Live-cell System Research Articles

Murine pituitary tumor-transforming gene functions as a securin protein in insulin-secreting cells

Human pituitary tumor-transforming gene 1 (PTTG1) encodes a securin protein critically important in regulating chromosome separation. Murine PTTG (mPTTG) is 66% homologous to human PTTG1 and PTTG-null (PTTG-/-) mice exhibit pancreatic beta-cell hypoplasia and abnormal nuclear morphology with resultant diabetes. As we show that ductal beta-cell neogenesis is intact in PTTG-/- mice, we explored mechanism for defective beta-cell replication. We tested whether mPTTG exhibits securin properties in mouse insulin-secreting insulinoma MIN6 cells, using a live-cell system to monitor mitosis in cells transfected with an enhanced green fluorescent protein (EGFP)-tagged mPTTG conjugate (mPTTG-EGFP). To fulfill the criteria for securin properties, the protein should undergo degradation immediately before the metaphase-to-anaphase transition when expression levels are low, and should inhibit metaphase-to-anaphase transition when expression levels are high. EGFP itself did not undergo degradation throughout mitosis and high levels of EGFP per se did not affect normal mitosis progression (n=25). However, mPTTG-EGFP was degraded 2 min before the metaphase-to-anaphase transition when expression levels were low (n=19), and high mPTTG-EGFP levels blocked metaphase-to-anaphase transition in 13 cells. mPTTG-EGFP inhibited MIN6 cell proliferation and caused apoptosis. Immunocoprecipitation demonstrated binding of mPTTG-EGFP and separase. These results show that mPTTG exhibits properties consistent with a murine securin in insulin-secreting mouse cells and mPTTG overexpression inhibits cell proliferation, suggesting that defective beta-cell proliferation observed in PTTG-/- mice is likely due to abnormal cell-cycle progression.

γ-Cleavage Is Dependent on ζ-Cleavage during the Proteolytic Processing of Amyloid Precursor Protein within Its Transmembrane Domain

beta-Amyloid precursor protein apparently undergoes at least three major cleavages, gamma-, epsilon-, and the newly identified zeta-cleavage, within its transmembrane domain to produce secreted beta-amyloid protein (Abeta). However, the roles of epsilon- and zeta-cleavages in the formation of secreted Abeta and the relationship among these three cleavages, namely epsilon-, zeta-, and gamma-cleavages, remain elusive. We investigated these issues by attempting to determine the formation and turnover of the intermediate products generated by these cleavages, in the presence or absence of known gamma-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that Abeta(46) is an intermediate precursor of secreted Abeta. Our co-immunoprecipitation data also reveal that, as an intermediate, Abeta(46) is tightly associated with presenilin in intact cells. Furthermore, we identified a long Abeta species that is most likely the long sought after intermediate product, Abeta(49), generated by epsilon-cleavage, and this Abeta(49) is further processed by zeta- and gamma-cleavages to generate Abeta(46) and ultimately the secreted Abeta(40/42). More interestingly, our data demonstrate that gamma-cleavage not only occurs last but also depends on zeta-cleavage occurring prior to it, indicating that zeta-cleavage is crucial for the formation of secreted Abeta. Thus, we conclude that the C terminus of secreted Abeta is most likely generated by a series of sequential cleavages, namely first epsilon-cleavage which is then followed by zeta- and gamma-cleavages, and that Abeta(46) produced by zeta-cleavage is the precursor of secreted Abeta(40/42).

Ubiquitination of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Permeabilized Cells Mediated by Cytosolic E1 and a Putative Membrane-bound Ubiquitin Ligase

The endoplasmic reticulum (ER) enzyme, 3-hydroxy-3-methylglutaryl-CoA reductase, catalyzes the production of mevalonate, a rate-controlling step in cholesterol biosynthesis. Excess sterols promote ubiquitination and subsequent degradation of reductase as part of a negative feedback regulatory mechanism. To characterize the process in more detail, we here report the development of a permeabilized cell system that supports reductase ubiquitination stimulated by the addition of sterols in vitro. Sterol-dependent ubiquitination of reductase in permeabilized cells is dependent upon exogenous cytosol, ATP, and either Insig-1 or Insig-2, two membrane-bound ER proteins shown previously to mediate sterol regulation of reductase degradation in intact cells. Oxysterols, but not cholesterol, promote reductase ubiquitination under our conditions. Finally, we show that ubiquitin-activating enzyme (E1) can efficiently replace cytosol to ubiquitinate reductase in response to sterol treatment, suggesting that other molecules required for ubiquitination of reductase, such as the ubiquitin-conjugating and -ligating enzymes (E2 and E3), are localized to ER membranes.

Effects of extracellular sodium on mu-opioid receptors coupled to potassium channels coexpressed in Xenopus oocytes.

Wild-type or mutant H297N or H297Q of the mu-opioid receptor were co-expressed with the inwardly rectifying potassium channel GIRK1 in oocytes from Xenopus laevis. Under voltage clamp, pairs of concentration response curves were generated using the agonist normorphine in a bathing medium containing 38.5 mM sodium or an identical medium in which the sodium was replaced by an equimolar concentration of choline. The maximum currents were greater in the presence of sodium by about 30% at wild-type receptors and by about 100% at the mutant receptors. The EC(50) values tended to increase somewhat as well, though these differences reached statistical significance only for the mutant H297Q. Flame photometry detected no change in the intracellular sodium or potassium concentrations of oocytes, suggesting that the effect of sodium was solely extracellular. Thus sodium, long known for its effects on in vitro ligand binding at mu-opioid receptors, also affects overall transduction as revealed in the Xenopus oocyte model of a complete, living cell system.

