Ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

One phase of a study on cryosurvival and cryoprotection of mammalian cells, in terms of ultrastructural alteration of rough endoplasmic reticulum (RER) within rat pancreatic acinar cells, is presented. Small (2–3 mm) squares of tissue, 0.7–0.9 mm in thickness, were compared as unfrozen controls, with (w) and without (wo) glycerol pretreatment (15% $\text{v}\text{v}$ in mammalian Ringer's solution) at 0 °C and 22 °C (to regulate glycerol permeability); as well as parallel frozen-thawed samples, after combinations of slow (3.8 °C/min) freezing (SF) and rapid (38 °C/sec) freezing (RF) with either slow (1.5 °C/min) thawing (ST) or rapid (8 °C/sec) thawing (RT). Regimens compared were SFRT, SFST, RFRT, and RFST, all w and wo glycerol pretreatment at 0 °C and 22 °C. Tissue from each treatment was prepared for electron microscopic observations. The results on rates of freezing and thawing and relative cryoprotection of intracellular and extracellular glycerol under conditions described are intended to serve as a correlative basis for subsequent parallel studies on function (protein synthesis) and ultrastructure of the frozen state. They now indicate the following: (1) Cryoinjury of RER, which occurred during all treatments compared, was manifested in irregularity, dilatation, vesiculation, and altered matrix density of cisternae, and ribosomal derangement or disjunction. Least injury was shown by some disorientation and dilatation with increasing degrees of damage involving accentuation of these and other alterations. Such ultrastructural alterations to RER are not unique to cryoinjury, since they have been induced by treatments and agents other than freeze-thawing in experimental pathology. (2) Cryoinjury is unique, however, in that it can be regulated to demonstrate a spectrum of degrees of injury to cells and their organelles, immediately after cryoexposure. Controlled cryoinjury is suggested as a research tool for studies on injury, in general, on an ultrastructural-functional level. (3) Glycerol is injurious or toxic during pretreatment. Toxicity, which resembles cryoinjury, is greater during 22 ° C (intracellular) than 0 °C (extracellular) glycerol pretreatment, especially with respect to dilatation of cisternae. (4) Extra-cellular glycerol is cryoprotective during both slow and rapid freezing followed by either slow or rapid thawing, while little or no cryoprotection is afforded when glycerol is located simultaneously in the intracellular and extracellular location. (5) Rate of freezing is more important than rate of thawing as a factor in cryosurvival. Rapid freezing is more injurious than slow freezing, in the absence of glycerol or in the presence of extracellular glycerol, with slight or no differences seen as a function of thawing rate. Neither rate of freezing nor rate of thawing is of serious consequence when glycerol is intracellular. (6) Rate of thawing has importance after slow freezing, when slow thawing is more injurious than rapid, but not after rapid freezing, either in the presence or absence of extracellular glyeerol.     

Compartmentation of the rough endoplasmic reticulum

It has become evident during recent years that a wide variety of proteins are synthesized on membrane-bound polysomes, very many of which are not ultimately secreted from the cell. The majority of proteins appear to go through some form of post-translational modification before the final appearance of an 'active' product, and in some cases the polypeptide chain may be modified before the completed protein molecule is released from the ribosome. This then raises the question concerning the possibility of the organization of the rough endoplasmic reticulum into individual domains, or compartments, each of which may have the responsibility of performing definite and well defined functions. During recent years the behaviour of two subfractions of the rough endoplasmic reticulum in a variety of cell types and under a variety of conditions has been studied in order to gain insight into a possible compartmentation of this organelle. Throughout the studies disruption of cells has been performed by nitrogen cavitation. This technique was chosen in order to provide conditions of homogenization which were extremely reproducible since shearing forces, mechanical damage and the effects of local heating were eliminated. Endoplasmic reticulum (ER) membranes isolated from the post-mitochondrial supernatant have been separated into subfractions by centrifugation on discontinuous sucrose gradients. By virtue of their high density imparted by the association of ribosomes, rough ER (RER) membranes penetrate 1.4 M sucrose accumulating above either 2.0 M sucrose (light rough -LR membranes) or a cushion of 2.3 M sucrose (heavy rough -HR membranes). Smooth (S) membranes, which are virtually devoid of ribosomes, collect above 1.4 M sucrose. The HR, LR and S subfractions in MPC-11 cells differ in a number of respects: RNA/protein and RNA/phospholipid ratios, polysome profiles and marker enzymes. When cells were homogenized in buffer containing 25 mM KCl then all three ER subfractions were observed, however, when the buffer contained 100 mM KCl then only the LR and S subfractions were observed in gradients, radioactivity equivalent to that in the HR fraction was not recovered in the other two subfractions. Four times as many light chain immunoglobulin polypeptides were found associated with polysomes of HR membranes compared to LR membranes. The nuclear associated ER (NER), though very active in protein synthesis, was only 20% as active in the synthesis of light chain as the combined LR/HR fraction. Studies with MPC-11 cells showed that the relative amounts of the three ER subfractions were related to the phase of the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antibodies to the Golgi complex and the rough endoplasmic reticulum

Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endoplasmic reticulum (RER) by injection into the popliteal lymph nodes. The antisera were then tested by indirect immunofluorescence on tissue culture cells or frozen, thin sections of tissue. There were may unwanted antibodies to cell components other than the RER or the Golgi complex, and these were removed by suitable absorption steps. These steps were carried out until the pattern of fluorescent labeling was that expected for the Golgi complex or RER. Electron microscopic studies, using immunoperoxidase labeling of normal rat kidney (NRK) cells, showed that the anti-Golgi antibodies labeled the stacks of flattened cisternae that comprise the central feature of the Golgi complex, many of the smooth vesicles around the stacks, and a few coated vesicles. These antibodies were directed, almost entirely, against a single polypeptide with an apparent molecular weight of 135,000. The endoplasmic reticulum (ER) in NRK cells is an extensive, reticular network that pervades the entire cell cytoplasm and includes the nuclear membrane. The anit-RER antibodies labeled this structure alone at the light and electron microscopic levels. They were largely directed against four polypeptides with apparent molecular weights of 29,000, 58,000, 66,000, and 91,000. Some examples are presented, using immunofluorescence microscopy, where these antibodies have been used to study the Golgi complex and RER under a variety of physiological and experimental condition . For biochemical studies, these antibodies should prove useful in identifying the origin of isolated membranes, particularly those from organelles such as the Golgi complex, which tend to lose their characteristic morphology during isolation.     

Heparin releases monosomes and polysomes from rough endoplasmic reticulum

Incubation of membrane-bound polyribosomes isolated from murine myeloma cells with heparin caused release of material which sedimented in the polysome, monosome and ribosomal subunit regions of linear sucrose gradients. The released material corresponded to approximately one half that which could be released by treatment with heparin plus Triton X-100. The action of heparin appeared to be related to its polyanionic nature. The use of heparin as a ribonuclease inhibitor in the separation and isolation of free and membrane-bound polysomes could cause artificial accumulation of detached polysomes in the free polysome fraction.     

Contribution of the endoplasmic reticulum to peroxisome formation

How peroxisomes are formed in eukaryotic cells is unknown but important for insight into a variety of diseases. Both human and yeast cells lacking peroxisomes due to mutations in PEX3 or PEX19 genes regenerate the organelles upon reintroduction of the corresponding wild-type version. To evaluate how and from where new peroxisomes are formed, we followed the trafficking route of newly made YFP-tagged Pex3 and Pex19 proteins by real-time fluorescence microscopy in Saccharomyces cerevisiae. Remarkably, Pex3 (an integral membrane protein) could first be observed in the endoplasmic reticulum (ER), where it concentrates in foci that then bud off in a Pex19-dependent manner and mature into fully functional peroxisomes. Pex19 (a farnesylated, mostly cytosolic protein) enriches first at the Pex3 foci on the ER and then on the maturing peroxisomes. This trafficking route of Pex3-YFP is the same in wild-type cells. These results demonstrate that peroxisomes are generated from domains in the ER.     

