Nuclear Phospholipid Research Articles

Running 'LAPS' Around nLD: Nuclear Lipid Droplet Form and Function.

The nucleus harbours numerous protein subdomains and condensates that regulate chromatin organization, gene expression and genomic stress. A novel nuclear subdomain that is formed following exposure of cells to excess fatty acids is the nuclear lipid droplet (nLD), which is composed of a neutral lipid core surrounded by a phospholipid monolayer and associated regulatory and lipid biosynthetic enzymes. While structurally resembling cytoplasmic LDs, nLDs are formed by distinct but poorly understood mechanisms that involve the emergence of lipid droplets from the lumen of the nucleoplasmic reticulum and de novo lipid synthesis. Luminal lipid droplets that emerge into the nucleoplasm do so at regions of the inner nuclear membrane that become enriched in promyelocytic leukemia (PML) protein. The resulting nLDs that retain PML on their surface are termed lipid-associated PML structures (LAPS), and are distinct from canonical PML nuclear bodies (NB) as they lack key proteins and modifications associated with these NBs. PML is a key regulator of nuclear signaling events and PML NBs are sites of gene regulation and post-translational modification of transcription factors. Therefore, the subfraction of nLDs that form LAPS could regulate lipid stress responses through their recruitment and retention of the PML protein. Both nLDs and LAPS have lipid biosynthetic enzymes on their surface suggesting they are active sites for nuclear phospholipid and triacylglycerol synthesis as well as global lipid regulation. In this review we have summarized the current understanding of nLD and LAPS biogenesis in different cell types, their structure and composition relative to other PML-associated cellular structures, and their role in coordinating a nuclear response to cellular overload of fatty acids.

Nuclear accumulation of KPNA2 impacts radioresistance through positive regulation of the PLSCR1-STAT1 loop in lung adenocarcinoma.

Lung adenocarcinoma (ADC) is the predominant histological type of lung cancer, and radiotherapy is one of the current therapeutic strategies for lung cancer treatment. Unfortunately, biological complexity and cancer heterogeneity contribute to radioresistance development. Karyopherin α2 (KPNA2) is a member of the importin α family that mediates the nucleocytoplasmic transport of cargo proteins. KPNA2 overexpression is observed across cancer tissues of diverse origins. However, the role of KPNA2 in lung cancer radioresistance is unclear. Herein, we demonstrated that high expression of KPNA2 is positively correlated with radioresistance and cancer stem cell (CSC) properties in lung ADC cells. Radioresistant cells exhibited nuclear accumulation of KPNA2 and its cargos (OCT4 and c‐MYC). Additionally, KPNA2 knockdown regulated CSC‐related gene expression in radioresistant cells. Next‐generation sequencing and bioinformatic analysis revealed that STAT1 activation and nuclear phospholipid scramblase 1 (PLSCR1) are involved in KPNA2‐mediated radioresistance. Endogenous PLSCR1 interacting with KPNA2 and PLSCR1 knockdown suppressed the radioresistance induced by KPNA2 expression. Both STAT1 and PLSCR1 were found to be positively correlated with dysregulated KPNA2 in radioresistant cells and ADC tissues. We further demonstrated a potential positive feedback loop between PLSCR1 and STAT1 in radioresistant cells, and this PLSCR1‐STAT1 loop modulates CSC characteristics. In addition, AKT1 knockdown attenuated the nuclear accumulation of KPNA2 in radioresistant lung cancer cells. Our results collectively support a mechanistic understanding of a novel role for KPNA2 in promoting radioresistance in lung ADC cells.

Nuclear phosphoinositide regulation of chromatin.

Phospholipid signaling has clear connections to a wide array of cellular processes, particularly in gene expression and in controlling the chromatin biology of cells. However, most of the work elucidating how phospholipid signaling pathways contribute to cellular physiology have studied cytoplasmic membranes, while relatively little attention has been paid to the role of phospholipid signaling in the nucleus. Recent work from several labs has shown that nuclear phospholipid signaling can have important roles that are specific to this cellular compartment. This review focuses on the nuclear phospholipid functions and the activities of phospholipid signaling enzymes that regulate metazoan chromatin and gene expression. In particular, we highlight the roles that nuclear phosphoinositides play in several nuclear-driven physiological processes, such as differentiation, proliferation, and gene expression. Taken together, the recent discovery of several specifically nuclear phospholipid functions could have dramatic impact on our understanding of the fundamental mechanisms that enable tight control of cellular physiology.

