Endothelial nitric oxide synthase and its negative regulator caveolin-1 localize to distinct perinuclear organelles.

Caveolin-1 is a member of a subset of intracellular proteins that regulate endothelial nitric oxide synthase (eNOS) activity. In caveolae, caveolin-1 inhibits eNOS activity via a direct interaction with the enzyme. Previous work has indicated that both eNOS and caveolin-1 are also localized at the perinuclear Golgi complex. Whether caveolin-1 is involved in eNOS regulation in this cell compartment is unknown. Here we studied the localization of eNOS and caveolin-1 in the perinuclear region of primary bovine aortic endothelial cells. By immunofluorescence microscopy we show that both eNOS and caveolin-1 co-localize with Golgi markers. On treatment of the cells with the microtubule-depolymerizing drug nocodazole, the Golgi complex is scattered and caveolin-1 is found in vesicles at the periphery of the cell, while eNOS is localized at large structures near the nucleus. The nocodazole-induced redistribution of eNOS is similar to that of cis-, medial-, and trans-Golgi markers, while the caveolin-1 redistribution resembles that of sec22, a marker for the intermediate compartment. The localization of eNOS and caveolin-1 at distinct perinuclear compartments that behave differently in the presence of nocodazole indicates that eNOS activity is not regulated by caveolin-1 in the Golgi complex.     

Structural Maturation of the Transmissible Gastroenteritis Coronavirus

During the life cycle of the transmissible gastroenteritis coronavirus (TGEV), two types of virus-related particles are detected in infected swine testis cells: large annular viruses and small dense viruses. We have studied the relationships between these two types of particles. Immunoelectron microscopy showed that they are closely related, since both large and small particles reacted equally with polyclonal and monoclonal antibodies specific for TGEV proteins. Monensin, a drug that selectively affects the Golgi complex, caused an accumulation of large annular viral particles in perinuclear elements of the endoplasmic reticulum-Golgi intermediate compartment. A partial reversion of the monensin blockade was obtained in both the absence and presence of cycloheximide, a drug that prevented the formation of new viral particles. After removal of monensin, the Golgi complex recovered its perinuclear location, and a decrease in the number of perinuclear large viral particles was observed. The release of small dense viral particles into secretory vesicles and the extracellular medium was also observed, as was a partial recovery of infectivity in culture supernatants. Small viral particles started to be seen between the third and the fourth Golgi cisternae of normally infected cells. All of these data strongly indicate that the large annular particles are the immature precursors of the small dense viruses, which are the infectious TGEV virions. The immature viral particles need to reach a particular location at the trans side of the Golgi stack to complete their morphological maturation.     

Penetration of Toxoplasma gondii into host cells induces changes in the distribution of the mitochondria and the endoplasmic reticulum.

Fluorescence microscopy, using dyes which specifically label mitochondria, endoplasmic reticulum and the Golgi complex, and transmission electron microscopy, were used to analyze the changes which occur in the organization of these structures during interaction of Toxoplasma gondii with host cells. In uninfected cells the mitochondria are long filamentous structures which radiate from the nuclear region toward the cell periphery. After parasite penetration they become shorter and tend to concentrate around the parasite-containing vacuole (parasitophorous vacuole) located in the cytoplasm of the host cell. The mitochondria of extracellular parasites, but not of those located within the parasitophorous vacuole, were also stained by rhodamine 123. Labeling with DiOC6, which binds to elements of the endoplasmic reticulum, in association with transmission electron microscopy, revealed a concentration of this structure around the parasitophorous vacuole. The membrane lining this vacuole was also stained, suggesting that components of the endoplasmic reticulum are also incorporated into this membrane. The Golgi complex, as revealed by staining with NBD-ceramide and electron microscopy, maintains its perinuclear position throughout the evolution of the intracellular parasitism.     

Reduction capacity of untreated and of repeatedly isoproterenol treated parotid gland of the mouse

Osmium impregnation was used to show possible differences of reduction capacity of perinuclear space, rough endoplasmic reticulum and the Golgi apparatus of unstimulated mouse parotid gland and in the gland after repeated pharmacological doses of isoproterenol. There were some significant differences between the staining of acinar and duct cells. In all intercalated and striated duct cells the staining is dense in the perinuclear space and in the rough endoplasmic reticulum. Osmiophility was not detected in the Golgi complex of intercalated duct cells. The staining was also lacking in the perinuclear space and endoplasmic reticulum of the acinar cells. The cis face of the Golgi complex and numerous transitional vesicles in the acinar cells showed variability of the reduction capacity of their membrane segments. In chronically treated acinar cells Os black was lacking in the Golgi cisternae, except that the numerous transitional vesicles were heavily stained. These results reveal characteristic differences of reduction capacity of endomembrane compartments in different parotid glandular cells, as well as between untreated and treated acinar cells.     

