LAUDERIA ANNULATA—A MARINE CENTRIC DIATOM WITH AN ELONGATE BILOBED NUCLEUS1

Lauderia annulata Cleve is probably unique among marine centric diatoms in possessing an elongate dumbbell-shaped nucleus. A lobe at, each end of the nucleus lies adjacent to each valve during interphase and each lobe resembles a typical eucaryotic nucleus. The central portion of the thin strand, which passes through the vacuole connecting the two lobes is Feulgen positive and is nuclear membrane bounded. A group of micro tubules occurs in this strand clustered excentrically between, the nuclear membrane and the tonoplast. Evidence for the coordinated functioning of both nuclear lobes is suggested, by the aggregation of chloroplasts around, both lobes when shade-adapted cells are exposed, to intense white or blue (400–500 nm) light.     

Effect of near ultraviolet and visible light on amoeba.

The near ultraviolet and visible light (VL) impinging at an intensity of 2-5 x 10(2) J s-1 m-2 for 2-5 h kills the mitotic and the early S-phase (0- to 15-min-old) amoebae. At the mid- and late S-period only a fraction of cells are killed by VL and G2 phase cells are quite resistant. Amoebae of all cell cycle stages show a delay in the first mitotic division. DNA synthesis, as measured by [3H]thymidine incorporation, is depressed in the VL-exposed early-S amoebae. A concurrent but temporary inhibition in [3H]leucine incorporation also occurs in these cells. However, no significant change in [3H]uridine incorporation has been found. To localize the site of lethal damage, nuclear transplantation studies were undertaken between the control amoebae and the amoebae treated with VL. The nucleus of a VL-exposed early S-phase cell recovers when transplanted immediately after VL exposure into an enucleate G2 cytoplasm but dies if grafted into an enucleat S-phase cytoplasm. The therapeutic effect of the G2 cytoplasm, although at a lower level, is also evident even when the treated early S-phase nucleus is implanted 20 h later, but not after 48 h, into the G2 cytoplasm. The amoeba cytoplasm shows resistance to VL-irradiation, can accept a control nucleus from any cell cycle stage, and function normally. The G2 nucleus also remains apparently unaffected to VL exposure and can survive when it is transfered to the control cytoplasm of any cell-cycle phase. All these findings are discussed in the light of the possible existence of a repair system against VL-induced damage in the G2-phase amoeba.     

Viruses of Entamoeba histolytica II. Morphogenesis of the Polyhedral Particle (ABRM2→HK-9)→HB-301 and the Filamentous Agent (ABRM)2→HK-9

The intracellular development of two morphologically different amoebal viruses has been studied by electron microscopy. One is a polyhedral agent which was observed as early as 24 hr after infection in the perinuclear cytoplasm. Subsequently, cell lysis occurred and particles were found in large number bound to membranes of disrupted amoebae. Other particles were found in phagocytic vacuoles suggesting a possible portal of entry into amoebae. The other virus is a filamentous particle which is first seen in small clusters in the nucleus after 24 hr of infection. The number of particles increases such that by 72 hr massive whorls of particles occupy a substantial part of the nucleus. After rupture of the nuclear membrane, clusters of filaments are widely dispersed throughout the cytoplasm. Still later, the cytoplasmic membrane disintegrates and clusters of filaments are found extracellularly, but free of cell membranes. The morphology of these agents is discussed in comparison with a variety of plant, animal, and bacterial viruses.     

Translocation of cAMP-dependent protein kinase to the nucleus during development of Dictyostelium discoideum

The distribution of the catalytic and regulatory subunits of the cAMP-dependent protein kinase between cytoplasm and nucleus was determined during the development of Dictyostelium discoideum. In vegetative amoebae approximately 2% of the subunits were in the nucleus. During development there was an approximately 5-fold increase in total soluble cAMP-dependent protein kinase and a 15- to 30-fold increase of enzyme in the nuclear fraction. There was a reverse translocation from nucleus to cytoplasm, when Tipped Aggregates were disrupted and the resultant amoebae incubated in single-cell suspension. The addition of cAMP to these single-cell suspensions brought about the reentry of the subunits into the nucleus. The findings are discussed in relation to the potential role of the cAMP-dependent protein kinase in the regulation of mRNA and protein synthesis.     

