopus laevis Egg Extracts

Publisher Summary This chapter describes the conditions for preparing cell-free extracts from Xenopus eggs, which allows the study of nuclear dynamics and has proved fruitful in elucidating the structural and functional requirements for nuclear reassembly, DNA replication, and nuclear disassembly. In the amphibian Xenopus laevis , egg fertilization is followed by a period of rapid and synchronous cell division, which occurs in the complete absence of transcription or of any increase in total mass of the egg. During oogenesis, the egg must synthesize and store a large reservoir of nuclear components for use during the rapid proliferation that occurs during early development. The existence of such a reservoir of nuclear components permits the development of systems for the study of nuclear dynamics in vitro . Xenopus sperm provides an abundant source of chromatin, which after removal of plasma and nuclear membranes, may be easily purified. The chapter diagrammatically presents the preparation of the S phase extract and membrane and cytosol fractions for nuclear assembly. The inhibition of DNA replication by aphidicolin prevents the subsequent activation of maturation promoting factor (MPF) and entry into mitosis, indicating the presence of a control system that monitors the replication state of DNA and regulates the activation of MPF.

The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro.

Cell-free extracts prepared from Xenopus eggs support chromosome decondensation and pronuclear formation on demembranated sperm heads. 32P-dCTP pulse-labelling studies demonstrate that DNA synthesis occurs in multiple bursts of 30-40 min in extracts containing pronuclei, each burst being followed by a period of 20-50 min during which no synthesis occurs. Density substitution with bromodeoxyuridine indicates that the synthesis in each burst is semiconservative and results from new initiations, and that, following multiple bursts of synthesis, reinitiation events can occur. Changes in nuclear morphology have been characterized in the extract by phase-contrast microscopy and by fluorescence microscopy following pulse labelling with biotin-11-dUTP and staining with anti-lamin antibodies. Lamin accumulation occurs as DNA decondenses and parallels the acquisition of membrane structures. Biotin-11-dUTP incorporation is first observed in small nuclei having decondensed DNA and an extensive lamina. While DNA synthesis is occurring nuclei remain relatively small, but rapid swelling accompanied by chromosome condensation occurs when biotin incorporation ceases. Nuclear swelling and chromatin condensation is followed by nuclear membrane breakdown, lamin dispersal and chromosome formation. Mitosis lasts for approximately 20 min. Nuclear reassembly is recognized by the appearance of membrane vesicles around small pieces of decondensed DNA, which parallels the appearance of lamin islands within a chromatin mass. These 'islands' incorporate biotin, indicating that DNA synthesis is occurring, and apparently fuse as larger S-phase nuclei are formed. Extensive protein synthesis occurs for at least 4 h in most extracts. This synthesis is required for the initiation of mitotic events and the reinitiation of DNA synthesis.

Specific attachment of nuclear-mitotic apparatus protein to metaphase chromosomes and mitotic spindle poles: Possible function in nuclear reassembly

NuMA protein is the largest, abundant, primate-specific chromosomal protein. The protein was purified from HeLa cells and monospecific monoclonal antibodies were prepared that react exclusively with NuMA protein in immunoblot analysis. These antibodies were used to define the intracellular location and properties of NuMA protein. Using indirect immunofluorescence, NuMA protein was detected only in the nucleus of interphase cells and on the chromosomes in mitotic cells. One class of monoclonal antibody called the 2E4-type antibody, caused NuMA protein (or a complex of proteins including NuMA) to be released from its binding site on metaphase or anaphase chromosomes. The separation of NuMA protein from chromosomes was observed either with the immunofluorescence assay or in electrophoretic analyses of proteins released from isolated metaphase chromosomes after reaction with 2E4 antibody. The immunofluorescence studies also showed that after release of the NuMA protein from chromosomes of metaphase or anaphase cells, the protein bound specifically to the polar region of the mitotic spindle. It was shown that exogenously added NuMA antigen/antibody complex bound only to the mitotic spindle poles of permeabilized primate cells and not to the spindle poles of other mammalian cells, thus demonstrating the specificity of the spindle-pole interaction. The antibody mediated transfer of NuMA from chromosomes to poles was blocked when the chromosomes were treated with cross-linking fixatives. Results suggest that the NuMA protein has specific attachment sites on both metaphase chromosomes and mitotic spindle poles (the site where post-mitotic nuclear assembly occurs). A model is proposed suggesting that a protein having such dual binding sites could function during nuclear reassembly to link mitotic chromosomes into the reforming nucleus.

Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs.

We have found that injection of bacteriophage lambda DNA into unfertilized Xenopus eggs causes the assembly around the DNA of structures resembling typical eucaryotic cell nuclei. These spherical structures begin to form 60-90 min after injection. They contain lambda DNA and are bounded by a phase-dense envelope. Immunofluorescent staining of the lambda-DNA-containing structures with anti-lamin antibody reveals the presence of the lamin nuclear proteins at the periphery of the structure, a pattern identical to that of embryonic nuclei. Electron microscopy reveals that the injected DNA is surrounded by a double bilayer nuclear membrane containing nuclear pore complexes. The "nuclei" containing lambda DNA respond to modulators of the Xenopus cell cycle in a manner that mimics the response of embryonic nuclei to these modulators during mitosis. These results suggest that nuclear reassembly and breakdown occur independently of specific DNA sequence information.

Human-specific nuclear protein that associates with the polar region of the mitotic apparatus: Distribution in a human/hamster hybrid cell

We describe the first example of a predominantly nuclear protein which during mitosis becomes part of the mitotic apparatus. This protein has been termed the nuclear-mitotic apparatus (NuMA) protein. In interphase cells NuMA protein is restricted to the nucleus and is a constituent of isolated nuclear matrices, but in mitotic cells it is observed by indirect immunofluorescence microscopy to be concentrated at the polar regions of the mitotic apparatus. This mitotic localization is dependent on the integrity of the spindle, since treatments which disrupt the spindle result in dispersion of NuMA protein throughout the cell. Comparison to the subcellar distribution of tubulin at different stages of the cell cycle indicates that NuMA protein is distinct from the previously identified components of the mitotic spindle. Its association with the nuclear matrix and its localization during mitosis to the site of nuclear reassembly suggest the interesting possibility that NuMA protein could be representative of a class of proteins involved in the early events of nuclear reassembly. NuMA is present in the nuclei and mitotic spindle of all types of human cells that have been examined, but proteins of similar molecular weight (300,000 daltons in dissociating solvents) or immunological specificity are not detected in cells of other species (including monkey). However, the NuMA protein is synthesized in a human/Chinese hamster hybrid cell containing a reduced number of human chromosomes. Immunofluorescence studies of this hybrid cell showed that the distribution of NuMA protein is equivalent to that in human cells. These results suggest that the human gene coding for NuMA protein, unlike other genes coding for human specific nuclear proteins, can be expressed in human/hamster hybrid cells and that the cell hybrid will be useful in further characterization of NuMA protein.