Abstract LB-175: ATM regulates radiation-induced translational control of gene expression

Abstract The cellular radioresponse is composed of processes regulated by constitutively expressed proteins and processes regulated by new gene expression. While processes regulated by constitutively expressed proteins, such as those participating in DNA repair and cell cycle arrest, have been extensively studied, the radiation-induced changes in gene expression are not well defined. Previously, we have demonstrated that translation plays an important role in regulating gene expression in response to radiation. In glioblastoma cells, treatment with radiation affects more genes at the translational level than at the transcriptional level. Specific transcripts were found to be recruited to or away from polysomes without global changes in the polysome profile of the cells. Here, we utilized microarray analysis of polysome-associated transcripts in the glioblastoma stem-like cell line NSC11 to demonstrate that the DNA damage response kinase ATM controls the translation of a subset of genes as part of the radioresponse. Cells were incubated with the ATM inhibitor KU-60019 at a dose of 500nM for one hour, exposed to 2 Gy, and harvested for polysome extraction one hour after irradiation. ATM inhibition blocked translational changes of approximately one third of genes that were affected by radiation. Gene enrichment analysis revealed that genes translationally regulated by ATM were significantly involved in a number of cellular processes, indicating that ATM-mediated translational control may have functional consequences on the cellular radioresponse. By GSEA, cells treated with radiation alone were highly enriched for terms involving mitochondrial structure and function, cellular respiration, and oxidative stress as compared to irradiated cells treated with the ATM inhibitor. Other significantly enriched categories of functional interest included the spliceosome, RNA catabolic processes, the small nuclear ribonucleoprotein complex, and phospholipid and phosphoinositide metabolism. Analysis of UTR regulatory elements revealed that a significant percentage of the genes translationally upregulated by ATM in irradiated cells were enriched for HuR binding sites, which suggests a potential mechanism for polysome recruitment of these transcripts. Overall, these findings uncover a novel network of post-transcriptional gene regulation orchestrated by ATM and provide a framework to identify potential new molecules and pathways that govern tumor cell radiosensitivity. Citation Format: Stacey L. Lehman, Amy Wahba, Kevin Camphausen, Philip J. Tofilon. ATM regulates radiation-induced translational control of gene expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-175.

CAMP stimulates diacylglycerol kinase‐θ phosphorylation in adrenocortical cells (605.18)

Diacylglycerol kinase theta (DGKθ) is an intercellular lipid kinase that coordinates nuclear phospholipid metabolism and cortisol production in the adrenal cortex by generating phosphatidic acid (PA), an agonist of the nuclear receptor steroidogenic factor 1 (SF1). We previously found that cAMP signaling induces the transcription of DGKθ and increases nuclear DGK activity. Moreover, mass spectrometric analysis of DGKθ isolated from H295R human adrenocortical cells has revealed that DGKθ is a phosphoprotein. However, the functional significance of DGKθ phosphorylation is unclear. Additionally, the mechanism by which cAMP signaling regulates DGKθ activity is not known. We show that dibutyryl cAMP increases the phosphorylation of DGKθ in H295R human adrenocortical cells. Alanine substitution at Ser‐354 reduces cAMP‐stimulated phosphorylation impairs the ability of DGKθ to activate SF1‐dependent transcription of CYP17. Cycloheximide sensitivity assays revealed that the S354A mutant is more stable than the wild type. Notably, we also identify Ser‐241 as a second site that is phosphorylated, where the S241A mutant abrogates DGKθ‐stimulated activation of a CYP17 reporter gene. Since Ser‐241 lies near to two proline residues, Pro‐244 and Pro‐245, that have been identified as key for optimal catalytic activity, we postulate that mutation of these prolines also alters the ability of DGKθ to be phosphorylated at this site. Indeed, P244L and P245L impair SF1‐dependent CYP17 luciferase activity. In summary, we identify DGKθ as an effector in the cAMP signaling pathway and a target of protein kinase A.Grant Funding Source: Supported by NIH DK084178.

