A sub-population of rat liver membrane-bound ribosomes that are detached in vitro by carcinogens and centrifugation.
ABSTRACT: The chemical-carcinogen-induced detachment of ribosomes from rat liver endoplasmic reticulum was studied in vitro. Incubation of postmitochondrial supernatant with 0.2 mM-diethylnitrosamine or N-2-acetylaminofluorene removed approx. 16% of membrane-bound ribosomes, measured as differences in RNA/protein values of membrane separated from unbound ribosomes by flotation. These ribosomes are also detached by exposure to high centrifugal forces (160000g) and are among those removed by NADPH-catalysed lipid peroxidation. Extensive lipid peroxidation prohibits any measurement. The ribosomes (polyribosomes) removed are not those detached from the membrane by exposure to high KC1 concentrations (loosely bound) or high KC1 concentrations in the presence of puromycin (tightly bound). It is concluded then that centrifugally labile and carcinogen-sensitive represent a previously unreported sub-population of membrane-bound ribosomes.
Project description:A rough-membrane fraction isolated from rat liver by a procedure designed to prevent membrane denaturation was subjected to the gradient treatment normally used to isolate free ribosomes. Under these conditions, at most 20% of the ribosomes were detached from membrane with less than 5% sedimenting into the free-polyribosome pellet.
Project description:Ferroptosis is an iron-dependent form of programmed cell death characterized by the accumulation of lipid-targeting reactive oxygen species that kill cells by damaging their plasma membrane. The lipid repair enzyme GSH peroxidase 4 (GPX4) protects against this oxidative damage and enables cells to resist ferroptosis. Recent work has revealed that matrix-detached carcinoma cells can be susceptible to ferroptosis and that they can evade this fate through the signaling properties of the ?6?4 integrin, which sustains GPX4 expression. Although these findings on ferroptosis are provocative, they differ from those in previous studies indicating that matrix-detached cells are prone to apoptosis via a process referred to as anoikis. In an effort to reconcile these discrepant findings, here we observed that matrix-detached epithelial and carcinoma cells cluster spontaneously via a mechanism that involves the cell adhesion protein PVRL4 (also known as Nectin-4). We found that this clustering process allows these cells to survive by stimulating a PVRL4/?6?4/Src signaling axis that sustains GPX4 expression and buffers against lipid peroxidation. In the absence of ?6?4, PVRL4-mediated clustering induced an increase in lipid peroxidation that was sufficient for triggering ferroptosis. When the clustering was inhibited, single cells did not exhibit a significant increase in lipid peroxidation in the absence of ?6?4, and they were more susceptible to apoptosis than to ferroptosis. These results indicate that ferroptosis induction depends on cell clustering in matrix-detached cells that lack ?6?4 and imply that the fate of matrix-detached cells can be determined by the state of their cell-cell interactions.
Project description:Because it has been proposed that the ribosome-membrane interaction is different in endoplasmic reticulum derived from a non-secretory and secretory cell we undertook a study to determine whether attachment of the ribosome to the membrane involved ribosomal RNA and if the rRNA in ribosomes derived from the two classes of cell possessed an altered susceptibility to RNAase (ribonuclease) hydrolysis. We found that brain ribosomes appeared to possess more regions accessible to nuclease attack, independent of whether a sequence-dependent RNAase (T(1)) or a sterically hindered RNAase bound to Enzite polymer was employed. These results were independent of whether the ribosomes were membrane-bound or detached from the endoplasmic reticulum membranes, but at high RNAase concentration these differences became negligible. No conclusions, however, could be drawn as to whether ribosomal RNA is involved in the attachment of the ribosome to the endoplasmic reticulum membrane, because of the presence of endogeneous membrane-associated RNAases. Analysis of the rRNA fragments by polyacrylamide-gel electrophoresis suggests that the sites available for attack by low concentrations of nuclease in bound-ribosomes derived from brain cortex are different from those of liver.
