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PubMed2000

Glycosylated nuclear lectin CBP70 also associated with endoplasmic reticulum and the Golgi apparatus: does the "classic pathway" of glycosylation also apply to nuclear glycoproteins?

The subcellular plurilocalization of some lectins (galectin-1, galectin-3, galectin-10, calreticulin, etc.) is an intriguing problem, implying different partners according to their localization, and involvement in a variety of cellular activities. For example, the well-known lectin, galectin-3, a lactose-binding protein, can act inside the nucleus in splicing events, and at the plasma membrane in adhesion, and it was demonstrated that galectin-3 interacts in the cytoplasm with Bcl-2, an antiapoptotic protein. Some years ago, our group isolated a nuclear lectin CBP70, capable of recognizing N-acetylglucosamine residues. This lectin, first isolated from the nucleus of HL60 cells, was also localized in the cytoplasm. It has been demonstrated that CBP70 is a glycosylated lectin, with different types of glycosylation, comparing cytoplasmic and nuclear forms. In this article, we have studied the localization of CBP70 in undifferentiated HL60 cells by electron microscopy, immunofluorescence analysis, and subcellular fractionation. The results obtained clearly demonstrated that CBP70 is a plurilocalized lectin that is found in the nucleus, at the endoplasmic reticulum, the Golgi apparatus, and mitochondria, but not at the plasma membrane. Because CBP70, a nuclear glycoprotein, was found to be associated also with the endoplasmic reticulum and the Golgi apparatus where the glycosylation take place, it raised the question: where does the glycosylation of nuclear proteins occur?

Cell Nucleolus↗

PubMed1979

Three-dimensional architecture of the golgi apparatus in Sertoli cells of the rat.

Glutaraldehyde-fixed testes were stained "en bloc" with the Ur-Pb-Cu technique of Thiéry and Rambourg ('76) or post-fixed and stained with the osmium tetroxide-potassium ferrocyanide method of Karnovsky ('71). Thin or thick (up to 3 micron) sections were examined with the Philips (301 or 400) EM or the high voltage EM. Stereopairs were prepared with photographs of tilted specimens (+/- 7 degrees). At low magnification, in thick sections (0.5-3 micron) stained with Ur-Pb-Cu, the whole Golgi apparatus formed a single network of interconnected wavy ribbon or platelike structures extending from the juxtanuclear region toward the apex of the cell. At higher magnifications, with the two staining techniques, this Golgi network showed two distinct types of regions: the "saccular region" corresponding to the conventional stack of saccules and the "intersaccular connecting region" made up of anastomotic tubules which bridge adjacent stacks. In the saccurlar regions, there was, on the cis-face of the stack, a tight polygonal meshwork of anastomotic tubules (osmiophilic element). Underlying it there were three to seven closely apposed saccules perforated with pores of various diameters, and finally, on the trans-face, a network of tubules was usually connected to the last saccule of the stack, which seemed to peel off" from the pile. The intersaccular connecting regions showed proximal and distal zones with regard to the associated stacks. The proximal zone was made up of superimposed and parallel polygonal networks of membranous tubules which were continuous with corresponding saccules of the stack. In the distal zone they interdigitated, intertwined, anastomosed and bridged adjacent saccular regions; others turned at right angles and established connections with tubular extensions arising at various levels of the same stack. While cisternae of endoplasmic reticulum were contiguous with tubules or saccules located on the transface of the Golgi apparatus, a close association between the ER cisternae and the cis-face of the stacks was not usually observed.

Animals↗

PubMed1984

Tridimensional architecture of the Golgi apparatus in the atrial muscle cell of the rat.

