Is thiamine pyrophosphatase a reliable marker of the neuronal Golgi apparatus? A critical analysis

The thiamine pyrophosphatase histochemical technique is believed to be a suitable approach to the selective staining of the Golgi vesicles of all animal cells, icluding the neuron. However, during the past decade a considerable number of data have been published, suggesting that the TPPase is in general a membrane-associated enzyme in the brain, which can be found in subcellular fractions other than Golgi lamellae. It has therefore become necessary to reconsider the view that thiamine pyrophosphatase is an exclusive marker enzyme of the Golgi apparatus.     

The Golgi body and thiamine pyrophosphatase localization in embryonic cells in culture

Chick embryo endoblast and segmented mesoderm cells were cultured and processed using the cytochemical technique for the detection of the enzyme thiamine pyrophosphatase. Reaction product was found in cisternae of the Golgi body and associated vesicles. Random, patchy distribution of deposit was observed on the dorsal surface of the monolayer cells and between overlapping processes, but not on the ventral surface which faces the substratum. Both cell types reacted similarly. The results suggest that new membrane of Golgi origin is formed at the dorsal but not the ventral surface of cells. There was no defined locus of insertion, at the tip of the advancing lamellipodia or elsewhere.     

Spatial distribution of thiamine pyrophosphatase and arylsulfatase activities in the area of the Golgi apparatus of the adenohypophyseal secretory cells in the white rat

Specimens of albino rat pituitary glands were processed consecutively for demonstration of thiamine pyrophosphatase (TPPase) and aryl sulfatase (ArSase) activities. To differentiate between the structures associated with each particular enzyme studied within a single cell, either somatotroph or mammotroph, ultrathin sections were exposed for 2 minutes to 2--50% H2SO4 which removed the reaction products for TPPase rather than those for ArSase. The comparative study of pairs of micrographs of the same area taken before and after the etching with H2SO4 has shown TPPase and ArSase to reside in the Golgi apparatus and GERL system, respectively. Transitional elements have also been discovered, thus supporting the idea that GERL may be a derivative of the Golgi apparatus.     

Golgi complex is brefeldin A resistant in multidrug resistant cells.

The multidrug resistance (MDR) is one of the main reasons for chemotherapeutic failures in cancer patients. The overexpression of mdr1 gene product, P-glycoprotein (Pgp), leads to the appearance of resistant tumor cells. In the previous paper (Erokhina, 1997) we have demonstrated that the first stages of Pgp-mediated MDR are accompanied by the reorganization of cytoskeleton elements and the vacuolar system. These data were true for two independently isolated sublines of Syrian hamster embryo fibroblasts transformed by Raus sarcoma virus. In this study, we continued the investigation of the properties of the vacuolar system in Pgp-expressing cells. Brefeldin A (BFA), which is not a Pgp substrate, affects different elements of the vacuolar system and blocks vesicular transport. Our data demonstrate that BFA has different effects on parental and resistant cells. In parental cells, the Golgi apparatus and vesicular transport are sensitive to BFA, while in resistant sublines, BFA affects the vesicular transport but not the Golgi apparatus structure. We discuss the existence of similar and different BFA targets in parental and resistant cells and their role in the evolution of multidrug resistance mechanisms.     

A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes

Summary The use of cerium chloride for the localization of thiamine-pyrophosphatase (TPPase) in rat liver parenchymal cells has been investigated and the results are compared with the classical lead capture method. A medium containing 3 mM cerium chloride gave the most uniform and consistent results with a homogenous electron dense reaction product in the first trans lamella of the Golgi complex and a weak staining of endoplasmic reticulum. The fine deposits of cerium phosphate filled completely the first trans Golgi cisterna. In contrast the reaction product of the lead-based method appeared clumpy and aggregated with an irregular distribution over both Golgi complex and endoplasmic reticulum. Higher and lower concentrations of cerium chloride than 3 mM gave inconsistent results. The present study demonstrates that the cerium-based method is superior to the classical lead-technique for the localization of TPPase.     