Lectin-binding pattern in normal human gastric mucosa. A light and electron microscopic study

Normal human gastric mucosal cells were examined by light and electron microscopy using lectins as a probe. The ABC method was used with biotinylated lectins for light microscopy and HRP-labeled lectins for electron microscopy. The human gastric mucosal cells revealed specific binding patterns for each lectin by light microscopy. Among the lectins tested, in particular, DBA gave a characteristic pattern. It specifically stained the supranuclear region of surface epithelial cells and the perinuclear region of parietal cells. By electron microscopy, the stacked cisternae and the vesicles of the Golgi apparatus of the surface epithelial cells were positive for the DBA staining. These results show that the DBA-positive supranuclear region observed by light microscopy corresponds to the Golgi apparatus. In the parietal cells, DBA, RCA and ConA bound to the intracellular secretory canaliculi which are invaginations of the cell membrane running around the nucleus in the cytoplasm. Therefore, the tubular perinuclear positive region observed by light microscopy corresponds to the membranes of the intracellular secretory canaliculi. In addition, the ConA reagent stained the endoplasmic reticulum, Golgi apparatus, nuclear envelope, and cell membrane of the parietal cell, which explains the diffuse cytoplasmic staining observed at the light microscopic level with this lectin. Lectins have proved to be very useful for the evaluation of in situ cytochemical aspects of the glycoconjugates characteristic to human gastric mucosal cells.     

Reduction capacity of untreated and of repeatedly isoproterenol treated parotid gland of the mouse

Summary Osmium impregnation was used to show possible differences of reduction capacity of perinuclear space, rough endoplasmic reticulum and the Golgi apparatus of unstimulated mouse parotid gland and in the gland after repeated pharmacological doses of isoproterenol. There were some significant differences between the staining of acinar and duct cells. In all intercalated and striated duct cells the staining is dense in the perinuclear space and in the rough endoplasmic reticulum. Osmiophility was not detected in the Golgi complex of intercalated duct cells. The staining was also lacking in the perinuclear space and endoplasmic reticulum of the acinar cells. The cis face of the Golgi complex and numerous transitional vesicles in the acinar cells showed variability of the reduction capacity of their membrane segments. In chronically treated acinar cells Os black was lacking in the Golgi cisternae, except that the numerous transitional vesicles were heavily stained.These results reveal characteristic differences of reduction capacity of endomembrane compartments in different parotid glandular cells, as well as between untreated und treated acinar cells.     

Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding.

The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane protein M (previously called E1). We tested this by analyzing the site of budding of four different coronaviruses in relation to the intracellular localization of their M proteins. Mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) grown in CrFK cells, all budded exclusively into smooth-walled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called budding compartment previously identified for MHV. Indirect immunofluorescence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense labeling in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and medial Golgi cisternae. The corresponding M proteins of the four viruses, when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase analysis of metabolically labeled cells showed that in each case the majority of the M glycoproteins carried oligosaccharide side chains with Golgi-specific modifications within 4 h after synthesis. Immunoelectron microscopy localized recombinant MHV M and IBV M to the same membranes as the respective proteins in coronavirus-infected cells, with the same cis-trans distribution over the Golgi complex. Our results demonstrate that some of the M proteins of the four viruses are transported beyond the budding compartment and are differentially retained by intrinsic retention signals; in addition to M, other viral and/or cellular factors are probably required to determine the site of budding.     

Islet cell autoantigen of 69 kDa is an arfaptin-related protein associated with the Golgi complex of insulinoma INS-1 cells

Islet cell autoantigen of 69 kDa (ICA69) is a cytosolic protein of still unknown function. Involvement of ICA69 in neurosecretion has been suggested by the impairment of acetylcholine release at neuromuscular junctions upon mutation of its homologue gene ric-19 in C. elegans. In this study, we have further investigated the localization of ICA69 in neurons and insulinoma INS-1 cells. ICA69 was enriched in the perinuclear region, whereas it did not co-localize with markers of synaptic vesicles/synaptic-like microvesicles. Confocal microscopy and subcellular fractionation in INS-1 cells showed co-localization of ICA69 with markers of the Golgi complex and, to a minor extent, with immature insulin-containing secretory granules. The association of ICA69 with these organelles was confirmed by immunoelectron microscopy. Virtually no ICA69 immunogold labeling was observed on secretory granules near the plasma membrane, suggesting that ICA69 dissociates from secretory granule membranes during their maturation. In silico sequence and structural analyses revealed that the N-terminal region of ICA69 is similar to the region of arfaptins that interacts with ARF1, a small GTPase involved in vesicle budding at the Golgi complex and immature secretory granules. ICA69 is therefore a novel arfaptin-related protein that is likely to play a role in membrane trafficking at the Golgi complex and immature secretory granules in neurosecretory cells.     