Regulation of Glucocorticoid Production by Nuclear Lipid Metabolism

Glucocorticoids are produced in response to activation of a cAMP signaling cascade that increases the interaction of the nuclear receptor steroidogenic factor 1 (SF1) with the promoters of multiple target genes. We have previously identified phosphatidic acid (PA) and sphingosine (SPH) as an agonist and antagonist for SF1, respectively. Based on the nuclear localization of SF1, we hypothesized that cAMP signaling regulates the ability of SF1 to activate target genes by modulating the nuclear concentrations of PA and SPH. We show that cAMP rapidly activates nuclear phospholipid and sphingolipid metabolism, where PA production is catalyzed by diacylglycerol kinase theta (DGKQ) and SPH biosynthesis by acid ceramidase (ASAH1). Ligand binding is facilitated by the ability of these lipid metabolizing enzymes to bind directly to the receptor. Silencing ASAH1 increases the capacity of adrenocortical cells to secrete cortisol, primarily by increasing the expression of genes required for steroid hormone production. In contrast, suppression of DGKQ expression attenuates the ability of SF1 to activate target gene transcription and impairs hormone biosynthesis. Collectively, we show that ASAH1 and DGKQ are novel coregulatory proteins that regulate SF‐1 function by directly binding to the receptor on SF‐1‐target gene promoters and identify a key role for nuclear lipid metabolism in regulating gene transcription.

Nuclear phospholipid scramblase 1 prolongs the mitotic expansion of granulocyte precursors during G-CSF-induced granulopoiesis

PLSCR1-/- mice exhibit normal, steady-state hematologic parameters but impaired emergency granulopoiesis upon in vivo administration of G-CSF. The mechanism by which PLSCR1 contributes to G-CSF-induced neutrophil production is largely unknown. We now report that the expansion of bone marrow myelocytes upon in vivo G-CSF treatment is reduced in PLSCR1-/- mice relative to WT. Using SCF-ER-Hoxb8-immortalized myeloid progenitors to examine the progression of G-CSF-driven granulocytic differentiation in vitro, we found that PLSCR1 prolongs the period of mitotic expansion of proliferative granulocyte precursors, thereby giving rise to increased neutrophil production from their progenitors. This effect of PLSCR1 is blocked by a ΔNLS-PLSCR1, which prevents its nuclear import. By contrast, mutation that prevents the membrane association of PLSCR1 has minimal impact on the role of PLSCR1 in G-CSF-induced granulopoiesis. These data imply that the capacity of PLSCR1 to augment G-CSF-dependent production of mature neutrophils from myeloid progenitors is unrelated to its reported activities at the endofacial surface of the plasma membrane but does require entry of the protein into the nucleus, suggesting that this response is mediated through the observed effects of PLSCR1 on gene transcription.

Nuclear Localization of Phospholipase D1 Mediates the Activation of Nuclear Protein Kinase Cα and Extracellular Signal-regulated Kinase Signaling Pathways

Recent studies highlight the existence of a nuclear lipid metabolism related to cellular proliferation. However, the importance of nuclear phosphatidylcholine (PC) metabolism is poorly understood. Therefore, we were interested in nuclear PC as a source of second messengers and, particularly, nuclear localization of PC-specific phospholipase D (PLD). In the present study we have identified the nuclear localization sequence (NLS) of PLD1 whose mutation abolished its nuclear import. Recently, we reported that caspase-mediated cleavage of PLD1 generates the N-terminal fragment (NF-PLD1) and C-terminal fragment (CF-PLD1). Here we show that CF-PLD1 but not NF-PLD1, is exclusively imported into the nucleus via its functional NLS, whereas only some portions of intact PLD1 were localized into the nucleus. The NLS of intact PLD1 or CF-PLD1 is required for interaction with importin-β, which is known to mediate nuclear import. The amount of intact PLD1 or CF-PLD1 translocated into nucleus is correlated with its binding affinity with importin-β. Ultimately, nuclear localization of intact PLD1 but not CF-PLD1 mediates the activation of nuclear protein kinase Cα and extracellular signal-regulated kinase signaling pathways. Taken together, we propose that nuclear localization of PLD1 via the NLS and its interaction with importin-β may provide new insights on the functional role of nuclear PLD1 signaling.