The distribution of secretory products in plant cells is affected by brefeldin A

The patterns of redistribution of two classes of Golgi derived cell surface antigens recognised by monoclonal antibodies JIM1 and JIM7, after the treatment of roots with Brefeldin A (BFA) are described. The results for these secretory products are compared with those previously reported for Golgi membrane epitopes recognised by JIM 84. The preliminary results described here demonstrate that the combination of immunocytochemical techniques with drug induced perturbation of secretory pathways will be invaluable in enhancing our understanding of the pathways of intracellular trafficking in plants.     

A cytochemical study of acid phosphatase,thiamine pyrophosphatase and horseradish peroxidase in the Golgi-GERL complex of hepatoma ascites cells

Data from studies of ascitic cells of Chang hepatoma have shown that acid phosphatase (ACPase) can be localized simultaneously within the trans portion of the Golgi apparatus and in tubules of the Golgi-endoplasmic reticulum-lysosome (GERL) system. Reaction products of thiamine pyrophosphatase (TPPase) were also present consistently within trans elements of the Golgi apparatus and within GERL tubules. These new findings indicate that a close physiological association may exist between the Golgi apparatus and GERL, a concept that is consistent with previous observations of fibroblasts. When horseradish peroxidase (PO) is injected intraperitoneally into ascites-bearing rats and the ascitic cells withdrawn at different time intervals, PO could be localized within vesicles and tubules in the GERL region but could not be detected within the Golgi apparatus. Bulk-phase endocytosis requires a long time and a high concentration of PO to occur. The presence of PO within GERL indicates that this organelle may play a role in transporting or processing of certain exogenous proteins.     

Glucocorticoid-regulated localization of cell surface glycoproteins in rat hepatoma cells is mediated within the Golgi complex

Glucocorticoid hormones regulate the post-translational maturation and sorting of cell surface and extracellular mouse mammary tumor virus (MMTV) glycoproteins in M1.54 cells, a stably infected rat hepatoma cell line. Exposure to monensin significantly reduced the proteolytic maturation and externalization of viral glycoproteins resulting in a stable cellular accumulation of a single 70,000-Mr glycosylated polyprotein (designated gp70). Cell surface- and intracellular-specific immunoprecipitations of monensin-treated cells revealed that gp70 can be localized to the cell surface only in the presence of 1 microM dexamethasone, while in uninduced cells gp70 is irreversibly sequestered in an intracellular compartment. Analysis of oligosaccharide processing kinetics demonstrated that gp70 acquired resistance to endoglycosidase H with a half-time of 65 min in the presence or absence of hormone. In contrast, gp70 was inefficiently galactosylated after a 60-min lag in uninduced cells while rapidly acquiring this carbohydrate modification in the presence of dexamethasone. Furthermore, in the absence or presence of monensin, MMTV glycoproteins failed to be galactosylated in hormone-induced CR4 cells, a complement-selected sorting variant defective in the glucocorticoid-regulated compartmentalization of viral glycoproteins to the cell surface. Since dexamethasone had no apparent global effects on organelle morphology or production of total cell surface-galactosylated species, we conclude that glucocorticoids induce the localization of cell surface MMTV glycoproteins by regulating a highly selective step within the Golgi apparatus after the acquisition of endoglycosidase H-resistant oligosaccharide side chains but before or at the site of galactose attachment.     

A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes. Comparison with the lead technique

The use of cerium chloride for the localization of thiamine-pyrophosphatase (TPPase) in rat liver parenchymal cells has been investigated and the results are compared with the classical lead capture method. A medium containing 3 mM cerium chloride gave the most uniform and consistent results with a homogeneous electron dense reaction product in the first trans lamella of the Golgi complex and a weak staining of endoplasmic reticulum. The fine deposits of cerium phosphate filled completely the first trans Golgi cisterna. In contrast the reaction product of the lead-based method appeared clumpy and aggregated with an irregular distribution over both Golgi complex and endoplasmic reticulum. Higher and lower concentrations of cerium chloride than 3 mM gave inconsistent results. The present study demonstrates that the cerium-based method is superior to the classical lead-technique for the localization of TPPase.     