A study of the change in DNA synthesis of S phase cells treated with N-methyl-N-nitrosourethane: a study using Amoeba proteus as a single cell model.

The present experiments using Amoeba proteus as a single cell model show that DNA synthesis continues during and after exposure of S phase cell to N-methyl-N'-nitrosourethane (MNU). At sublethal dose levels which caused long division delays, division and growth abnormalities and mutations, the amount of [3h] thymidine ([3h]Tdr) incorporated was decreased by 20-30%; at dose levels which killed all S phase cells it was inhibited by up to 90%. There was a direct correlation between the dose of MNU used and the degree of inhibition of [3H]Tdr incorporated. The effect was rapid, mainly taking place within 20 min of treatment. Amoeba heterokaryons (HKs) were used to examine the rate of DNA synthesis of treated and untreated nuclei in the same cytoplasm, i.e. where the nuclei would have the same [h]tdr intake, the same thymidine kinase (TK) activity and the same endogenous precursor pools. Direct comparison of the nuclear DNA synthetic activity in this way revealed less difference between treated and untreated nuclei than comparisons made using the nuclear grain counts from treated and untreated amoebae. This suggested that much of the decrease in [3H]Tdr incorporation by MNU-treated S phase cells was due to a change in the cytoplasm and/or the cell membrane, rather than to nuclear damage. Thus MNU-treated nuclei were able to synthesize DNA at a near normal rate when they could draw on the resources of untreated cytoplasm, while the rate of DNA synthesis of control nuclei decreased when they occupied cytoplasm which had been exposed to high doses of MNU. These studies suggest that nuclear sites of damage were only involved when lethal doses of MNU had been used.     

Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space

Apoptosis-inducing factor (AIF) is a mitochondrial intermembrane flavoprotein that is translocated to the nucleus in response to proapoptotic stimuli, where it induces nuclear apoptosis. Here we show that AIF is synthesized as an approximately 67-kDa preprotein with an N-terminal extension and imported into mitochondria, where it is processed to the approximately 62-kDa mature form. Topology analysis revealed that mature AIF is a type-I inner membrane protein with the N-terminus exposed to the matrix and the C-terminal portion to the intermembrane space. Upon induction of apoptosis, processing of mature AIF to an approximately 57-kDa form occurred caspase-independently in the intermembrane space, releasing the processed form into the cytoplasm. Bcl-2 or Bcl-XL inhibited both these events. These findings indicate that AIF release from mitochondria occurs by a two-step process: detachment from the inner membrane by apoptosis-induced processing in the intermembrane space and translocation into the cytoplasm. The results also suggest the presence of a unique protease that is regulated by proapoptotic stimuli in caspase-independent cell death.     

Identification of a Golgi-associated protein that undergoes mitosis dependent phosphorylation and relocation

By means of a monoclonal antibody (BH3), we have identified a 57-kD protein (p57) that in interphase is restricted largely to the perinuclear region of the cell. Double label immunofluorescence microscopy suggests localization of p57 to the Golgi complex and associated membranous structures. Protease protection experiments and chemical extractability indicate that p57 is a peripheral membrane protein exposed to the cytoplasm. p57 displays unique behavior during mitosis. At the end of G2 or in early prophase, p57 leaves the perinuclear region and accumulates very rapidly within the nucleus, at a time when the nuclear envelope is still intact and before nuclear lamina disassembly. This relocation of p57 coincides with its hyperphosphorylation on serine and threonine residues. After nuclear envelope breakdown p57 becomes uniformly distributed throughout the mitotic cytoplasm until in late telophase when it returns to its perinuclear location and is once again excluded from the nucleus. The behavior of p57 during mitosis suggests that it may play a role in the cellular reorganization evident during mitotic prophase.     

Monitoring the disruption of nuclear envelopes in interphase cells with GFP-beta-galactosidase.