Project description:Achieving efficient and biocompatible detachment between adhered wet materials (i.e., tissues and hydrogels) is a major challenge. Recently, photodetachable topological adhesion has shown great promise as a strategy for conquering this hurdle. However, this photodetachment was triggered by UV light with poor biocompatibility and penetration capacity. This study describes near-infrared (NIR) light-detached topological adhesion based on polyacrylic acid coated upconverting nanoparticles (UCNP@PAA) and a photodetachable adhesive (termed Cell-Fe). Cell-Fe is a coordinated topological adhesive consisting of carboxymethylcellulose and Fe<sup>3+</sup> that can be photodecomposed by UV light. To prepare a substrate for NIR-detached topological adhesion, UCNP@PAA and Cell-Fe were mixed and brushed on the surface of the model adherent. The UCNP@PAA can harvest NIR light and convert it into UV light, triggering the decomposition of the Cell-Fe and inducing the detachment. This NIR-detached topological adhesion is also feasible in deep tissue because of the ability of NIR light to penetrate tissue.
Project description:Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.
Project description:Harvested fruit undergo carbon and energy deprivation. However, the events underlying this energy-related stress in detached fruit and their involvement in cell damage have not yet been elucidated. We showed that supplementing detached sweet oranges with additional carbon or energy sources reduced peel damage, while inhibitors of energy metabolism increased it. We investigated the effect of an exogenous source of carbon (glycerol), energy (ATP), and an inhibitor of energy metabolism 2-deoxy-D-glucose (DeOGlc)?+?sodium iodoacetate (IAc), on the transcriptome of harvested fruit flavedo (outer peel part). ATP and Gly induced common, but also specific, alternative modes of energy metabolism by reducing the stress caused by energy shortage. They also induced shifts in energy metabolism that led to the production of the intermediates required for plant defense secondary metabolites to form. ATP and Gly triggered changes in the expression of the genes involved in cell lesion containment through a defined pathway involving hormones and redox-mediated signaling. DeOGlc?+?IAc had a contrasting effect on some of these mechanisms. These chemicals altered the biological processes related to membrane integrity and molecular mechanisms involving reactive oxygen species (ROS) production, and lipid and protein degradation.
Project description:Background:Integrin-mediated adhesion is normally required for cytokinetic abscission, and failure in the process can generate potentially oncogenic tetraploid cells. Here, detachment-induced formation of oncogenic tetraploid cells was analyzed in non-transformed human BJ fibroblasts and BJ expressing SV40LT (BJ-LT) ± overactive HRas. Results:In contrast to BJ and BJ-LT cells, non-adherent BJ-LT-Ras cells recruited ALIX and CHMP4B to the midbody and divided. In detached BJ and BJ-LT cells regression of the cytokinetic furrow was suppressed by intercellular bridge-associated septin; after re-adhesion these cells divided by cytofission, however, some cells became bi-nucleated because of septin reorganization and furrow regression. Adherent bi-nucleated BJ cells became senescent in G1 with p21 accumulation in the nucleus, apparently due to p53 activation since adherent bi-nucleated BJ-LT cells passed through next cell cycle and divided into mono-nucleated tetraploids; the two centrosomes present in bi-nucleated BJ cells fused after furrow regression, pointing to the PIDDosome pathway as a possible mechanism for the p53 activation. Conclusions:Several mechanisms prevent detached normal cells from generating tumor-causing tetraploid cells unless they have a suppressed p53 response by viruses, mutation or inflammation. Importantly, activating Ras mutations promote colony growth of detached transformed cells by inducing anchorage-independent cytokinetic abscission in single cells.