Domains of rough endoplasmic reticulum (a review)

The rough endoplasmic reticulum isolated from several eukaryotic cell lines can be separated into subfractions. These subfractions possess different properties indicating that they represent separate domains of the endoplasmic reticulum system.     

Topography of glycosylation reactions in the rough endoplasmic reticulum membrane

The translocation of UDP-glucose and GDP-mannose from an external to a luminal compartment has been examined in rat liver vesicles derived from the rough endoplasmic reticulum (RER). RER vesicles with the same topographical orientation as in vivo were incubated with a mixture of [3H]UDP-glucose and UDP-[14C]glucose to demonstrate that the intact sugar nucleotide was translocated into the lumen of the vesicles. The translocation of UDP-glucose was dependent on temperature and was saturable at high concentrations of the sugar nucleotide. The transfer of glucose to endogenous acceptors was dependent on the translocation of UDP-glucose into the lumen of the RER since leaky vesicles resulted in both a decrease in transport and transfer of glucose to endogenous acceptors. Preliminary results suggest that the mechanism of UDP-glucose transport into RER-derived vesicles is via a coupled exchange with luminal UMP. Using the same experimental approach to detect translocation of UDP-glucose into the lumen of RER vesicles, we were unable to detect transport of GDP-mannose. Incubation of leaky vesicles with GDP-mannose resulted in stimulation of the amount of mannose transferred to endogenous acceptors, in marked contrast to that observed for UDP-glucose and UDP-N-acetylglucosamine. These results suggest that whereas UDP-glucose is translocated across the RER membrane in vitro, GDP-mannose is not transported. In addition, these results tentatively suggest that there is asymmetric synthesis of the lipid-linked oligosaccharides within the membrane of the RER.     

Alpha-COPI coatomer protein is required for rough endoplasmic reticulum whorl formation in mosquito midgut epithelial cells

Background

One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.

Methodology/Principal Findings

Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta''), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.

Conclusions

alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases.     

Cyclic AMP-dependent protein kinase in canine pancreatic rough endoplasmic reticulum

A canine pancreas homogenate was subfractionated by several differential centrifugation steps. The distribution of cAMP-dependent protein kinase in the various fractions was monitored by assaying [3H]cAMP binding and photo-cross-linking of the regulatory subunits of the enzyme (RI and RII) with radiolabeled 8-azido-cAMP. The distribution of the kinase was also compared to that of markers for the plasma membrane, the endoplasmic reticulum and the cytosol. While our results confirm previous studies suggesting the presence of cyclic AMP-dependent protein kinase in the cytosol and Golgi, a significant amount of the total [3H] cAMP binding and photolabeled R subunits (both RI and RII) were found in rough microsomes (RM). The association is relatively resistant to extraction with EDTA, low and high ionic strength solutions. These extractions unmasked several new phosphorylation substrates in the "stripped" RM that were inaccessible in the RM, possibly because they were covered by ribosomes or peripheral membrane proteins. RII with a molecular mass of 52 kDa (RII-52 kDa) was the predominant RII found in the cytosolic fraction, whereas RII-52 kDa and RII with a molecular mass of 54 kDa (RII-54 kDa) were approximately equally enriched in the RM fraction. The mobility of the RII-52 kDa-photolabeled band could be shifted to the mobility of the RII-54 kDa band by phosphorylation with purified catalytic subunit and ATP, indicating that they represent "dephospho" and "phospho" forms of RII, respectively. A more precise localization to the rough endoplasmic reticulum was accomplished by isopycnic floatation in sucrose gradients. The enzyme cobanded at the density of rough microsomes and shifted to the lower density of "stripped" microsomes after treatment with puromycin/high salt, which specifically removes ribosomes.     

Protein fatty acyltransferase is located in the rough endoplasmic reticulum

The fatty acid acylation of polypeptides was studied in vivo and in vitro by incorporation of radiolabeled palmitic acid into Semliki Forest viral polypeptides. Utilizing a cell-free system for acylation protein fatty acyltransferase was characterized as an integral membrane protein. No acylation activity was detected in the cytosol. During subcellular fractionation of a variety of mammalian or avian cells the enzyme was localized to the rough endoplasmic reticulum. Therefore this posttranslational hydrophobic modification starts earlier in the biosynthesis of acylated polypeptides than previously believed.     