The nuclear envelope of eukaryotic cells provides a barrier separating nucleus from cytoplasm, thereby regulating the exchange of macromolecules between both compartments. However, in cells exposed to severe forms of stress this barrier may break down, resulting in the mixing of nuclear and cytoplasmic contents. We show here that the fusion protein GFP-beta-galactosidase can be used to evaluate the intactness of nuclear envelopes in HeLa cells that have been exposed to heat and oxidative stress. GFP-beta-galactosidase is restricted to the cytoplasm of interphase cells, but enters the nucleus when nuclear membranes are disrupted. For comparison, we have analyzed the barrier function of nuclear membranes with antibodies against lamin B. Treatment of fixed cells with digitonin permeabilizes the plasma membrane, but leaves nuclear envelopes intact. Consequently, after digitonin incubation antibodies to lamin B can bind their antigen only if nuclear membranes are damaged. For various heat and oxidative stress conditions, we have compared the distribution of GFP-beta-galactosidase with the accessibility of lamin B to antibodies. Our results demonstrate that nuclear envelopes are permeable to antibodies whenever GFP-beta-galactosidase enters the nucleus. GFP-beta-galactosidase is therefore a useful tool for evaluating the disintegration of the nuclear envelope and identifying cells in which a mixing of nuclear and cytoplasmic material takes place.     

Intranuclear Virus-like Bodies in the Amoeboflagellate Naegleria gruberi*

SYNOPSIS. Intranuclear virus-like bodies were seen in cultures of the EG strain of Naegleria gruberi from soil. Introduction of these virus-like particles into cultures seemed to coincide with use of chicken embryo extract as a supplement in culture media used to maintain axenic amoebae in the laboratory; the appearance of the virus-like units is triggered by transfer of axenic lines of N. gruberi EG into monobacterial culture medium. The particles, ～ 100 nm in diameter, are mainly restricted to the nucleus of the cell. Passage of particles from the nucleoplasm into the cytoplasm is suggested by theit association with tubular projections from the nuclear membrane, and particles have been seen in the cytoplasm of the amoebae. The virus-like bodies resemble reovirus.     

Molecular evidence for the nuclear localization of FADD

The Fas-associated death domain (FADD) adaptor protein FADD/Mort-1 is recruited by several members of the tumor necrosis factor receptor (TNFR) superfamily during cell death activated via death receptors. Since most studies have focused on the interaction of FADD with plasma membrane proteins, FADD's subcellular location is thought to be confined to the cytoplasm. In this report, we show for the first time that FADD is present in both the cytoplasm and the nucleus of cells, and that its nuclear localization relies on strong nuclear localization and nuclear export signals (NLS and NES, respectively) that reside in the death-effector domain (DED) of the protein. Specifically, we found that a conserved basic KRK35 sequence of the human protein is necessary for FADD's nuclear localization, since disruption of this motif leads to the confinement of FADD in the cytoplasm. Furthermore, we show that the leucine-rich motif LTELKFLCL28 in the DED is necessary for FADD's nuclear export. Functionally, mutation of the NES of FADD and its seclusion in the nucleus reduces the cell death-inducing efficacy of FADD reconstituted in FADD-deficient T cells.     

Ultrastructure of the Giant Amoeba Pelomyxa palustris*

SYNOPSIS. The ultrastructure of the herbivorous amoeba Pelomyxapalustris was studied. Nuclear division is not understood in this amoeba, and evidence for the method of nuclear division was sought. This species typically has many spheroidal nuclei which are similar within a given cell. However, some amoebae from our collections differed from this common type in both the number and structure of their nuclei. This suggested stages associated with nuclear division. One current hypothesis of nuclear division in this organism is that of nuclear budding. Our evidence is more in accord with this method than with mitosis. The cytoplasm contained no mitochondria, Golgi bodies, contractile vacuoles or crystals. Most amoebae had 2 types of bacteria (bacteroids or endosymbionts) in their cytoplasm; a separate vesicle enclosed each of these. Characteristically, only 1 type of bacterium (B_n) surrounded the nucleus. Another type (B) was found elsewhere in the cytoplasm. Also in the cytoplasm were the following: food vacuoles enclosing various algae, relatively clear vacuoles and vesicles, glycogen, various electron-opaque particles, and occasional microtubules. The plasmalemma was smooth, lacking the external fringe which characterizes other large fresh-water amoebae.     

