The Golgi apparatus and lysosomes of rat pancreatic acinar cells following refeeding

Summary The short term effects of refeeding on the Golgi apparatus and lysosomes of the rat exocrine pancreas were evaluated by ultrastructural, morphometric and cytochemical methods. Ten minutes after refeeding, there was a significant enlargement of Golgi cisternae and a significant increase, compared with the controls, in the number of condensing vacuoles and lysosomes. These modifications were accompanied by the appearance of acid phosphatase activity in stacked Golgi cisternae (as well as GERL) of some cells. One hour after refeeding, there were about the same numbers of condensing vacuoles and lysosomes as in the control; Golgi cisternae were still significantly enlarged, compared with the controls, but they were no longer reactive for acid phosphatase. In both fasting and refed animals, acid phosphatase activity was demonstrable in tubular lysosomes.The data are interpreted in terms both of membrane disposal and recycling, leading to enhanced formation of zymogen granules, during physiologically stimulated secretion.     

4Pi-microscopy of the Golgi apparatus in live mammalian cells

We report the applicability of 4Pi-microscopy to live mammalian cells. Controlled interference of the counterpropagating wavefronts is possible despite the slight variations in cellular refractive index. Superresolved 3D-fluorescence imaging is exemplified with the first representation of the Golgi apparatus in a live cell at approximately 100 nm resolution.     

Biochemical sub-fractionation of the mammalian Golgi apparatus

We have exploited the breakdown of the Golgi apparatus that occurs during mitosis to isolate subfractions using immuno-affinity methods. Rat liver Golgi stacks were treated with mitotic cytosol from HeLa cells, and the fragments were then incubated with antibodies immobilized on magnetic beads. Antibodies against the cis -Golgi marker, GM130, bound membranes that were depleted in the trans -Golgi network marker, TGN38, whereas antibodies against the cytoplasmic tail of TGN38 did the reverse. A range of other Golgi enzymes, SNAREs and tethers were also tested and were found to bind to anti-GM130 antibodies to an extent that reflected their proximity to cis -cisternae as determined by other techniques. This method should provide a useful complement to the immuno-EM methods presently used to map the Golgi apparatus .     

Modular organization of the mammalian Golgi apparatus

The Golgi apparatus is essential for post-translational modifications and sorting of proteins in the secretory pathway. In addition, it further performs a broad range of specialized functions. This functional diversity is achieved by combining basic morphological modules of cisternae into higher ordered structures. Linking cisternae into stacks that are further connected through tubules into a continuous Golgi ribbon greatly increases its efficiency and expands its repertoire of functions. During cell division, the different modules of the Golgi are inherited by different mechanisms to maintain its functional and morphological composition.     

Mitotic disassembly of the mammalian Golgi apparatus

A cell-free system that mimics mitotic fragmentation of Golgi stacks has provided a working model for the disassembly process. Two distinct pathways, one COP-dependent and one COP-independent, act on Golgi stacks to give rise to two types of end products: transport vesicles and larger, more heterogeneous vesicles and tubules. We suggest that both mitotic end products result from enhanced fission of Golgi membranes under conditions where membrane fusion is generally inhibited.     

Mitotic division of the mammalian Golgi apparatus

Successful cell reproduction requires faithful duplication and proper segregation of cellular contents, including not only the genome but also intracellular organelles. Since the Golgi apparatus is an essential organelle of the secretory pathway, its accurate inheritance is therefore of importance to sustain cellular function. Regulation of Golgi division and its coordination with cell cycle progression involves a series of sequential events that are subjected to a precise spatiotemporal control. Here, we summarize the current knowledge about the underlying mechanisms, the molecular players and the biological relevance of this process, particularly in mammalian cells, and discuss the unsolved problems and future perspectives opened by the recent studies.     

Ultrastructural features of cytofilaments within mammalian endothelial cells

Summary A submicroscopic study of the endothelium lining the blood capillaries and arterioles contained in the Cyon's nerve was made. The cytoplasm of some endothelial cells was found containing bundles of thin filaments. These measure about 60 Å in diameter, and do not show any cross striation, nor contacts with other cytoplasmic components. They are oriented parallel to each other and to the cell surface. No attachment plate of cytofilaments to the plasma membrane was seen. The filamentous structures were mostly found within the supranuclear cytoplasm. The endothelial cells in question never showed contacts with axons.In the light of these findings it can be advanced the view that the cytofilaments present in endothelial cells are supportive in function; namely they may confer a higher elasticity to these cells, subject to continuous pressure and morphological variations.

