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Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei.

ABSTRACT: (1) The removal of the nuclear envelope from isolated rat-liver nuclei by washing with Triton X-100 (TX-100) was assessed by electron microscopy. All the envelope was removed by 0.04% (w/v) TX-100. (2) After this removal, phosphorylation of inositol lipids and diacylglycerol (DAG) from [gamma-32P]ATP still occurs, despite the near complete absence of detectable (by mass assay) DAG and PtdIns. This suggests that the majority of these two lipids in nuclei are present in the nuclear membrane, but the small amounts remaining after extraction, defined as intranuclear, are available for phosphorylation by lipid kinases (36% for DAG and 24% for PtdIns respectively, when expressed as a percentage of incorporation of intact nuclei). (3) PtdIns(4,5)P2 did not follow the same pattern as PtdIns and DAG; after removal of the nuclear membrane, 40% of the mass of this lipid was left in the nucleus. Moreover, a similar amount of PtdIns(4,5)P2 was also resistant to extraction with even higher concentrations of detergent, suggesting that PtdIns(4,5)P2 has a discrete intranuclear location, probably bound to nuclear proteins. (4) Addition of exogenous substrates, PtdIns, PtdIns(4)P and DAG, to membrane-depleted nuclei resulted in reconstitution of the majority of lipid phosphorylations from [gamma-32P]ATP (70%, 90% and 94% of intact nuclei respectively), suggesting a predominantly intranuclear location for the respective kinases. (5) Nuclei also showed phosphomonoesterase and phosphatidic acid hydrolase activity; dephosphorylation of pre-radiolabelled PtdIns(4)P, PtdIns(4,5)P2 and phosphatidic acid was observed when [gamma-32P]ATP was removed. However, some of the radioactivity was apparently resistant to these enzymes, suggesting the existence of multiple pools of these lipids. (6) Addition of excess non-radiolabelled ATP to nuclei pre-labelled with [gamma-32P]ATP resulted in an initial increase in the label in PtdIns(4,5)P2, implying a precursor-product relationship between the radiolabelled pools of PtdIns(4)P and PtdIns(4,5)P2. This was confirmed by analysis of the incorporation of 32P into the 4'-phosphate group of PtdIns(4)P and the individual 4'- and 5'-phosphate groups of PtdIns(4,5)P2. The data from these experiments also indicated that PtdIns(4,5)P2 can be produced from a pre-existing pool of PtdIns(4)P, as well as de novo from PtdIns. (7) Taken together our data suggest that isolated rat-liver nuclei have an intranuclear inositol lipid metabolism mechanism utilizing enzymes and substrates equivalent to those found in cytosol and plasma membrane, and that there may be some, but not complete, compartmentalization of the components of the nuclear inositol cycle.

SUBMITTER: Vann LR

PROVIDER: S-EPMC1218831 | biostudies-other | 1997 Oct

REPOSITORIES: biostudies-other

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Project description:The nucleus of mammalian cells displays a distinct spatial segregation of active euchromatic and inactive heterochromatic regions of the genome1,2. In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery1,2. Genome-wide chromosome conformation capture (Hi-C) analyses show this segregation as a plaid pattern of contact enrichment within euchromatin and heterochromatin compartments3, and depletion between them. Many mechanisms for the formation of compartments have been proposed, such as attraction of heterochromatin to the nuclear lamina2,4, preferential attraction of similar chromatin to each other1,4-12, higher levels of chromatin mobility in active chromatin13-15 and transcription-related clustering of euchromatin16,17. However, these hypotheses have remained inconclusive, owing to the difficulty of disentangling intra-chromatin and chromatin-lamina interactions in conventional nuclei18. The marked reorganization of interphase chromosomes in the inverted nuclei of rods in nocturnal mammals19,20 provides an opportunity to elucidate the mechanisms that underlie spatial compartmentalization. Here we combine Hi-C analysis of inverted rod nuclei with microscopy and polymer simulations. We find that attractions between heterochromatic regions are crucial for establishing both compartmentalization and the concentric shells of pericentromeric heterochromatin, facultative heterochromatin and euchromatin in the inverted nucleus. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. In addition, our models allow us to rule out mechanisms of compartmentalization that involve strong euchromatin interactions. Together, our experiments and modelling suggest that attractions between heterochromatic regions are essential for the phase separation of the active and inactive genome in inverted and conventional nuclei, whereas interactions of the chromatin with the lamina are necessary to build the conventional architecture from these segregated phases.

Project description:Swimming suspensions of Chlamydomonas eugametos were pelleted and homogenized, and the metabolism of inositol polyphosphates by cellular homogenates or supernatants was investigated. Ins(1,4,5)P3 was dephosphorylated under physiological conditions to yield a single InsP2, Ins(1,4]2. In the presence of ATP it was phosphorylated to give Ins(1,3,4,5)P3 as the only InsP4. The Ins(1,4,5)P3 3-kinase activity was predominantly soluble, was not detectably affected by calmodulin or Ca2+, and had a Km for Ins(1,4,5)P3 of 50 microM (two orders of magnitude higher than its mammalian counterpart). Ins(1,3,4,5)P4 was dephosphorylated by the cellular supernatants to Ins(1,3,4)P3 and Ins(1,4,5)P3, and could be phosphorylated to Ins(1,3,4,5,6)P4. No Ins(1,3,4)P3 6-kinase activity could be detected, and experiments with [3H]Ins(1,4,[32P]5)P3 revealed that Ins(1,3,4,5,6)P5 is formed from Ins(1,4,5)P3 with little loss of the 5-phosphate, i.e. the predominant route of synthesis is probably by a direct 6-phosphorylation of Ins(1,3,4,5)P4. Similar experiments with an (NH4)2SO4 fraction of turkey erythrocyte cytosol gave essentially the same result, i.e. direct phosphorylation of Ins(1,3,4,5)P4 in the 6 position is the predominant route of synthesis of InsP5 from that InsP4 in vitro. No InsP6 formation was detected in any of these experiments, but labelling of intact C. eugametos with [3H]inositol revealed that the cells do synthesize InsP6. The lipids of C. eugametos cells contain PtdIns, PtdIns(4)P and PtdIns(4,5)P2 [Irvine, Letcher, Lander, Drøbak, Dawson & Musgrave (1989) Plant Physiol. 64, 888-892]. Further examination of 32P-labelled lipids revealed that about 20% of the PtdInsP was the PtdIns(3)P isomer, and about 1% or less of the PtdInsP2 was the PtdIns(3,4)P2 isomer. The overall inositide metabolism of C. eugametos resembles that of a mammalian cell more closely than it does that of a plant cell or slime mould, and this suggests firstly that the known metabolism of inositol polyphosphates arose at an early time in eukaryotic evolution, and secondly that Chlamydomonas might prove a useful organism for genetic and comparative studies of inositide enzymology.

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Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei.

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