Impairment of nuclear pores in bovine herpesvirus 1-infected MDBK cells

Herpesvirus capsids originating in the nucleus overcome the nucleocytoplasmic barrier by budding at the inner nuclear membrane. The fate of the resulting virions is still under debate. The fact that capsids approach Golgi membranes from the cytoplasmic side led to the theory of fusion between the viral envelope and the outer nuclear membrane, resulting in the release of capsids into the cytoplasm. We recently discovered a continuum from the perinuclear space to the Golgi complex implying (i) intracisternal viral transportation from the perinuclear space directly into Golgi cisternae and (ii) the existence of an alternative pathway of capsids from the nucleus to the cytoplasm. Here, we analyzed the nuclear surface by high-resolution microscopy. Confocal microscopy of MDBK cells infected with recombinant bovine herpesvirus 1 expressing green fluorescent protein fused to VP26 (a minor capsid protein) revealed distortions of the nuclear surface in the course of viral multiplication. High-resolution scanning and transmission electron microscopy proved the distortions to be related to enlargement of nuclear pores through which nuclear content including capsids protrudes into the cytoplasm, suggesting that capsids use impaired nuclear pores as gateways to gain access to the cytoplasmic matrix. Close examination of Golgi membranes, rough endoplasmic reticulum, and outer nuclear membrane yielded capsid-membrane interaction of high identity to the budding process at the inner nuclear membrane. These observations signify the ability of capsids to induce budding at any cell membrane, provided the fusion machinery is present and/or budding is not suppressed by viral proteins.     

Correlation of Golgi localization of ZW10 and centrosomal accumulation of dynactin

ZW10 participates in the termination of the spindle checkpoint during mitosis by interacting with dynamitin, a subunit of the dynein accessory complex dynactin. We previously showed that ZW10 is attached to the endoplasmic reticulum through RINT-1 in interphase HeLa cells and involved in membrane transport between the endoplasmic reticulum and Golgi. Although a recent study demonstrated that ZW10 is localized in the Golgi in COS7 cells, the mechanism that regulates ZW10 localization remains unknown. In this study we showed a correlation between the Golgi localization of ZW10 and the centrosomal accumulation of dynactin. The amounts of ZW10 associated with dynactin were larger in cells where ZW10 was present in the Golgi than those where ZW10 was not in the Golgi. The targeting of ZW10 to the perinuclear Golgi region was found to depend on the perinuclear accumulation of dynactin, suggesting that dynactin regulates ZW10 localization.     

Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.     

Giardia lamblia: identification and characterization of Rab and GDI proteins in a genome survey of the ER to Golgi endomembrane system

To investigate the complexity of the endomembrane transport system in the early diverging eukaryote, Giardia lamblia, we characterized homologues of the GTP-binding proteins, Rab1 and Rab2, involved in regulating vesicular trafficking between the endoplasmic reticulum and Golgi in higher eukaryotes, and GDI, which plays a key role in the cycling of Rab proteins. G. lamblia Rab1, 2.1, and GDI sequences largely resemble yeast and mammalian homologues, are transcribed as 0.66-, 0.62-, and 1.4-kb messages, respectively, and are expressed during growth and encystation. Western analyses detected an abundant Rab/GDI complex at approximately 80 kDa, and free GDI (60 kDa) in both trophozoites and encysting cells. Immunoelectron microscopy with antibody to Rab1 localized Rab with ER, encystation secretory vesicles, and lysosome-like peripheral vesicles. GDI associated with these structures, and with small vesicles found throughout the cytoplasm, consistent with GDI's key role in Rab cycling between organelles within the cell.     