The tridimensional structure of the Golgi apparatus of atrial muscle cells has been studied in thin and thick sections with low- and high-voltage electron microscopes. Cardiac tissue was inpregnated with osmium, stained to demonstrate phosphate activity (i.e., nicotinamide adenine dinucleotide phosphatase or NADPase; thiamine pyrophosphatase or TPPase; cytidine monophosphatase or CMPase) or postfixed and stained with potassium ferrocyanide-reduced osmium. At low power, in thick (3-10 micron) sections, the cisosmiophilic element and the NADPase- and the TPPase-positive saccules each appeared as a continuous irregular ribbon that formed, at the two poles of the nucleus, two conical masses connected to each other by beltlike bands encircling the nucleus. At higher magnifications, the continuous Golgi apparatus showed saccular regions along its length connected by short intersaccular tubular regions. In the saccular regions, the following five superimposed elements formed a stack: (1) the cis-osmiophilic network of anastomosed tubules; (2) a chromophobic, dilated saccule perforated with numerous pores; (3) a thin NADPase-positive saccule showing few pores; (4) a thin TPPase-positive saccule perforated with numerous minute pores; and (5) a CMPase-positive transelement that showed saccular and tubular regions and was often partly separated from the overlying saccule. In the intersaccular tubular regions, membranous tubules connected and bridged saccules of two adjacent saccular regions. Secretory granules usually appeared in this region as dilations of the tubules connected to all elements of the Golgi stack except the cis-osmiophilic element.

Animals↗

PubMed1986

Ultrastructural localization of concanavalin A-binding sites in the Golgi apparatus of various types of neurons in rat dorsal root ganglia: functional implications.

The localization of concanavalin A (con A) binding sites has been determined at the electron-microscopic level in the six types of neurons (A1, A2, A3, B1, B2, C) of rat dorsal root ganglia. In all ganglion cells, con A stained the plasma membrane, the nuclear envelope, the cisternae of the rough endoplasmic reticulum, and the matrix of some multivesicular bodies. In contrast, the con A reactivity of the Golgi apparatus varied according to cell type. In type B1 and B2 cells and possibly in type A3 cells, the lectin was exclusively located in three or four saccules on the cis side of the Golgi stacks, whereas the TPPase-positive saccules and the trans sacculotubular elements were unstained with con A. In type A1, A2, and C neurons, all Golgi saccules as well as the trans sacculotubular elements were stained with the lectin. These results suggest that different types of glycoproteins were produced in these two groups of neurons. In the type A1, A2, and C cells, the persistence of the lectin reactivity in the TTPase-positive saccules or sacculotubular elements on the trans side of the Golgi stacks suggests the presence of glycoproteins with oligosaccharide side chains rich in alpha-D-mannosyl residues in terminal positions. In contrast, the disappearance of the con A reactivity in equivalent elements of the Golgi stacks in type B1, B2, and A3 cells suggests the addition at this level of other sugar residues characteristic of complex oligosaccharide side chains. The majority of the vesicular elements associated with the Golgi apparatus, as well as lysosomes, were unstained with con A.

Animals↗

PubMed1987

Tridimensional architecture of the Golgi apparatus and its components in mucous cells of Brunner's glands of the mouse.

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in thin and thick sections of mucous cells of mouse Brunner's glands by using low- and high-voltage electron microscopes and a stereoscopic approach. In thick sections of glands impregnated with osmium or treated to detect nicotinamide adenine dinucleotide phosphatase (NADPase) or thiamine pyrophosphatase (TPPase) activity, the Golgi apparatus appeared, at low magnification, as a continuous network located in the supranuclear region. At higher magnifications and in thin sections of tissue postfixed with reduced osmium and stained with lead citrate or treated to demonstrate phosphatase activity, the following components were observed: on the cis-face of the Golgi stacks, an osmiophilic tubular network referred to as the cis-element; a cis-saccular-compartment composed of a distended porous saccule slightly reactive for NADPase and three or four underlying NADPase-positive, flattened, poorly fenestrated saccules; a trans-saccular-compartment consisting of four to six TPPase-positive saccules or sacculo-tubular elements, prosecretory granules, and "peeling off" trans-tubular networks. The saccules of the cis-compartment were often perforated by large pores in register. The cavities thus formed in the stacks were called wells and were pan-shaped with a mouth directed toward the cis-face of the stacks and a bottom closed by TPPase-positive saccules. The wells always contained 80-nm vesicles. The saccules of the trans-compartment were involved in the formation of secretory granules according to the following proposed sequence of transformation. The secretion product appeared initially as a granular material evenly distributed throughout a slightly distended, poorly fenestrated saccule. These saccules appeared to transform into fenestrated elements with irregular pores and with parts of them taking on the appearance of a tubular network; they were thus referred to as sacculotubular elements. The secretory material initially distributed throughout these elements accumulated in nodular dilatations randomly distributed along the tubular portions of the elements. The dilatations, considered as prosecretory granules, increased in size as they drained the secretory material from the rest of the sacculotubular elements. Such prosecretory granules, large and irregular in shape, "peeled off" from the stacks of saccules with residual saccular or tubular structures still attached to them, some of the latter forming trans-tubular networks. The prosecretory granules detached from such membranous residues, condensed, and finally transformed into spherical secretion granules.