Targeting of rough endoplasmic reticulum membrane proteins and ribosomes in invertebrate neurons

The endoplasmic reticulum (ER) is divided into rough and smooth domains (RER and SER). The two domains share most proteins, but RER is enriched in some membrane proteins by an unknown mechanism. We studied RER protein targeting by expressing fluorescent protein fusions to ER membrane proteins in Caenorhabditis elegans. In several cell types RER and general ER proteins colocalized, but in neurons RER proteins were concentrated in the cell body, whereas general ER proteins were also found in neurites. Surprisingly RER membrane proteins diffused rapidly within the cell body, indicating they are not localized by immobilization. Ribosomes were also concentrated in the cell body, suggesting they may be in part responsible for targeting RER membrane proteins.     

Accumulation of unglycosylated liver secretory glycoproteins in the rough endoplasmic reticulum

Previous studies in this laboratory (1) have shown that tunicamycin-treatment inhibits the secretion of three secretory glycoproteins--alpha 2-macroglobulin, ceruloplasmin, and alpha 1-protease inhibitor in human hepatoma (Hep G2) cell cultures. In the present study, we have investigated (i) their site of accumulation within the endoplasmic reticulum/Golgi pathway, and (ii) the solubility characteristics of these unglycosylated proteins. Using percoll density gradient centrifugation, we found that tunicamycin-treatment markedly inhibited the transport of alpha 2-macroglobulin, ceruloplasmin and alpha 1-protease inhibitor from the rough endoplasmic reticulum. However, there was no detectable changes in their solubility properties as both the glycosylated and unglycosylated species were associated with the 100,000 xg supernatant fraction following disruption of the microsomal fraction (i) with 0.2% Triton X-100 and (ii) by repeated freeze-thaw cycles. Also no evidence of protein aggregation was detected by liquid chromatography of the unglycosylated proteins on Bio-Gel A-1.5 column.     

Intracellular polycationic molecules cause reversible swelling of the rough endoplasmic reticulum

The microinjection of polycationic but not anionic molecules causes swelling of the rough endoplasmic reticulum (RER) in salivary gland cells of a fly larva. Ca-EGTA buffers, lanthanum chloride, lysozyme, bovine serum albumin, cationic and anionic ferritin were microinjected into salivary gland cells and their effects observed by light and electron microscopy. Immediately after the microinjection of polycationic molecules, the cytoplasm changed from transparent to opaque as the RER became swollen. Binding of polycationic molecules to the RER may cause the membrane to become permeable to some solute and swell due to osmotic forces.     

Proteomics analysis of rough endoplasmic reticulum in pancreatic beta cells

Pancreatic beta cells have well‐developed ER to accommodate for the massive production and secretion of insulin. ER homeostasis is vital for normal beta cell function. Perturbation of ER homeostasis contributes to beta cell dysfunction in both type 1 and type 2 diabetes. To systematically identify the molecular machinery responsible for proinsulin biogenesis and maintenance of beta cell ER homeostasis, a widely used mouse pancreatic beta cell line, MIN6 cell was used to purify rough ER. Two different purification schemes were utilized. In each experiment, the ER pellets were solubilized and analyzed by 1D SDS‐PAGE coupled with HPLC‐MS/MS. A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments and 737 proteins found in all three experiments. GO analysis revealed a comprehensive profile of known and novel players responsible for proinsulin biogenesis and ER homeostasis. Further bioinformatics analysis also identified potential beta cell specific ER proteins as well as ER proteins present in the risk genetic loci of type 2 diabetes. This dataset defines a molecular environment in the ER for proinsulin synthesis, folding and export and laid a solid foundation for further characterizations of altered ER homeostasis under diabetes‐causing conditions. All MS data have been deposited in the ProteomeXchange with identifier PXD001081 ( http://proteomecentral.proteomexchange.org/dataset/PXD001081 ).