Cyclic-AMP-dependent protein kinase type II is associated with the Golgi complex and with centrosomes

The subcellular distribution of the type II enzyme of cAMP-dependent protein kinase (cAMP-dPK II) in epithelial and fibroblastic cells was determined by indirect immunofluorescence microscopy. In interphase cells both regulatory (R II) and catalytic (C) subunits were concentrated in a perinuclear area. By comparison of the R II distribution with the location of a bona fide Golgi membrane constituent, this area was identified as the Golgi complex. The cytochemical localization of R II was confirmed by subcellular fractionation. In addition, cAMP-dPK II was associated with microtubule-organizing centers, in particular with mitotic spindle poles. These distributions of cAMP-dPK II probably represent important factors in mediating the effects of cAMP on basic cellular activities ranging from secretion and proliferation to cell shape and motility.     

Phospholipase D2 is localized to the rims of the Golgi apparatus in mammalian cells

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid, a molecule known to have multiple physiological roles, including release of nascent secretory vesicles from the trans-Golgi network. In mammalian cells two forms of the enzyme, PLD1 and PLD2, have been described. We recently demonstrated that PLD1 is localized to the Golgi apparatus, nuclei, and to a lesser extent, plasma membrane. Due to its low abundance, the intracellular localization of PLD2 has been characterized only indirectly through overexpression of chimeric proteins. Using antibodies specific to PLD2, together with immunofluorescence microscopy, herein we demonstrate that a significant fraction of endogenous PLD2 localized to the perinuclear Golgi region and was also distributed throughout cells in dense cytoplasmic puncta; a fraction of which colocalized with caveolin-1 and the plasma membrane. On treatment with brefeldin A, PLD2 translocated into the nucleus in a manner similar to PLD1, suggesting a potential role in nuclear signaling. Most significantly, cryoimmunogold electron microscopy demonstrated that in pituitary GH(3) cells >90% of PLD2 present in the Golgi apparatus was localized to cisternal rims and peri-Golgi vesicles exclusively. The data are consistent with a model whereby PLD2 plays a role in Golgi vesicular transport.     

Heterogeneous distribution of the cAMP receptor protein RII in the nervous system: evidence for its intracellular accumulation on microtubules, microtubule-organizing centers, and in the area of the Golgi complex

The cellular and subcellular distribution of the regulatory subunit RII of cAMP-dependent protein kinase was studied by light and electron microscopy immunocytochemistry in tissue sections from rat brain and in primary cultures of brain cells. RII immunoreactivity was present in most neurons, although at variable concentration. In addition, RII was also detectable in other cell types including glia, neuroepithelial cells, and cells of mesenchymal origin. In the cell cytoplasm, RII immunoreactivity was concentrated at certain sites. An accumulation of RII immunoreactivity was found in all RII-positive cells at the Golgi area, precisely at a region directly adjacent to one of the two major faces of the Golgi complex. RII was also highly concentrated in some microtubule-rich cell processes such as cilia and neuronal dendrites, but was below detectability in most axons. In neurons, its concentration in dendrites is consistent with the previously demonstrated high affinity interaction between RII and the dendritic microtubule-associated protein 2. In addition, RII was accumulated at basal bodies of cilia and at centrosomes, i.e., sites known to act as microtubule organizers. RII-labeled centrosomes, however, were visible only in cells where the Golgi complex had a pericentrosomal organization, and not in cells where the Golgi complex was perinuclear such as in neurons and glia in situ. We hypothesize that centrosomal RII is bound to the pericentriolar microtubule-organizing material and that this material remains associated with the trans region of the Golgi complex when the latter is no longer associated with the centrosome. Our results suggest a key but not obligatory role of cAMP in the Golgi-centrosomal area, the headquarters of cell polarity, mobility and intracellular traffic, and in the function of a subpopulation of microtubules.     

Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia

Giardia lamblia, which belongs to the earliest identified lineage to diverge from the eukaryotic line of descent, is one of many protists reported to lack a Golgi apparatus. Our recent finding of a developmentally regulated secretory pathway in G. lamblia makes it an ideal organism with which to test the hypothesis that the Golgi may be more readily demonstrated in actively secreting cells. These ultrastructural studies now show that a regulated pathway of transport and secretion of cyst wall antigens via a novel class of large, osmiophilic secretory vesicles, the encystation-specific vesicles (ESV), is assembled during encystation of G. lamblia. Early in encystation, cyst antigens are localized in simple Golgi membrane stacks and concentrated within enlarged Golgi cisternae which appear to be precursors of ESV. This would represent an unusual mechanism of secretory vesicle biogenesis. Later in differentiation, cyst antigens are localized within ESV, which transport them to the plasma membrane and release them by exocytosis to the nascent cell wall. ESV are not observed after completion of the cyst wall. In contrast to the regulated transport of cyst wall proteins, we demonstrate a distinct constitutive lysosomal pathway. During encystation, acid phosphatase activity is localized in endoplasmic reticulum, Golgi, and small constitutive peripheral vacuoles which function as lysosomes. However, acid phosphatase activity is not detectable in ESV. These studies show that G. lamblia, an early eukaryote, is capable of carrying out Golgi-mediated sorting of proteins to distinct regulated secretory and constitutive lysosomal pathways.     