Phospholipase C-δ1 Expression Is Linked to Proliferation, DNA Synthesis, and Cyclin E Levels

Phospholipase C-δ1 Expression Is Linked to Proliferation, DNA Synthesis, and Cyclin E Levels

Molecular characterization of phospholipid hydroperoxide glutathione peroxidases from Hydra vulgaris

Apparent full-length cDNA sequences coding respectively for mitochondrial (HvGPx41) and nuclear (HvGPx42) phospholipid hydroperoxide glutathione peroxidase were isolated from Hydra vulgaris. The cDNA sequences share total identity in their 3′-end and differ in their 5′-end. The protein-coding regions of the HvGPx41 and HvGPx42 cDNA encode polypeptides of 190 and 168 amino acids, including a TGA-encoded selenocysteine, respectively. Phylogenetic analysis showed that the HvGPx41 and HvGPx42 are clustered together along with other phospholipid hydroperoxide glutathione peroxidases (PHGPx) from several organisms. A tertiary structure model generated for the H. vulgaris PHGPx displayed the thioredoxin fold. Hydrae exposed to starvation, metal and oxidative stress responded by regulating their PHGPx mRNA transcription. These results indicated that the PHGPx gene is affected by the cellular stress response and (anti)oxidative processes triggered by stressor and contaminant exposure. Hence the expression of PHGPx mRNA in hydra may have potential use as molecular biomarkers for assessing stress, toxicity and pro-oxidant quality of chemicals and aquatic environmental quality.

Phospholipase-C β1 is predominantely expressed in the granular layer of rat cerebellar cortex

The beta1 isoform of phospholipase-C is exclusively present in the nucleus of several hematopoietic and non-hematopoietic cell lines and primary cells of different species. When present, it represents the key enzyme for initiating the nuclear phospholipid breakdown that is involved in the cellular response to proliferating and differentiating stimuli. We have studied the expression of this enzyme isoform in the rat cerebellar cortex. We demonstrate that phospholipase-C beta1 (PLCbeta1) is predominantly expressed in the neurons of the granular layer, while it is virtually absent in the molecular and Purkinje cell layers of rat cerebellar cortex. This pattern of expression is partially different from that of the mouse cerebellar cortex, where not only granular cells, but also Purkinje cells express PLCbeta1. The high level of synaptic inputs that converge on granular cells may imply a constantly active nuclear phospholipid metabolism that may not be strictly required for the appropriate cellular responses of the other cell types of rat cerebellar cortex.

Hydrolysis of nuclear phospholipids in relation with proliferative state in uterine stromal cells

The current study examined the metabolism of phospholipid (PL) in the whole cell homogenate and in the nuclear fraction in proliferative and non-proliferative uterine stromal cells (U III cells). Growth arrested cells were obtained either from contact-inhibited confluent cells or from proliferative cells treated with aristolochic acid (AR) for 2 days. Fatty acid composition and fatty acid amount of both total and nuclear PL were not significantly different between proliferative, confluent and AR-treated cells. In contrast, marked differences were observed in the incorporation of [ 3H]AA, with greater incorporation in proliferative cells than in confluent or AR-treated cells, particularly in nuclear PL. Considering endogenous level of arachidonic acid (AA) in total and nuclear PL, we found that AA turnover in nuclear PL was especially high compared to that in total PL and that this difference was accentuated in proliferative cells compared to non-proliferative cells. Interestingly, [ 3H]AA incorporation and AA turnover in proliferative, confluent and AR-treated cells vary accordingly to the expression, activity and/or content of pancreatic phospholipase A 2 (PLA 2-I) in the nuclear compartment of these cells that we reported in previous studies. The changes in metabolism of nuclear PL during cell proliferation are consistent with an enhanced PL hydrolysis that could involve PLA 2-I.

Electrospray ionization mass spectrometry analyses of nuclear membrane phospholipid loss after reperfusion of ischemic myocardium

The role of nuclear membrane phospholipids as targets of phospholipases resulting in the generation of nuclear signaling messengers has received attention. In the present study, we have exploited the utility of electrospray ionization mass spectrometry to determine the phospholipid content of nuclei isolated from perfused hearts. Rat heart nuclei contained choline glycerophospholipids composed of palmitoyl and stearoyl residues at the sn-1 position with oleoyl, linoleoyl, and arachidonoyl residues at the sn-2 position. Diacyl molecular species were the predominant molecular subclass in the choline glycerophospholipids, with the balance of the molecular species being plasmalogens. In the ethanolamine glycerophospholipid pool from rat heart nuclei approximately 50% of the molecular species were plasmalogens, which were enriched with arachidonic acid at the sn-2 position. A 50% loss of myocytic nuclear choline and ethanolamine glycerophospholipids was observed in hearts rendered globally ischemic for 15 min followed by 90 min of reperfusion in comparisons with the content of these phospholipids in control perfused hearts. The loss of nuclear choline and ethanolamine glycerophospholipids during reperfusion of ischemic myocardium was partially reversed by the calcium-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL), suggesting that the loss of nuclear phospholipids during ischemia/reperfusion is mediated, in part, by iPLA2. Western blot analyses of isolated nuclei from ischemic hearts demonstrated that iPLA2 is translocated to the nucleus after myocardial ischemia. Taken together, these studies have demonstrated that nuclear phospholipid mass decreases after myocardial ischemia by a mechanism that involves, at least in part, phospholipolysis mediated by iPLA2.—Williams, S. D., F-F. Hsu, and D. A. Ford. Electrospray ionization mass spectrometry analyses of nuclear membrane phospholipid loss after reperfusion of ischemic myocardium. J. Lipid Res. 2000. 41: 1585–1595.