Live Cell Imaging of ERK and MEK: simple binding equilibrium explains the regulated nucleocytoplasmic distribution of ERK

In response to epidermal growth factor (EGF), the mitogen-activated protein kinase ERK2 translocates into the nucleus. To probe the mechanisms regulating the subcellular localization of ERK2, we used live cell imaging to examine the interaction between MEK1 and ERK2. Fluorescence resonance energy transfer (FRET) studies show that MEK1 and ERK2 directly interact and demonstrate that this interaction in the cytoplasm is largely responsible for cytoplasmic retention of ERK2. Stimulation with EGF caused loss of FRET as ERK separated from MEK and moved into the nucleus. FRET was recovered as ERK returned to the cytosol, indicating ERK reassociation with MEK in the cytoplasm. The EGF-induced transit of ERK through the nucleus was complete within 20 min, and there was no significant movement of MEK into the nucleus. Fluorescence recovery after photobleaching experiments was used to assess the rate of movement of MEK and ERK. The steady-state rate of ERK entry into the nucleus in resting cells was energy-independent and greater than the rate of ERK entry upon EGF stimulation. This suggests that the rate constant for ERK transport across the nuclear membrane is not limiting nuclear entry. Thus, we suggest that the movement of ERK into and out of the nucleus in response to agonist occurs primarily by diffusion and is controlled by interactions with binding partners in the cytosol and nucleus. No evidence of ERK dimerization was detected by FRET methods; the kinetics for nucleocytoplasmic transport were unaffected by mutations in the ERK putative dimerization domain.     

A relationship between nuclear DNA and RNA synthetic activities and the changes produced by n-methyl-n-nitroso urethane: A study using Amoeba proteus as a single cell model

Changes caused by a carcinogen generally vary from one cell to another even among similar types of cells. The following work investigates the degree to which damage (inhibition of division, lethality, or inherited cellular changes) caused by N-methyl-N-nitroso urethane (MNU) alters at different times during the cell cycle, and relates fluctuations in the sensitivity of cells to changes in their DNA and RNA synthetic activities—possibly in the configuration of their DNA—at the time of treatment.Studies on amoebae exposed to MNU for short periods at 50 different times in their cell cycle led to the following conclusions: amoebae are sensitive to MNU at all ages, but the dose needed to produce lethal damage to young and old cells varies by a factor of 3. Cells are most sensitive at the time of division and during the peak of DNA synthesis. Smaller changes are found during the G₂ phase, some of which occur at times of intensive RNA synthesis. Transfer of nuclei between treated and control cells proved that the changing sensitivity of the cells, as shown by both inherited changes and lethal damage, was dependent on changes in their nuclei. Though the cytoplasm could be affected directly by MNU, i.e. in the absence of a nucleus, supralethal doses 2–6 times whole cell dose were required to either kill the cell or to cause a recognizable change in the offspring of viable cells. Experiments with cells having altered nuclear/cytoplasmic ratios showed that the relative insensitivity of older cells was not due to the increased volume of their cytoplasm. However, a possible involvement of cytoplasm in the repair of nuclear damage is suggested by the ability of control cytoplasm to alleviate some nuclear damage, particularly in S phase cells.     

Proteins in nucleocytoplasmic interactions. I. The fundamental characteristics of the rapidly migrating proteins and the slow turnover proteins of the Amoeba proteus nucleus

By the transplantation of amino acid-³H-labeled nuclei between cells and the subsequent isolation of nuclei for quantitative assay, we have confirmed that all the nuclear proteins of Amoeba proteus are divisible into two classes that are sharply defined by their physiological behavior. About 40% of the proteins in the nucleus rapidly migrates back and forth between the nucleus and the cytoplasm. These rapidly migrating proteins (RMP) are 25–50 times more concentrated in the nucleus than in the cytoplasm, and migration into the nucleus therefore occurs against a high concentration differential. The remaining 60% of nuclear proteins has been classified as slow turnover proteins (STP) since (as reported in a following paper) virtually all of them ultimately undergo turnover. Turnover in this context means loss of label from the nucleus, by either protein breakdown or protein migration to the cytoplasm. Isolation of nuclei in the detergent Triton X-100 results in a 20% loss of nuclear proteins but conclusions about RMP and STP were not found to be significantly affected by this loss.     

Dynamics of proteasome distribution in living cells.