Uptake of a fluorescent marker in plant cells is sensitive to brefeldin A and wortmannin

We assessed FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl)pyridinium dibromide] as a fluorescent endocytosis marker in intact, walled plant cells. At 4 degrees C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26 degrees C, FM1-43 labeled cytoplasmic vesicles and then the vacuole. Fluorimetric quantitation demonstrated dye uptake temperature sensitivity (approximately 65% reduction at 16 degrees C, >90% at 4 degrees C). FM1-43 uptake in suspension cells was stimulated more than twofold by brefeldin A and inhibited approximately 0.4-fold by wortmannin. FM1-43 delivery to the vacuole was largely inhibited by brefeldin A, although overall uptake was stimulated, and brefeldin A treatment caused the accumulation of large prevacuolar endosomal vesicles heavily labeled with FM1-43. Three-dimensional time lapse imaging revealed that FM1-43-labeled vacuoles and vesicles are highly dynamic. Thus, FM1-43 serves as a fluorescent marker for imaging and quantifying membrane endocytosis in intact plant cells.     

The occurrence of the Golgi apparatus in mouse oocytes. Demonstration of thiamine pyrophosphatase activity

Location of thiamine pyrophosphatase activity as a marker of the Golgi apparatus was studied ultracytochemically in mouse oocytes with germinal vesicle (OGV), oocytes at metaphase I (OMI) and oocytes at metaphase II (OMII), and further in cells of the respective cumulus oophorus serving as comparative objects. TPPase activity in cumulus oophorus cells and in OGV was found exclusively in the Golgi apparatus. In OMI the reaction product of TPPase activity was observed in isolated smooth vesicles, and in only one case in structures identifiable as the Golgi apparatus. In OMII the occurrence of TPPase activity was also recorded in isolated smooth vesicles in cortical cytoplasm and further, exceptionally, in smooth concentrically arranged vesicles or tubules. The TPPase activity was not present in vesicular complexes. The results have shown that after the resumption of meiosis the occurrence of the reaction product of TPPase activity drops abruptly due to the reduction of the Golgi apparatus. Changes affecting the Golgi apparatus after the resumption of meiosis are related to the loss of the nucleus after the germinal vesicle breakdown.     

Sulfation in the Golgi lumen of Madin-Darby canine kidney cells is inhibited by brefeldin A and depends on a factor present in the cytoplasm and on Golgi membranes

Madin-Darby canine kidney cells are more resistant than most other cell types to the classical effects of brefeldin A (BFA) treatment, the induction of retrograde transport of Golgi cisternae components to the endoplasmic reticulum. Here we show that sulfation of heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), and proteins in the Golgi apparatus is dramatically reduced by low concentrations of BFA in which Golgi morphology is unaffected and secretion still takes place. BFA treatment seems to reduce sulfation by inhibition of the uptake of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) into the Golgi lumen, and the inhibitory effect of BFA was similar for HSPGs, CSPGs, and proteins. This was different from the effect of chlorate, a well known inhibitor of PAPS synthesis in the cytoplasm. Low concentrations of chlorate (2-5 mm) inhibited sulfation of CSPGs and proteins only, whereas higher concentrations (15-30 mm) were required to inhibit sulfation of HSPGs. Golgi fractions pretreated with BFA had a reduced capacity for the synthesis of glycosaminoglycans (GAGs), but control level capacity could be restored by the addition of cytosol from various sources. This indicates that the PAPS pathway to the Golgi lumen depends on a BFA-sensitive factor that is present both on Golgi membranes and in the cytoplasm.     

The thiamine pyrophosphatase technique as an indicator of the morphology of the Golgi apparatus in the neurons

Summary Detailed studies have been made on the morphology of the TPPase-positive material in various components of the cerebellum. The various types of nerve fibers in various layers of the cerebellum and the glomeruli are free of any TPPase-positive Golgi material. The stellate and basket cells have a thick plate-like Golgi complex. The Purkinje cells showed either a Golgi network or discrete masses of TPPase-positive material of various sizes and shapes. The Bergman glial cells showed a delicate Golgi network. The Golgi type II cells showed a thick or thin TPPase-positive network. The granule cells showed only discrete vesicles, granules, and comma-shaped masses found at any one pole of each cell. The cells of the deep cerebellar nuclei also showed a network. The results of this study are compared with similar studies made on the spinal cord, olfactory bulb, Ammon's horn, fascia dentata, cerebral cortex, and ganglion cells.     

