Rosiglitazone ameliorates palmitic acid-induced endoplasmic reticulum stress and steroidogenic capacity in granulosa cells

Granulosa cells play essential roles in follicular development, oocyte maturation and sex hormone secretion. The exposure of granulosa cells to palmitic acid (PA), the main component of dietary saturated fat, inhibits cell viability. However, the mechanism underlying PA-induced cytotoxicity in granulosa cells has not been deeply investigated. Rosiglitazone (RSG) is a member of the thiazolidinedione family and is reported to protect cells from cytotoxicity and endoplasmic reticulum (ER) stress in other cell types, but whether RSG protects granulosa cells remain unknown. In this study, KGN cell line and primary granulosa cells were used as models of granulosa cells to explore the effects of PA and RSG and the underlying mechanisms. The results showed that PA inhibits cell viability and estradiol secretion through inducing ER stress and cAMP/PKA/CREB pathway. CCAAT/enhancer-binding protein homologous protein (CHOP), an ER stress marker, was demonstrated to participate in PA-induced cytotoxicity. RSG treatment rescued granulosa cells from PA-induced cell death and ER stress. Moreover, RSG was identified to ameliorate ER stress induced by tunicamycin in granulosa cells. In addition, RSG treatment rescued granulosa cells from PA-induced decrease of estrogen secretion by cAMP/PKA/CREB pathway. In conclusion, RSG can protect granulosa cells against PA-induced cytotoxicity by inhibiting ER stress, and can recover steroidogenic capacity, indicating a potential use of RSG in the treatment of granulosa cell dysfunction.     

BiP and calreticulin form an abundant complex that is independent of endoplasmic reticulum stress

BiP is found in association with calreticulin, both in the presence and absence of endoplasmic reticulum stress. Although the BiP-calreticulin complex can be disrupted by ATP, several properties suggest that the calreticulin associated with BiP is neither unfolded nor partially or improperly folded. (1) The complex is stable in vivo and does not dissociate during 8 hr of chase. (2) When present in the complex, calreticulin masks epitopes at the C terminus of BiP that are not masked when BiP is bound to an assembly-defective protein. And (3) overproduction of calreticulin does not lead to the recruitment of more BiP into complexes with calreticulin. The BiP-calreticulin complex can be disrupted by low pH but not by divalent cation chelators. When the endoplasmic reticulum retention signal of BiP is removed, complex formation with calreticulin still occurs, and this explains the poor secretion of the truncated molecule. Gel filtration experiments showed that BiP and calreticulin are present in distinct high molecular weight complexes in which both molecules interact with each other. The possible functions of this complex are discussed.     

The islet-specific glucose-6-phosphatase-related protein, implicated in diabetes, is a glycoprotein embedded in the endoplasmic reticulum membrane

The islet-specific glucose-6-phosphatase-related protein (IGRP) has no known catalytic activity, but is of interest because it is the source of the peptide autoantigen targeted by a prevalent population of pathogenic CD8(+) T cells in non-obese diabetic mice. To better understand the potential roles of this protein in diabetes mellitus, we examine the subcellular localization and membrane topography of human IGRP. We show that IGRP is a glycoprotein, held in the endoplasmic reticulum by nine transmembrane domains, which is degraded in cells predominantly through the proteasome pathway that generates the major histocompatibility complex class I-presented peptides.     

A novel membrane associated carbonyl reducing enzyme is present in smooth endoplasmic reticulum of mouse liver.

Evidence supporting the existence of two distinct carbonyl (metyrapone) reducing enzymes which differ in subcellular localization and immunological homology has been provided. A soluble enzyme, designated as carbonyl reductase (EC 1.1.1.184) is located in the cytosol. The distribution of the second, membrane associated, MPON-reductase shows an excellent linear correlation to NADPH-cytochrome c reductase and, on the other hand, is reciprocal to the RNA/protein ratio of submicrosomal preparations. This indicates that the membrane associated MPON-reductase is exclusively located in the smooth endoplasmic reticulum. Using antibodies against the purified membrane associated enzyme the extent of immunological crossreaction corresponds well to the specific activities of MPON-reductase in the granular fractions, thus further confirming the localization of this enzyme within this organelle. The absence of antigenic crossreaction to cytosolic MPON-reductase indicates differences also in terms of the immunological relationship between the two enzymes.     