Proteasomes are proteolytic complexes involved in non-lysosomal degradation which are localized in both the cytoplasm and the nucleus. The dynamics of proteasomes in living cells is unclear, as is their targeting to proteins destined for degradation. To investigate the intracellular distribution and mobility of proteasomes in vivo, we generated a fusion protein of the proteasome subunit LMP2 and the green fluorescent protein (GFP). The LMP2-GFP chimera was quantitatively incorporated into catalytically active proteasomes. The GFP-tagged proteasomes were located within both the cytoplasm and the nucleus. Within these two compartments, proteasomes diffused rapidly, and bleaching experiments demonstrated that proteasomes were transported slowly and unidirectionally from the cytoplasm into the nucleus. During mitosis, when the nuclear envelope has disintegrated, proteasomes diffused rapidly throughout the dividing cell without encountering a selective barrier. Immediately after cell division, the restored nuclear envelope formed a new barrier for the diffusing proteasomes. Thus, proteasomes can be transported unidirectionally over the nuclear membrane, but can also enter the nucleus upon reassembly during cell division. Since proteasomes diffuse rapidly in the cytoplasm and nucleus, they may perform quality control by continuous collision with intracellular proteins, and degrading those proteins that are properly tagged or misfolded.     

Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import

Because influenza virus replicates in the nucleus and buds from the plasma membrane, its ribonucleoproteins (RNPs) must undergo bidirectional transport across the nuclear membrane. Export from the nucleus to the cytoplasm was found to depend on the viral matrix protein (M1). M1 associated with newly assembled viral RNPs (vRNPs) in the nucleus and escorted them to the cytoplasm through the nuclear pores. In contrast, during entry of the virus into a new host cell, M1 protein dissociated from the RNPs, allowing them to enter the nucleus. Amantadine, an antiviral agent that induces an early block in influenza A infection, was found to block the dissociation event and thereby to prevent import of incoming RNPs into the nucleus. Together, these results showed that M1 modulates the directionality of vRNP transport into and out of the nucleus.     

Ultrastructure of the ovary of Dermatobia hominis (Diptera: Cuterebridae). I. Development during the 3rd larval instar

The ultrastructure and distribution of gonial and somatic cells in the ovary of Dermatobia hominis was studied during the 3rd larval instar. In larvae weighing between 400 and 500 mg, the ovary is partially divided into basal and apical regions by oblong somatic cells that penetrate from the periphery; these cells show ovoid nucleus and cytoplasm full of microtubules. In both regions, gonial cells with regular outlines, large nucleus and low electron-density cytoplasm are scattered among the interstitial somatic cells. These later cells have small nucleus and electrondense cytoplasm. Clear somatic cells with small nucleus and cytoplasm of very low electron-density are restrict to the apical region of the gonad. Degenerating interstitial somatic cells are seen in the basal portion close to the ovary peduncle. During all this larval period the morphological features of the ovary remain almost the same. At the end of the period there is a gradual deposition of glycogen in the cytoplasm of the somatic cells, increase in the number and density of their mitochondria plus nuclear modification as membrane wrinkling and chromatin condensation in masses.     

20 S proteasomes are imported as precursor complexes into the nucleus of yeast

The mechanism by which yeast 20 S proteasomes are imported into the nucleus is still unresolved. Here, we provide the first evidence that 20 S proteasomes are imported as precursor complexes into the nucleus. By using the srp1-49 mutant which is deficient in nuclear import of cargos with classical nuclear localization sequences (cNLS), we show that proteasome precursor complexes associate with importin/karyopherin alphabeta, the cNLS receptor, and that they accumulate inside the cytoplasm. Reconstitution assays revealed that only precursor complexes are targeted to the nuclear envelope (NE) by karyopherin alphabeta. In support, the green fluorescent protein (GFP)-labelled maturation factor Ump1, marking precursor complexes, mainly localizes to the nucleus and around the NE. Our data suggest that nuclear 20 S proteasomes are finally matured inside the nucleus.     

Electron microscopic studies of giant nucleus-like structure formed by lambda DNA introduced into the cytoplasm of Xenopus laevis fertilized eggs and embryos

When bacteriophage lambda DNA was injected into the cytoplasm of the fertilized egg of Xenopus laevis, giant nucleus-like structures were assembled around the injected DNA. These nucleus-like structures survived during cleavage and were partitioned into blastomeres at the blastula stage. The nucleus-like structures formed in the uncleaved fertilized eggs and the blastula cells were both surrounded by a bilayer nuclear membrane with nuclear pore complexes. The ultrastructural features of the lambda DNA-induced nucleus-like structure were considerably different from those of the normal blastula nucleus: although the nuclear pore complexes appeared to be normal, the 'nucleoplasm' was much too homogeneous as compared with that of the normal nucleus.