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Riassunto Lo studio ultrastrutturale dell'endotelio di piccoli vasi sanguigni contenuti nel nervo cardio-aortico depressore di Cyon del coniglio ha consentito di rilevare la presenza in alcune cellule endoteliali di esili filamenti raggruppati in fasci più o meno numerosi. Detti citofilamenti presentano uno spessore di circa 60 Å, occupano per lo più il citoplasma sopranucleare e sono orientati parallelamente tra loro e rispetto alla superficie cellulare. Essi non mostrano struttura periodica né rapporto alcuno, se non di semplice contiguità, con altre componenti citoplasmatiche. Non presentano, inoltre, punti di attacco sulla membrana cellulare, né connessioni con fibre nervose. Numerose le vescicole pinocitotiche osservate lungo i bordi superficiale e basale delle cellule endoteliali in oggetto.In base a tali reperti si avanza l'ipotesi che i citofilamenti endoteliali svolgano una funzione di sostegno nell'ambito del citoplasma, e siano capaci di conferire un più elevato grado di elasticità all'endotelio vasale, soggetto a continue variazioni di pressione e di forma.

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Dedicated to Prof. Wolfgang Bargmann on his 60th birthday.—This investigation was supported in part by a grant from the Italian C.N.R.

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The author wishes to thank Dr. Pasquale Romeo for his kind help in the preparation of the paper, and Mr. Ciro Paesano for making the photographic prints.     

Association of a dynamin-like protein with the Golgi apparatus in mammalian cells

Dynamins are a family of 100-kD GTPases comprised of at least three distinct gene products and multiple alternatively spliced variants. Homologies with the shibire gene product in Drosophila melanogaster and with Vps1p and Dnm1p in Saccharomyces cerevisiae suggest that dynamins play an important role in vesicular transport. Morphological studies have localized brain dynamin to coated pits and tubular invaginations at the plasma membrane, where it is believed to facilitate the formation of endocytic vesicles. Because similar membrane-budding events occur at the Golgi apparatus and multiple dynamin isoforms exist, we have studied the distribution of dynamins in mammalian cells. To this end, we generated and characterized peptide-specific antibodies directed against conserved regions of the dynamin family. By immunoblot analysis, these antibodies reacted specifically with a 100-kD protein in fibroblasts that sedimented with membranes and microtubules in vitro in a manner similar to brain dynamin. By immunofluorescence microscopy, these antibodies strongly labeled the Golgi complex in cultured fibroblasts and melanocytes, as confirmed by double labeling with a Golgi-specific antibody. Furthermore, Western blot analysis showed significant enrichment of a 100-kD dynamin band in Golgi fractions isolated from the liver. To substantiate these findings, we use a specific antidynamin antibody to immunoisolate Golgi membranes from subcellular Golgi fractions, as determined by EM and immunoblot analysis. This study provides the first morphological and biochemical evidence that a dynamin-like protein associates with the Golgi apparatus in mammalian cells, and suggests that dynamin-related proteins may have multiple cytoplasmic distributions. The potential contributions of dynamin to the secretory and endocytic pathways are discussed.     

Changes of the Golgi apparatus and lysosomes during decidualization in mice.

The Golgi apparatus of the endometrial stromal cells of pregnant mice increases in size simultaneously with the differentiation of stromal cells into decidual cells. The activity of acid phosphatase in this organelle increases during this stage. On the other hand, the involuting decidual cells show morphological and cytochemical signs of Golgi regression (dilated cisternae, lack of enzymatic activity) together with the finding of numerous, pleomorphic lysosomes that have intense cytochemical label. These results confirm morphological data suggesting that decidual cell death occurs by autophagic degeneration.     

Asymmetrical distribution of clathrin in the Golgi apparatus of polypeptide-secreting cells

Using an antibody revealed by the protein A-gold technique, we have studied the distribution of clathrin antigenic sites in the Golgi area of pancreatic B-cells. Golgi compartments showing an immunolabelling comprised extensive segments of cisternae, typical coated vesicles, dilated extremities of cisternae with condensing secretory material, and newly formed secretory granules. Most of the labelled membranes were observed at the trans Golgi pole while little immunoreactivity was found on the cis pole.     

Ultrastructural identification of sulphated glycoconjugates in the Golgi apparatus in human colonic absorptive cells

The subcellular localization of sulphated glycoconjugates was determined at the ultrastructural level by using the high iron diamine (HID) technique for sulphate groups in the absorptive cells of human colonic mucosa. Stained material was observed on the apical plasma membrane, in intracytoplasmic vesicles and in the Golgi complex. In this organelle, the last two or three cisternae of the trans side and the trans-Golgi network (TGN) were labelled, as well as a variable number of coated and noncoated vesicles facing the trans side and surrounding trans-Golgi network. These findings point to the trans side of the Golgi apparatus and trans-Golgi network as the subcompartments functionally involved in the sulphation of glycoconjugates.     