Ca-pumps in smooth muscle: one in plasma membrane and another in endoplasmic reticulum

For several years it has been debated whether the Ca-pump in smooth muscle is located in the plasma membrane or in the endoplasmic reticulum (alias sarcoplasmic reticulum). Experimental evidence using skinned smooth muscle cells and subcellular membrane fractions isolated from a number of smooth muscles is reviewed here to hopefully resolve this issue. The inescapable conclusion is that there are two modes of nonmitochondrial ATP-dependent Ca-transport. The first one, unaffected by oxalate, is localized in the plasma membranes and the second, potentiated by oxalate, is localized in the endoplasmic reticulum. Clear experiments to delineate the roles of the two pumps in the excitation-contraction cycle of the smooth muscle remain to be conducted.     

The endoplasmic reticulum membrane is permeable to small molecules

The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of approximately 5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER.     

The smooth endoplasmic reticulum develops in mesophyll cells of early-spring ephemerals

A study was made of the mesophyll cell ultrastructure of early-spring ephemerals Scilla sibirica, Crocus vernus, Galanthus caucasicus, G. plicatus, Leucojum vernum, Muscari szovitsianum, Ornithogalum balansae of summer-vegetating. Scilla scilloides and of S. sibirica growing in greenhouse. The highly developed smooth endoplasmic reticulum (SER) was a distinctive feature of the early-spring ephemeral mesophyll cell during leaf growth (cell expansion). Direct connections of SER-compartments with the tonoplast were found (SER and vacuole cavities were continuous). On running through the cytosol the SER tubules were in close contact with the plasma membrane. SER was not found during the same period of cell expansion in summer-vegetating Scilla scilloides and S. sibirica growing in greenhouse, as well as in mature cells of all investigated plants. The role of SER in the adaptation of early-spring ephemerals to drastic temperature fluctuations is discussed.     

Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum membrane

Components of the plant cell secretory pathway, including the endoplasmic reticulum and Golgi apparatus, are in constant motion. The photoactivation of GFP has been used to determine that proteins within the membrane of the ER flow as the ER is remodelled. Measurement of the rate at which activated GFP moves away from the activation spot shows that this motion is much faster than would be expected if membrane components moved simply by diffusion. Treatment with latrunculin to depolymerize the actin cytoskeleton stops ER remodelling and reduces the rate of GFP movement to that expected from diffusion alone. This suggests that myosin binds directly or indirectly to ER membrane proteins and actively moves them around over the actin scaffold. Tracking of Golgi body movement was used to demonstrate that they move at the same rate and in the same direction as do photoactivated ER surface proteins. Golgi bodies, therefore, move with, and not over, the surface of the ER. These observations support the current theory of continuity between Golgi bodies and discrete ER exit sites in the ER membrane.     

Syntaxin 11 is an atypical SNARE abundant in the immune system

Several classes of proteins have been identified that mediate and regulate membrane dynamics throughout the eukaryotic cell. One class of membrane-trafficking proteins, referred to as soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), have been implicated in mediating membrane fusion. Here we characterize syntaxin 11, an atypical syntaxin family member lacking a transmembrane domain. Syntaxin 11 was found to be enriched in tissues of the immune system including thymus, spleen and lymphnodes; however, lower levels of the protein are found in other tissues. Using immunofluorescence and electron microscopy techniques, we demonstrate that syntaxin 11 associates with intermediate compartment (IC) and post-Golgi membranes through a putative palmitoylation domain, as well as through formation of the 100-kDa complex with, as of yet, unidentified proteins. The coiled-coil forming H3 domain is required for the formation of the 100-kDa complex, and this complex can be dissociated upon addition of alphaSNAP. Thus, while the precise function of syntaxin 11 remains to be elucidated, it may be particularly important in regulating membrane dynamics of the immune system.     

