The secretion of Mullerian inhibiting substance by cultured isolated Sertoli cells of the neonatal calf

Sertoli cells have been insolated from the newborn calf testis using a combination of mechanical and enzymatic disruption. Testicular fragments, previously chopped into 1-mm pieces, are digested in an enzyme mixture consisting of hyaluronidase, collagenase, trypsin and DNAse, followed by a second digestion in trypsin and DNAse. Isolation of the resulting cellular fractions by sedimentation with unit gravity produces an aliquot of Sertoli cells which is over 95% pure when examined by light and electron microscopy. Cultures of these cells grow rapidly and produce Mullerian Inhibiting Substance as evidenced by their ability to cause the involution of the Mullerian duct of the female fetal rat when co-cultured in an organ-culture assay system.     

Extraction of Mullerian inhibiting substance from newborn calf testis.

Using a dissociative solvent and a protease inhibitor, Mullerian inhibiting substance, a testicular substance responsible for regression of the Mullerian ducts in the mammalian male embryo, has been extracted from newborn calf testis. Data are presented which demonstrate that fractions with biological activity for Mullerian inhibiting substance can be extracted from whole tissue and that activity can be blocked by antisera raised to extracted testes components. Following extraction in guanidine hydrochloride the extract was fractionated by density gradient sedimentation, gel filtration chromatography, and ion-exchange chromatography. Fractions were subjected to amino acid and carbohydrate analyses and peptide constituents were determined by SDS gel electrophoresis. Fractions were dialyzed, concentrated, filtered, and added to an organ culture assay to detect Mullerian inhibiting substance activity, which was found (1) in the guanidine extract, (2) in a protein fraction of the cesium chloride gradient, (3) in constituents eluted with K_av values between 0.19 and 0.38 on gel filtration chromatography using a Bio-Gel A-0.5 M column, and (4) in constituents eluted between 0.15 and 0.20 M NaCl on ion-exchange chromatography using a DEAE Bio-Gel A-50 ion exchanger. Sequentially this scheme effected a 30-fold purification of a fraction with Mullerian inhibiting substance activity. Biological activity was lost when positive extracts were absorbed with antiserum raised to guanidine extract. The strong dissociative conditions employed in the gradient and extraction procedures make it likely that the distribution of activity obtained in the density gradient procedure was due to a macromolecule, and not to an interaction between an active low molecular weight component and an inactive macromolecule acting as a carrier. Further fractionation on the Bio-Gel column using a dissociative solvent also indicated that the active component was a macromolecule. Amino acid and carbohydrate analyses indicate that the active fractions are composed of proteins and glycoproteins.     

The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.     

Inheritance of the endoplasmic reticulum and Golgi apparatus

Yeast and mammalian cells use a variety of different mechanisms to ensure that the endoplasmic reticulum and Golgi apparatus are inherited by both daughter cells on cell division. In yeast, endoplasmic reticulum inheritance involves both active microtubule and passive actin-based mechanisms, while the Golgi is transported into the forming daughter cell by an active actin-based mechanism. Animal cells actively partition the endoplasmic reticulum and Golgi apparatus, but association with the mitotic spindle-rather than the actin cytoskeleton-appears to be the mechanism     

Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.     

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.     

Immunocytochemical localization of phaseolin and phytohemagglutinin in the endoplasmic reticulum and Golgi complex of developing bean cotyledons

Development of legume seeds is accompanied by the synthesis of storage proteins and lectins, and the deposition of these proteins in protein-storage vacuoles (protein bodies). We examined the subcellular distribution, in developing seeds of the common bean, Phaseolus vulgaris L., of the major storage protein (phaseolin) and the major lectin (phytohemagglutinin, PHA). The proteins were localized using an indirect immunocytochemical method in which ultrathin frozen sections were immunolabeled with rabbit antibodies specific for either PHA or phaseolin. Bound antibodies were then localized using goat-anti-rabbit immunoglobulin G adsorbed onto 4- to 5-nm colloidal gold particles. The sections were post-fixed with OsO₄, dehydrated, and embedded in plastic on the grids. Both PHA and phaseolin exhibited a similar distribution in the storage-parenchyma cells, being found primarily in the developing protein bodies. Endoplasmic reticulum and Golgi complexes (cisternal stacks and associated vesicles) also were specifically labeled for both proteins, whereas the cytosol and other organelles, such as mitochondria, were not. We interpret these observations as supporting the hypothesis that the transport of storage proteins and lectins from their site of synthesis, the rough endoplasmic reticulum, to their site of deposition, the protein bodies, is mediated by the Golgi complex.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - PBS phosphate-buffered saline - PHA phytohemagglutinin     

Bidirectional membrane traffic between the endoplasmic reticulum and Golgi apparatus

Membrane traffic between the endoplasmic reticulum and Golgi apparatus is a highly regulated process that uses distinct anterograde and retrograde pathways. These pathways link two organelles that together function as a dynamic membrane system specialized for the biosynthesis and sorting of membrane to be used throughout the cell. The nature and underlying biochemical control of membrane transport along these pathways is thought to be tied to a common regulatory system involving assembly and disassembly of cytosolic proteins on membranes.     