Ultrastructural identification of sulphated glycoconjugates in the Golgi apparatus in human colonic absorptive cells

Summary The subcellular localization of sulphated glycoconjugates was determined at the ultrastructural level by using the high iron diamine (HID) technique for sulphate groups in the absorptive cells of human colonic mucosa. Stained material was observed on the apical plasma membrane, in intracytoplasmic vesicles and in the Golgi complex. In this organelle, the last two or three cisternae of the trans side and the trans-Golgi network (TGN) were labelled, as well as a variable number of coated and noncoated vesicles facing the trans side and surrounding trans-Golgi network. These findings point to the trans side of the Golgi apparatus and trans-Golgi network as the subcompartments functionally involved in the sulphation of glycoconjugates.     

Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells

Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme, beta 1,4 galactosyltransferase (GalT) and the medial enzyme, N- acetylglucosaminyltransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. GalT was found in the trans-cisterna and trans- Golgi network but, contrary to expectation, NAGT I was found both in the medial- and trans-cisternae, overlapping the distribution of GalT. About one third of the NAGT I and half of the GalT were found in the shared, trans-cisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.     

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.     

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.     

Ultrastructural localization and distribution of Nardilysin in mammalian male germ cells

Background

NRD convertase, also termed Nardilysin, is a Zn⁺⁺ metalloendopeptidase that specifically cleaves the N-terminus of arginine and lysine residues into dibasic moieties. Although this enzyme was found located within the testis, its function in male reproduction is largely unknown. In addition, the precise distribution of this enzyme within germ cells remains to be determined.

Methods

To answer these questions, we developed an immuno-gold electron microscopy analysis to detect Nardilysin at ultrastructural level in mice. In addition, we performed a quantitative analysis of these gold particles to statistically estimate the distribution of Nardilysin in the different subcellular compartments of differentiating late spermatids/spermatozoa.

Results

Expression of Nardilysin in wild-type mice was restricted to germ cells and markedly increased during the last steps of spermiogenesis. In elongated spermatids, we found the enzyme mainly localized in the cytoplasm, more precisely associated with two microtubular structures, the manchette and the axoneme. No labelling was detected over the membranous organelles of the spermatids. To test whether this localization is dependent of the functional microtubules organization of the flagella, we analysed the localization into a specific mouse mutant ebo/ebo (ébouriffé) known to be sterile due to an impairment of the final organization of the flagellum. In the ebo/ebo, the enzyme was still localized over the microtubules of the axoneme and over the isolated cytoplasmic microtubules doublets. Quantification of gold particles in wild-type and mutant flagella revealed the specific association of the enzyme within the microtubular area of the axoneme.

Conclusions

The strong and specific accumulation of Nardilysin in the manchette and axoneme suggests that the enzyme probably contributes either to the establishment of these specific microtubular structures and/or to their functional properties.

     

Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells

Partitioning in dextran–poly(ethylene)glycol (PEG) aqueous–aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum<mitochondria<Golgi apparatus<lysosomes and endosomes<plasma membranes. Salt concentrations and temperature affect partitioning behavior and must be precisely standardized. In some cases, it is more fortuitous to combine aqueous two-phase partition with other procedures to obtain a more highly purified preparation. A procedure is described for preparation of Golgi apparatus from transformed mammalian cells that combines aqueous two-phase partition and centrifugation. Also described is a periodic NADH oxidase, a new enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions for measurement of activity.     

Maturation of the Golgi apparatus in urothelial cells

The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane formation. We have studied the distribution and organization of the GA in normal mouse urothelial cells and in the superficial urothelial cells that undergo differentiation following cyclophosphamide-induced regeneration, in correlation with urothelial cell differentiation. In normal urothelium, immature basal cells have a simple GA, which is small and distributed close to the nucleus. In intermediate cells, the GA starts to expand into the cytoplasm, whereas the GA of terminally differentiated umbrella cells is complex, being large and spread over the whole basal half of the cytoplasm. During early stages of regeneration after cyclophosphamide treatment, the GA of superficial cells is simple and no markers of urothelial differentiation (uroplakins or asymmetric unit membranes, discoidal or fusiform vesicles, apical surface covered with microvilli) are expressed. At a later stage, the GA expands and, in the final stage of regeneration, when cells express all markers of terminal urothelial differentiation, the GA become complex once again. Our results show that: (1) GA distribution and organization in urothelial cells is differentiation-dependent; (2) the GA matures from a simple form in partially differentiated cells to a complex form in terminally differentiated superficial cells; (3) major rearrangements of GA distribution and organization correlate with the beginning of asymmetric unit membrane production. Thus, GA maturation seems to be crucial for asymmetric unit membrane formation. The work was supported by the Ministry of Education and Sport, Government of Republic of Slovenia, Slovenia (grant no. 3311-04-831450).