The nuclear associated endoplasmic reticulum and membrane biogenesis in MPC-11 cells

The incorporation of 3H-glucosamine, 3H-choline and 14C-fucose into subcellular fractions of MPC-11 cells was studied. After a 20 min period of labelling with both 3H-glucosamine and 3H-choline, greatest incorporation was observed in nuclear-associated endoplasmic reticulum (NER). 14C-fucose, however, was incorporated to a greater extent in endoplasmic reticulum (ER) membranes. Pulse-chase experiments with 3H-glucosamine showed a loss of radioactivity from NER and a simultaneous increase in the ER fraction. In comparison to NER, ER membranes were poorly labeled with 3H-glucosamine after a 20 min pulse. Following a 2 h incubation there was a 12 fold increase in radioactivity in ER membranes in comparison to a 1.2 fold increase in NER. There were no individual differences between subfractions of ER membranes with respect to 3H-glucosamine content after the pulse, or following the 2 h incubation. The results indicate that the NER is a major, early site of the synthesis of 3H-glucosamine labeled membrane glycoproteins, and that these proteins migrate into other ER membranes early after their synthesis.     

Surface membrane proteins of endoplasmic reticulum in brain and liver cells]

A comparative study of proteins adsorbed on outer surface of microsomal membranes was carried out. Electrophoretic differences between endoplasmic reticulum proteins from liver and brain cells were revealed. These differences were not observed in the presence of sodium dodecyl sulphate. Proteins of brain microsomes are shown to bind in vitro with membranes of brain endoplasmic reticulum to a higher extent than with liver microsomal membranes.     

Contacts of the endoplasmic reticulum membrane with plasmalemma in plant cells

It is shown by electron microscopy that, in maize root cells, there is close contact between the membrane of the endoplasmic reticulum and the plasmalemma. A qualitative preliminary comparison is conducted between these contacts and high-permeability intercellular contacts in animals.     

Microtubule-associated smooth endoplasmic reticulum in the frog's brain

Summary Electron microscopic studies of neural processes in the cerebellum, optic tectum, and cerebral hemisphere of the frog reveal a distinctive system of SER cisternae lying at intervals (commonly 1–2 m apart) perpendicular to the long axis of axons and dendrites, interconnected by tubular, longitudinally orientated SER elements, and in direct continuity with the outer membrane of mitochondria. The transverse cisternae are fenestrated, with a single mierotubule (or rarely, two) passing through the centre of each 50–75 nm fenestration. Extensions of the SER-microtubule complex may be located parasynaptically in axon terminals and dendrites. The SER of dendritic spines also appears to be continuous with the fenestrated cisternae.Possible roles for the specialized SER (particularly of the parasynaptic extensions), such as calcium ion sequestration and ATP or monoamine oxidase transport, are discussed.Thanks are due to Profs. E. G. Gray and J. Z. Young for helpful discussion and to Mrs. N. Morgan and Mr. R. Boddy for technical assistance.     

Reticulon 3 is involved in membrane trafficking between the endoplasmic reticulum and Golgi

Reticulons (RTNs) constitute a family of endoplasmic reticulum (ER)-associated proteins with a reticular distribution. Despite the implication of their neuronal isoforms in axonal regeneration, the function of their widely expressed isoforms is largely unknown. In this study, we examined the role of the ubiquitously expressed RTN3 in membrane trafficking. Ectopically expressed RTN3 exhibited heterogeneous patterns; filamentous, reticular, and granular distributions. The ER morphology changed accordingly. In cells where RTN3 displayed a filamentous/reticular distribution, protein transport between the ER and Golgi was blocked, and Golgi proteins were dispersed. In contrast, ERGIC-53, a marker for the ER-Golgi intermediate compartment, accumulated at the perinuclear region, and remained there even after cells were treated with agents that induce redistribution of Golgi proteins to the ER, indicating an inhibition of Golgi-to-ER transport of ERGIC-53. These results suggest that RTN3 plays a role in membrane trafficking in the early secretory pathway.     