Diabetes affects phospholipid content in the nuclei of the rat liver.

The aim of the present study was to examine the effect of acute streptozotocin diabetes on the content of different phospholipids and the incorporation of blood-borne 14C-palmitic acid into the phospholipid moieties in the rat liver nuclei. Diabetes was produced by intravenous administration of streptozotocin, and determinations were carried out two and seven days thereafter. Phospholipids were extracted from isolated nuclei and separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. Following that, they were quantified and radioactivity was measured. It was found that, in comparison to non-diabetic controls, two-day diabetes reduced the total content of phospholipids in the nuclei by 9.6%. The content of phospholipids in the nuclei by 9.6%. The content of phosphatidylcholine and phosphatidylserine was reduced and the content of the remaining phospholipids was stable. The specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin, based on radioactivity incorporated from 14C-palmitic acid, was elevated. Seven-day diabetes resulted in a reduction of the total phospholipid content in the nuclei by 39.4%. This was accounted for by a reduction in the content of each phospholipid fraction with the exception of cardiolipin. The specific activity of each phospholipid fraction, was elevated in this group. It is concluded that insulin is involved in the regulation of the nuclear phospholipid content.

Translocation of diacylglycerol kinase α to the nuclear matrix of rat thymocytes and peripheral T-lymphocytes

The cytosolic α-diacylglycerol kinase (DGK) was translocated to and tightly associated with the nuclear matrix when rat thymocytes and peripheral T-lymphocytes were stimulated with concanavalin A or anti-T-cell receptor antibody. This translocation occurred rather slowly and was completed in 3–4 h after cell stimulation. We also detected significant accumulation of nuclear phosphatidic acid interpreted as being formed by the translocated enzyme. The enzyme translocation is not directly linked to phosphoinositide turnover and protein phosphorylation, since phorbol myristate acetate and calcium ionophore did not affect the cellular DGKα and since we detected no covalent modification of the enzyme molecule. Although the mechanisms underlying the enzyme translocation remain unknown, our results indicate that DGKα participates in nuclear phospholipid metabolism occurring at the intermediate stage of lymphocyte activation.

Decrease in nuclear phospholipids associated with DNA replication.

Lipid metabolism in nuclei is very active and appears involved in the transduction of signals to the genome in response to agonists acting at the plasma membrane level. However, the precise topology of nuclear lipid metabolism and the relationship between nuclear lipids and crucial events of the cell function, such as DNA replication, have not been fully elucidated. By using a recently developed cytochemical method for detecting phospholipids inside the nucleus of intact cells at the electron microscope level, we have analyzed the changes in intranuclear phospholipids in DNA-replicating versus resting cells, which are both present in the same sample of regenerating liver after partial hepatectomy. The pattern of DNA synthesis in replicating cells has been monitored by electron microscope immunocytochemistry after bromodeoxyuridine (BrdU) labeling. The data obtained, which allow a fine localization and a quantitative analysis of both DNA synthesis and phospholipid distribution, indicate a significant reduction in the phospholipids detectable inside the nucleus in all steps of the S phase. This could depend on an increased nuclear phospholipid hydrolysis, whose products should in turn activate some of the enzymes involved in the control of DNA replication.

Effect of membrane fatty acid changes on the radiation sensitivity of human lymphoid cells.