Immunocytochemical localization of native chondroitin-sulfate in tissues and cultured cells using specific monoclonal antibody

Chondroitin-sulfate containing proteoglycan (CSPG) of the extracellular matrix (ECM) was visualized in chick tissues and cell cultures with a monoclonal antibody, CS-56. Cultured cells of various origins contained dense punctate layers of CSPG on both the substrate and the cell surface, as determined by immunofluorescent and immunogold staining. Under culture conditions the CSPG-containing matrix was usually excluded from stable cell-to-substrate focal contacts. The substrate-attached CSPG exhibited remarkable chemical stability but could be successfully removed by pronase or chondroitinases ABC and AC. Incubation of living cells with CS-56 antibodies resulted in the clustering of surface CSPG into patches, indicating that the surface-bound CSPG is free to move laterally along the plasma membrane. The unique properties of the CSPG-containing ECM revealed by CS-56 antibodies and their relationships to specific types of cell contacts are discussed.     

Dynamic nucleation of Golgi apparatus assembly from the endoplasmic reticulum in interphase hela cells

Models of Golgi apparatus biogenesis and maintenance are focused on two possibilities: one is self-assembly from the endoplasmic reticulum, and the other is nucleation by a stable template. Here, we asked in three different experimental situations whether assembly of the Golgi apparatus might be dynamically nucleated. During microtubule depolymerization, the integral membrane protein p27 and the peripheral Golgi protein GM130, appeared in newly formed, scattered Golgi elements before three different Golgi apparatus cisternal enzymes, whereas GRASP55, a medial peripheral Golgi protein, showed, if anything, a tendency to accumulate in scattered Golgi elements later than a cisternal enzyme. During Golgi formation after brefeldin A washout, endoplasmic reticulum exit of Golgi resident enzymes could be completely separated from that of p27 and GM130. p27 and GM130 accumulation was onto newly organized perinuclear structures, not brefeldin A remnants, and preceded that of a cisternal enzyme. Reassembly was completely sensitive to guanosine 5'-diphosphate-restricted Sar1p. When cells were microinjected with Sar1pWT DNA to reverse a guanosine 5'-diphosphate-restricted Sar1p endoplasmic reticulum-exit block phenotype, GM130 and p27 collected perinuclearly with little to no exit of a cisternal enzyme from the endoplasmic reticulum. The overall data strongly indicate that the assembly of the Golgi apparatus can be nucleated dynamically by GM130/p27 associated structures. We define dynamic nucleation as the first step in a staged organelle assembly process in which new component association forms a microscopically visible structure onto which other components add later, e.g. Golgi cisternae.     

Mutations blocking the transport of the influenza virus hemagglutinin between the rough endoplasmic reticulum and the Golgi apparatus.

Mutants ts1 and ts227 of fowl plague virus have a temperature-sensitive defect in the transport of the hemagglutinin from the rough endoplasmic reticulum to the Golgi apparatus. The primary structure of the hemagglutinin of the mutants and of a number of revertants derived from them has been analysed by nucleotide sequencing. The transport block of the hemagglutinin of ts227 can be attributed to a single amino acid exchange. It involves the replacement of aspartic acid at position 457 by asparagine thereby introducing a new glycosylation site which appears to be located in a cryptic position in the lower part of the hemagglutinin stalk. Attachment of carbohydrate to this site is temperature-dependent. At permissive temperature only a small fraction of the monomers (approximately 30%) is glycosylated in this position, whereas at nonpermissive temperature this is the case with all subunits. The data suggest that under the latter conditions the new oligosaccharide interferes by steric hindrance with the trimerization of the hemagglutinin. The hemagglutinin of ts1 has an essential amino acid exchange at position 275 where serine is replaced by glycine. This substitution may increase the flexibility of the molecule in the hinge region between the globular domain and the stalk. The exchange of a conserved glutamic acid residue at position 398 that is involved in the interaction between different monomers contributes also to the structural instability of the ts1 hemagglutinin. These observations support the notion that the transport of the hemagglutinin from the rough endoplasmic reticulum to the Golgi apparatus depends on trimer assembly.     

Crossing the divide--transport between the endoplasmic reticulum and Golgi apparatus in plants

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.     

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune‐dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species‐dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA‐dependent secretory pathway. This led to a general inhibition of protein secretion by PDT‐treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin‐based PDT. Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro‐apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.     