Relationship between ciliary rootlets and smooth endoplasmic reticulum

Summary In ciliated cells of thymic cysts in Nude mice, ciliary rootlets are constantly and closely related to smooth endoplasmic reticulum and clear vesicles. This special association suggests that this structure does not play only an anchoring role but must be involved in the general metabolism of the cilium.This work was supported by Grant 10,013 from the Fonds de la Recherche Fondamentale Collective, Bruxelles, Belgium.Chargé de recherche au Fonds de la Recherche Scientifique.     

Rapid in vitro formation of smooth endoplasmic reticulum aggregates within peptide-producing islet cells

We report here that heptanol (3.5 mM) induces in vitro a rapid formation of smooth endoplasmic reticulum aggregates (SERA) within isolated islets of Langerhans. SERA appeared after only 15 min of exposure to the alkanol and increased in number during the first 30 min of incubation. At that time, SERA represented 2% and 6% of the volume of B- and non-B-cells, respectively. Removal of heptanol resulted in the rapid disappearance of SERA, whereas reintroduction of the alkanol rapidly induced these structures again. SERA formation was seen in different types of endocrine and nonendocrine islet cells. In the insulin-producing B-cells, SERA formation was not modified by conditions known to alter the secretory activity and the microtubular-microfilament network or to inhibit protein synthesis. By contrast, SERA formation was inhibited by low temperature and by conditions depleting the energy sources of the cells. Similar observations were made in the presence of either octanol (1 mM) or nonanol (1 mM) but not of shorter chain alkanols, alkanes, oxidative derivates of either heptanol or octanol, and of other unrelated lipid-soluble compounds. Incubations in the presence of long-chain alkanols provide, therefore, a unique model to study in vitro the formation and disposal of smooth endoplasmic reticulum, as well as a system in which rapid membrane biogenesis is amenable to direct experimental testing.     

Analysis of the proteins in thymocyte plasma membrane and smooth endoplasmic reticulum by sodium dodecylsulfate-gel electrophoresis

We have purified the plasma membranes and membranes of endoplasmic reticulum from calf and rabbit thymocytes and from calf mediastinal lymph node lymphocytes. We disrupted the cells by the “nitrogen cavitation method” and prepared a microsomal isolate by differential centrifugation. We fractionated this by isopycnic ultracentrifugation in dextran gradients into membrane vesicles, PM₁ and PM₂, most likely derived from plasma membrane and a fraction, ER, most likely originating from endoplasmic reticulum. More than 80% of the microsomal 5′-nucleotidase and acid p-nitrophenylphosphatase concentrates in the PM₁ and PM₂ fractions; alkaline p-nitrophenylphosphatase, another presumptive PM marker, is concentrated in the PM₁ fraction. These data are confirmed by the lacroperoxidase radioiodination of intact rabbit thymocytes followed by subcellular fractionation. The specific content of phospholipids (822 nmoles/mg protein) and cholesterol (1032 nmoles/mg protein) is highest in PM₁ and PM₂ plasma membrane fractions. NADH-oxidoreductase, our endoplasmic reticulum marker, is clearly enriched in gradient pellet.The membrane proteins were separated by electrophoretic molecular sieving in sodium dodecylsulfate-polyacrylamide gel electrophoresis, containing dithiothreitol (sodium dodecylsulfate-polyacrylamide gel electrophoresis). We numbered the 10 major protein components of the “microsomal fraction” (apparent molecular weights between 280000 and 15000) from 1–10 according to their decreasing molecular weights. Of these proteins, those with higher molecular weight, predominantly glycoproteins, appear in the PM₁ fraction, while the endoplasmic reticulum fraction contains mainly low molecular weight components.