To test the influence of changes in membrane fatty acid composition on the radiation response of mammalian cells, human LDV cells were cultured in medium containing delipidated serum supplemented with either oleic or linoleic acid. Analysis of lipid extracts of the cells by gas liquid chromatography showed that, after 3 or more days growth in oleic or linoleic acid supplemented media, there were substantial overall increases in the proportion of oleate and linoleate, respectively, in the cellular lipid. Smaller absolute changes were measured in nuclear phospholipid, which was found to be low in unsaturated phospholipid compared to the rest of the cell. Fluorescence polarization measurements using diphenylhexatriene indicated an increased membrane fluidity in cells grown in the presence of excess linoleic acid and to a lesser extent for oleic acid. The clonogenic capacity of the cells after irradiation in air or nitrogen was not altered by any of these membrane compositional changes. The lack of effect on radiation sensitivity, in contrast to that reported for bacteria (E. coli K1060), is consistent with the fact that little or no change was brought about in the nuclear membrane composition, since evidence from partial cell irradiation experiments indicates that the cell nucleus is the sensitive target for cell killing by ionizing radiation. The ability of the cell to maintain a low level of unsaturated phospholipids in its nuclear membrane may be an important general defence mechanism against free radical damage to chromatin mediated by lipid peroxidation.

Cholinephosphotransferase activities in microsomes and neuronal nuclei isolated from immature rabbit cerebral cortex: the use of endogenously generated diacylglycerols as substrate

A neuronal nuclear fraction (N1) and a microsomal fraction (P3) were isolated from homogenates of cerebral cortices of 15-day-old rabbits. A nuclear envelope fraction (E) was prepared from N1. To assay cholinephosphotransferase, diacylglycerols were first generated in the membranes of these subfractions using a phospholipase C (Bacillus cereus) preincubation. With levels of endogenous diacylglycerols producing maximal specific cholinephosphotransferase activities, an activity ratio of 1:1:5 was found for N1, P3, and E, respectively. An independent neuronal nuclear cholinephosphotransferase, concentrated in nuclear membranes, is indicated. With regard to changes in pH and concentrations of MgCl2 and CDP-choline, N1 and P3 activities responded in a similar manner. However, in contrast to P3, N1 activities we much more profoundly inhibited at low levels of Triton X-100 (0.01-0.02 w/v%) and N1 showed quite significant levels of cholinephosphotransferase activity in the absence of a phospholipase C preincubation. Choline phosphotransferase in N1 and P3 showed Km values for CDP-choline (0.028 and 0.031 mM, respectively) which were much lower than corresponding literature values determined using exogenous diacylglycerols as substrates for this enzyme. The presence of cholinephosphotransferase in neuronal nuclear membranes reflects a rather exceptional nuclear autonomy. This may be related to a need to maintain nuclear phospholipid in the absence of a well-developed endoplasmic reticulum at early stages of neuronal development or to synthesize phospholipid in response to functions unique to the nucleus.

RNA transport in isolated myeloma nuclei. Transport from membrane-denuded nuclei.

Nuclei prepared from MOPC-21 cells were treated with the nonionic detergents Triton X-100 or Nonidet P-40. Chemical analysis revealed that nearly 90% of the nuclear phospholipid was removed by detergent treatment. The membrane-denuded nuclei remained intact with preservation of nuclear pore complexes as demonstrated by electron microscopy. Ribonucleic acid transport from detergent-treated nuclei proceeded at the same rate and to the same extent as in control nuclei. Normal nuclear restriction of nucleic acids was unaltered by removal of the nuclear membranes. The effect of temperature on transport of RNA from freshly isolated myeloma nuclei with intact nuclear envelopes was studied. No temperature transition was associated with the transport process. These data indicate that the transport of macromolecules from isolated myeloma nuclei is independent of the nuclear membrane.

On the attachment of the nuclear pore complex.

Electron microscope examination of isolated rat liver nuclei after treatment with the detergent Triton X-100 revealed the complete removal of both the inner and outer membranes of the nuclear envelope. The envelope-denuded nuclei did not show any change in either shape or internal ultrastructure. Most strikingly, the nuclear pore complexes, which in untreated nuclei appear to be integral components of the nuclear envelope, were retained in their characteristic location at the distal ends of the channels leading through the peripheral heterochromatin. Determination of the chemical composition of detergent-treated nuclei showed that over 95% of the nuclear phospholipid was solubilized, thus corroborating the morphological absence of nuclear membranes. Furthermore, detergent treatment also solubilized approximately 10% of the nuclear protein. Analysis of the solubilized protein by polyacrylamide gel electrophoresis in the presence of SDS indicated that these proteins belong to a few specific classes which presumably represent the major polypeptides of the nuclear membranes. The total absence of the nuclear envelope on both morphological and biochemical grounds supports the idea that the nuclear pore complex does not require the membranes either for attachment to the nucleus or for maintenance of its own structural integrity.