Project description:Senescence is genetically-controlled and activated in mature tissues during ageing. However, immature plant tissues also display senescence-like symptoms when continuously exposed to adverse energy-depleting conditions. We used detached dark-held immature inflorescences of Arabidopsis thaliana to understand the metabolic reprogramming occurring in immature tissues transitioning from rapid growth to precocious senescence. Macroscopic growth of the detached inflorescences rapidly ceased upon placement in water in the dark at 21M-BM-0C. Inflorescences were completely de-greened by 120 h of dark incubation and by 24 h had already lost 24% of their chlorophyll and 34% of their protein content. Comparative transcriptome profiling at 24 h revealed that inflorescences response at 24 h had a large carbon-deprivation component. Genes that positively regulate developmental senescence (ANAC092) and shade avoidance syndrome (PIF4 and PIF5) were up-regulated within 24 h. Mutations in these genes delayed de-greening of the inflorescences. Their up-regulation was suppressed in dark-held inflorescences by glucose treatment, which promoted macroscopic growth and development and inhibited de-greening of the inflorescences. Detached inflorescences held in the dark for 4 days were still able to re-initiat development to produce siliques upon being brought out to light indicating the transcriptional reprogramming at 24 h was adaptive and reversible. Our results suggest that the response of detached immature tissues to dark storage involves interactions between carbohydrate status sensing and light deprivation signaling and that the dark adaptive response of the tissues appears to utilize some of the same key regulators as developmental senescence. Detached arabidopsis inflorescences were harvested and either immediately snap-frozen in liquid nitrogen and stored at -80M-BM-0C, or placed at 21M-BM-0C on blotting paper moistened with water inside a 2-L black plastic container for RNA extraction and hybridization on Affymetrix microarrays. Ten inflorescences from 10 independent plants were pooled for each time point (0 h or 24 h).
Project description:Exposure of cancer cells to anticancer agents in cultures induces detachment of cells that are usually considered dead. These drug-induced detached cells (D-IDCs) may represent a clinical problem for chemotherapy since they may survive anoikis, enter the circulation, invade other tissues and resume proliferation, creating a metastasis, especially in tissues where the bioavailability of anticancer agents is not enough to eliminate all cancer cells. In this study we evaluated the antiproliferative effect of menadione?:?sodium orthovanadate (M?:?SO) combination on A549 lung cancer cells as well as the ability of M?:?SO to induce cell detachment. In addition, we followed the fate and chemosensitivity of M?:?SO-induced detached cells. Using transwell chambers, we found that a fraction of the M?:?SO-induced detached cells were viable and, furthermore, were able to migrate, re-attach, and resume proliferation when re-incubated in drug-free media. The total elimination of A549 detachment-resistant cells and M?:?SO-induced detached cells were successfully eliminated by equivalent M?:?SO concentration (17.5??M?:?17.5??M). Thus, M?:?SO prevented cell migration. Similar results were obtained on DBTRG.05MG human glioma cells. Our data guarantee further studies to evaluate the in vivo occurrence of D-IDCs, their implications for invasiveness and metastasis and their sensitivity to anticancer drugs.
Project description:A major difficulty in studying quantitative changes in free and membrane-bound ribosomes in a tissue under different physiological conditions is that membrane-bound ribosomes are not usually recovered quantitatively in a conventional microsomal fraction. This problem was resolved for developing chick liver by determining the conditions for the isolation of a microsomal fraction containing the highest practicable yield of rough vesicles, and then separating it into free-ribosome- and rough-vesicle-containing fractions. With the aid of a marker enzyme for the microsomal membranes and the RNA content of the recovered membrane-bound ribosomes, it was possible to correct for the recovery of rough vesicles and hence to determine the concentration of membrane-bound ribosomes in the homogenate. Despite the fact that morphological studies have suggested that most of the cellular ribosomes are not bound to membrane in chick liver cells at the earliest developmental age studied (6 days of egg incubation), 49% of the total ribosomes were found to be membrane-bound by using the new fractionation technique. This fraction increased (to 66%) during development. The discrepancy between the cell-fractionation and morphological approaches could not be attributed to artifacts of the separation method but rather to difficulties inherent in the morphological approach.