Transcription in tight quarters

Muller et al. (page 33) have developed a live-cell system that allows analysis of chromatin condensation during transcription from a natural promoter. Using a tandem array of mouse mammary tumor virus (MMTV) promoters driving ras reporter genes, the authors demonstrate that normal transcription can occur in chromatin that is much more condensed than the transcribed chromatin of previous model systems such as lampbrush chromosomes or polytene puffs. Furthermore, instead of the looping out of DNA observed in these systems, the authors observe a linear unraveling that produces domains of variable packing density. Figure Length of transcription arrays increases over time (in minutes) after drug addition. In response to steroid hormone treatment, the MMTV promoter is first activated by the glucocorticoid receptor, and then down-regulated over a six-hour period. Expression of GFP-tagged glucocorticoid receptor allows visualization of the MMTV array in live cells. Muller et al. found that chromatin decondensation and recondensenation paralleled the transcriptional activity of the promoter, and that both the establishment and maintenance of the decondensed state required transcription by DNA polymerase II activity. Transcription in this system can occur at high chromatin packing densities, a phenomenon previously seen only with artificial promoters that have high densities of transcription-factor binding sites. It remains unclear how the transcriptional machinery interacts with the densely packed chromatin, but these questions should be easier to address now that a live-cell system is available. ▪

Identification of genotoxic stress in human cells by fluorescent monitoring of p53 expression

The tumor suppressor protein p53 is induced upon DNA damage essentially by post-translational regulatory mechanisms, which lead to a substantial increase of p53 levels. To exploit this essential property of p53, we developed a novel reporter system for monitoring accumulation and subcellular translocation of p53 protein, which is able to function as a simple test for detecting mutagenic and genotoxic stress in human cells. For this purpose, we constructed a plasmid with a specific translational TP53::EGFP gene fusion and selected stable transfected clones in the human cell line HEK293, in which p53 is functionally stabilized due to the expression of the transgenic adenoviral E1A oncoproteins. HEK293- TP53::EGFP clones may be used as a living cell system for monitoring not only of the induction of p53 protein in the cell, but also of its subcellular localization. Using this human reporter cell system, we examined levels of p53 by fluorescence microscopy and by FACS analysis following treatment with several classes of genotoxic and carcinogenic compounds. All tested DNA damaging agents caused a significant increase of intracellular p53-EGFP levels in a concentration-dependent manner. On the other hand, non-genotoxic carcinogens and stress conditions that cannot damage DNA were not able to induce p53-EGFP accumulation. The induction effect caused by genotoxic stress was found to be dependent on the endogenous p53 status, because it was not observed in p53-deficient cell lines. This corroborates the notion that p53 may be used as an universal sensor for genotoxic stress and demonstrates the usefulness of HEK293-p53-EGFP cells as a reporter system for identification of mutagens and genotoxic carcinogens in human cells by means of visualizing and monitoring intracellular p53 levels and localization.

Genomic footprinting using nucleases.

Gene expression is regulated by complex mechanisms involving dynamic interactions between cis-acting elements and trans-acting factors in a highly structured chromatin environment. Investigations of protein/DNA interactions in vitro may not have relevance to a living cell system. To analyze events occurring at the DNA level in a living cell, Church et al. introduced the genomic footprinting procedure (). The procedure was developed further () but ligation-mediated PCR (LM-PCR) was the breakthrough that rendered the technique accessible to many laboratories ().

Characterization of microbial system for degradation of bacterial endospores

In order to obtain microorganisms or enzymes responsible for lysis of bacterial spores (endospores), microbial cells capable of utilizing damaged spores as a sole carbon source were screened among 200 samples in nature. Such microbial cells were enriched in a cluster containing at least five kinds of bacterial strains. One of the strains was isolated from the cluster and designated strain S-2. During the growth of cells of strain S-2 or in the cluster, the turbidity (emulsified white in color) of the medium containing spores was eliminated with a concomitant loss of spore viability. The finding of spore-degrading bacteria suggests that spores are not always in a long-lived form for microorganisms to survive in living cell system.

Electron microscopic evidence of an immobilized living cell system

Whole cells of Rhodopseudomonas capsulata were immobilized by entrapment in Ca 2+ alginate gel. The cell growth inside the matrix was observed by transmission electron microscopy. Colonies enlarged upon incubation, giving some evidence of an immobilized living cell system. This observation was correlated by measurements of bacteriochlorophyll, nitrogen, and protein content.

Injected nuclei in frog oocytes provide a living cell system for the study of transcriptional control.

Human HeLa nuclei injected into Xenopus oocytes synthesise RNA continuously for up to 1 month, and some of this RNA is translated into HeLa proteins. Nuclear proteins extracted from other cells enter the injected nuclei. This living cell system can be used for the study of transcriptional controls.

Freeze-etch study of Pseudomonas aeruginosa: localization within the cell wall of an ethylenediaminetetraacetate-extractable.

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg(2+), the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system.