Structural integrity of the Golgi stack is essential for normal secretory functions of rat parotid acinar cells: effects of brefeldin A and okadaic acid.

We examined the effects of specific inhibitors, brefeldin A (BFA) and okadaic acid (OA), on the ultrastructural organization of the Golgi apparatus and distributions of amylase, Golgi-associated proteins, and cathepsin D in the rat parotid acinar cells. BFA induced a rapid regression of the Golgi stack into rudimentary Golgi clusters composed of tubulovesicules, in parallel with a redistribution of the Golgi-resident proteins and a coat protein (beta-COP) into the region of the rough endoplasmic reticulum (rER) or cytosol. The rapid disruption of the Golgi stack could also be induced by the effect of OA. However, redistribution of the Golgi proteins in rER or cytosol could not be observed and beta-COP was not dispersed but was retained on the rudimentary Golgi apparatus. These findings suggested that the mechanism of OA in inducing degeneration of the Golgi stack was markedly different from that of BFA. In addition, missorting of amylase, a Golgi protein, and cathepsin D into incorrect transport pathways is apparent in the course of the disruption of the Golgi stack by OA. These Golgi-disrupting effects are reversible and the reconstruction of the stacked structure of the Golgi apparatus started immediately after the removal of inhibitors. In the recovery processes, missorting was also observed until the integrated structure of the Golgi apparatus was completely reconstructed. This suggested that the integrated structure of the Golgi apparatus was quite necessary for the occurrence of normal secretory events, including proper sorting of molecules.     

The localization of thiamine pyrophosphatase activity in the acinar cells of stimulated and non-stimulated sublingual glands of the rat

Summary The distribution of thiamine pyrophosphatase (TPPase) activity in the acinar cells of the rat sublingual gland has been studied at various stages of the secretory cycle following stimulated secretion. The rats were stimulated to secrete by an intraperitoneal injection of isoproterenol and pilocarpine. In non-stimulated glands, TPPase activity is detected mainly in 3–4 cisternae at the inner concave side of the Golgi complex and in some adjacent condensing vacuoles as in other cells. In the acinar cells 1 to 2 h after stimulation, however, reaction product for the same enzyme activity is detected in the cisternae at the outer aspect, as well as the inner aspect, of the Golgi complex and even in the cisternae of the endoplasmic reticulum (ER). About 4 h after stimulation, TPPase activity becomes concentrated in 3–4 disternae at the inner concave side of the Golgi complex as in the acinar cells under non-stimulated conditions. Morphological observations of the acinar cells 1 to 2 h after the stimulation have indicated that the reorganization of the Golgi complex and ER is a major event which occurs at this stage. It is possible that this cellular event is related to the occurrence of TPPase activity in those sites which normally show negative reaction in non-stimulated state.     

Polycystin-1 distribution is modulated by polycystin-2 expression in mammalian cells

Mutations in PKD1 and PKD2, the genes that encode polycystin-1 and polycystin-2 respectively, account for almost all cases of autosomal dominant polycystic kidney disease. Although the polycystins are believed to interact in vivo, the two proteins often display dissimilar patterns and gradients of expression during development. In an effort to understand this apparent discrepancy, we investigated how changes in polycystin-2 expression can affect the subcellular localization of polycystin-1. We show that, when polycystin-1 is expressed alone in a PKD2 null cell line, it localizes to the cell surface, as well as to the endoplasmic reticulum. When co-expressed with polycystin-2, however, polycystin-1 is not seen at the cell surface and co-localizes completely with polycystin-2 in the endoplasmic reticulum. The localization of a polycystin-1 fusion protein was similarly affected by changes in its level of expression relative to that of polycystin-2. This phenomenon was observed in populations as well as in individual COS-7 cells. Our data suggest that the localization of polycystin-1 can be regulated via the relative expression level of polycystin-2 in mammalian cells. This mechanism may help to explain the divergent patterns and levels of expression observed for the polycystins, and may provide clues as to how the function of these two proteins are regulated during development.