Immunocytochemical localization of BiP to the rough endoplasmic reticulum: evidence for protein sorting by selective retention

Immunoglobulin heavy chain binding protein (BiP) (also known as GRP 78) is a protein of the endoplasmic reticulum (ER) which has been shown to be involved in post-translational processing of nascent membrane and secretory proteins. To determine BiP's location in the exocytic pathway, we localized BiP at the electron microscopic level in mouse myeloma cell lines by immunoperoxidase cytochemistry. BiP was found to be present within the cisternal spaces of the RER and nuclear envelope but was not detected in the cisternae of the Golgi complex. BiP reaction product was also found within transitional elements of the RER but was absent from smooth-surfaced vesicles found between the ER and the Golgi complex. Immunoperoxidase staining of BiP was reduced or absent in regions of a smooth ER membrane system in myelomas that contained endogenous murine retrovirus A particles. All compartments of the exocytic pathway, including the virus-containing smooth ER, stained for IgG, a secretory protein. These observations suggest that BiP is selectively retained in the cisternae of the ER and is not free to enter Golgi-directed transport vesicles. These studies suggest that BiP's subcellular localization may occur by selective interaction with component(s) of the ER.     

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.     

Traffic between the plant endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond

Significant advances have been made in recent years that have increased our understanding of the trafficking to and from membranes that are functionally linked to the Golgi apparatus in plants. New routes from the Golgi to organelles outside the secretory pathway are now being identified, revealing the importance of the Golgi apparatus as a major sorting station in the plant cell. This review discusses our current perception of Golgi structure and organization as well as the molecular mechanisms that direct traffic in and out of the Golgi.     

The Golgi apparatus maintains its organization independent of the endoplasmic reticulum

Under artificial conditions Golgi enzymes have the capacity to rapidly accumulate in the endoplasmic reticulum (ER). These observations prompted the idea that Golgi enzymes constitutively recycle through the ER. We have tested this hypothesis under physiological conditions through use of a procedure that captures Golgi enzymes in the ER. In the presence of rapamycin, which induces a tight association between FKBP (FK506-binding protein) and FRAP (FKBP-rapamycin-associated protein), an FKBP-tagged Golgi enzyme can be trapped when it visits the ER by an ER-retained protein fused to FRAP. We find that although FKBP-ERGIC-53 of the ER-Golgi intermediate compartment (ERGIC) rapidly cycles through the ER (30 min), FKBP-Golgi enzyme chimeras remain stably associated with Golgi membranes. We also demonstrate that Golgi dispersion upon nocodazole treatment mainly occurs through a mechanism that does not involve the recycling of Golgi membranes through the ER. Our findings suggest that the Golgi apparatus, as defined by its collection of resident enzymes, exists independent of the ER.     

Studies on the mechanism of different collagen glucosyltransferase reactions (Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum) in chick embryo liver.

1. The galactosylhydroxylysylglucosyltransferase (GGT) specific to collagen is located in the RER (rough endoplasmic reticulum), SER (smooth endoplasmic reticulum) and Golgi apparatus for the chick embryo liver. 2. The UDP-glucose collagen glucosyltransferase activities in chick embryo liver were solubilized by Nonidet P-40. 3. The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparation of Golgi apparatus CF2, smooth endoplasmic reticulum CF4 and rough endoplasmic reticulum CF8. 4. For the three fractions, data obtained in experiments were consistent with a sequential ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, collagen and UDP-glucose substrate, with different values for Km and Vmax.     

Anterograde and retrograde traffic between the rough endoplasmic reticulum and the Golgi complex

The transfer of newly synthesized membrane proteins moving from the rough endoplasmic reticulum (RER) to the Golgi complex has been studied by electron microscopy in HEp-2 cells transfected with cDNAs for chimeric proteins. These proteins consist of a reporter enzyme, horseradish peroxidase (HRP), anchored to the transmembrane domains of two integral membrane proteins, the transferrin receptor and sialyl- transferase. The chimeras are distributed throughout the nuclear envelope, RER, vesicular tubular clusters (VTCs) and a network of tubules in the cis-Golgi area. At 20 degrees C tubules containing chimera connect the RER to the VTCs and to the cis-Golgi network. On transfer to 37 degrees C in the presence of dithiothreitol (DTT), the chimeras are seen to move from the RER and through the Golgi stack. With this temperature shift the direct connections with the RER are lost and free vesicles form; some of these vesicles contain HRP reaction product which is much more concentrated than in the adjacent RER while others lack reaction product entirely. In cells expressing SSHRPKDEL, DAB reaction product remains distributed throughout the RER, the VTCs, and the cis-Golgi network for prolonged periods in the presence of DTT and almost all of the vesicles which form at 37 degrees C are DAB-positive. Together these observations demonstrate that all three chimeras are transported from the RER to the cis-Golgi in free, 40-60-nm vesicles at 37 degrees C. They also suggest that the retrograde traffic which carries SSHRPKDEL back to the RER is probably mediated by vesicles with a similar morphology but which, in cells expressing membrane-anchored chimeras, lack detectable reaction product.