Lectin-binding pattern in normal human gastric mucosa

Summary Normal human gastric mucosal cells were examined by light and electron microscopy using lectins as a probe. The ABC method was used with biotinylated lectins for light microscopy and HRP-labeled lectins for electron microscopy. The human gastric mucosal cells revealed specific binding patterns for each lectin by light microscopy. Among the lectins tested, in particular, DBA gave a characteristic pattern. It specifically stained the supranuclear region of surface epithelial cells and the perinuclear region of parietal cells. By electron microscopy, the stacked cisternae and the vesicles of the Golgi apparatus of the surface epithelial cells were positive for the DBA staining. These results show that the DBA-positive supranuclear region observed by light microscopy corresponds to the Golgi apparatus. In the parietal cells, DBA, RCA and ConA bound to the intracellular secretory canaliculi which are invaginations of the cell membrane running around the nucleus in the cytoplasm. Therefore, the tubular perinuclear positive region observed by light microscopy corresponds to the membranes of the intracellular secretory canaliculi. In addition, the ConA reagent stained the endoplasmic reticulum, Golgi apparatus, nuclear envelope, and cell membrane of the parietal cell, which explains the diffuse cytoplasmic staining observed at the light microscopic level with this lectin. Lectins have proved to be very useful for the evaluation of in situ cytochemical aspects of the glycoconjugates characteristic to human gastric mucosal cells.     

Prolonged osmification of stomach epithelium

Summary Osimium impregnation was used upon the mice stomach epithelium to show possible differences in staining during differentiation. In the cells of the stratified gastric epithelium of 14-day-old mice embryos Os black was completely lacking in the Golgi complex. In some but not all cells the staining appears in the perinuclear space and in the endoplasmic reticulum. In the mucoid cells 1 and 8 days after the birth the osmiophility is not uniformly distributed throughout the endomembrane segments, except the cis face of the Golgi complex which is heavily stained. Our results indicate on the variability of the reduction capacity of particular endomembrane segments during differentiation and among the cells at the definite developmental stage.     

Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions

During infection of sac- cells by murine coronavirus MHV A59 the intracellular sites at which progeny virions bud correlate with the distribution of the viral glycoprotein E1. Budding is first detectable by electron microscopy at 6 to 7 hours post infection in small, smooth, perinuclear vesicles and tubules in a region transitional between the rough endoplasmic reticulum and the Golgi apparatus. At later times the rough endoplasmic reticulum becomes the major site of budding and accumulation of progeny virus particles. Indirect immunofluorescence microscopy shows that E1 is confined at 6 hours post infection to the perinuclear region while at later times it also accumulates in the endoplasmic reticulum. At 6 hours post infection the second viral glycoprotein, E2, is distributed throughout the endoplasmic reticulum and is not restricted to the site at which budding begins. Core protein, the third protein in virions, can be detected 2 hours before E1 is detectable and budding begins, and at 6 hours post infection it is distributed throughout the cytosol. We conclude that the time and the site at which the maturation of progeny virions occurs is determined by the accumulation of glycoprotein E1 in intracellular membranes. Only rarely do progeny virions bud directly into the cisternae of the Golgi apparatus but at least some already budded virions are transported to the Golgi apparatus where they occur in structures some of which also contain TPPase, a trans Golgi marker.     

Prolonged osmification of stomach epithelium

Osmium impregnation was used upon the mice stomach epithelium to show possible differences in staining during differentiation. In the cells of the stratified gastric epithelium of 14-day-old mice embryos Os black was completely lacking in the Golgi complex. In some but not all cells the staining appears in the perinuclear space and in the endoplasmic reticulum. In the mucoid cells 1 and 8 days after the birth the osmiophility is not uniformly distributed throughout the endomembrane segments, except the cis face of the Golgi complex which is heavily stained. Our results indicate on the variability of the reduction capacity of particular endomembrane segments during differentiation and among the cells at the definite developmental stage.