Animals↗

PubMed1988

Formation of secretion granules in the Golgi apparatus of pancreatic acinar cells of the rat.

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in sections of pancreatic acinar cells by using stereopairs of electron microscope photographs. Pancreatic tissue fixed in glutaraldehyde was postfixed in reduced osmium, and the sections were stained with lead citrate. Tissues were also treated to demonstrate phosphatase activity (i.e., nicotinamide adenine dinucleotide phosphatase, NADPase; thiamine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase). The following stacked components were observed along the branching, anastomotic, continuous, ribbonlike Golgi apparatus. 1) On the cis-face of the Golgi stack there was a tubular membranous network known to be osmiophilic and referred to as the cis-osmiophilic tubular network or cis-element. 2) A first, poorly fenestrated saccule, unreactive for the phosphatases tested, was slightly distended in places and contained a fluffy granulofilamentous material. 3) The subjacent three or four saccules, reactive for NADPase and/or TPPase, showed dilated portions containing a granulofilamentous secretory material similar to that filling the rest of the saccule. They also showed nondilated portions perforated with large fenestrations, some of which were in register and formed wells containing 80-nm vesicles. The dilated portions of these saccules were present at random along the length of the saccules and were not located exclusively at their edges. 4) The remaining one or two elements of the stack, CMPase positive, showed dilated spheroidal portions or prosecretory granules containing a homogeneous secretory material and flattened fenestrated regions free of secretory material and having the appearance of networks of narrow membranous tubules. 5) Lastly on the trans-aspect of the stack there were detached prosecretory granules reactive for CMPase and surrounded by a corona of small vesicles, and smooth-surfaced spherical CMPase-negative granules having a denser content that were identified as fully formed secretion granules; there were also occasional free trans-tubular networks strongly reactive for CMPase that appeared to undergo fragmentation and numerous small vesicles free from acid-phosphatase activity. These various images were interpreted as indicating that prosecretory granules formed in relation to two or three fenestrated saccules on the trans-side of the stack. Such granules, following their detachment from the trans-face of the stack, their separation from trans-tubular networks, and condensation of their content, yielded mature secretion granules.

Animals↗

PubMed1989

Formation of secretion granules in the Golgi apparatus of plasma cells in the rat.

The three-dimensional structure of the components of the Golgi apparatus was analyzed in plasma cells of rat duodenum. The spheroidal juxtanuclear Golgi apparatus was formed by a continuous ribbonlike structure composed of the following stacked elements. On the cis-face of the Golgi stack, there was a tubular membranous network referred to as the cis-element and/or a slightly dilated saccule perforated with small pores. The two or three subjacent saccules, which showed few pores, were slightly dilated and contained a fluffy granulofilamentous material. They were also perforated in register by cavities or wells containing 80-nm vesicles. The next one or two underlying elements were fenestrated saccules showing flattened portions as well as distended portions containing a homogeneous material denser than that seen in the overlying saccules. The last two or three elements of the stack showed a partially separated or "peeling off" configuration. These last elements consisted of prosecretory granules attached to flattened, empty-looking saccules showing buds at their surface; detached, more-or-less fenestrated, flattened saccules; and shrivelled residual trans-tubular networks. In the trans-region of the stack, in addition to numerous small vesicles, short membranous tubules, detached prosecretory granules, and denser fully formed secretion granules were also seen. These images were interpreted to indicate that secretory material present in the trans-saccules flows toward the dilated portions which become prosecretory granules. The trans-most elements seemingly peel off the stack to yield prosecretory granules and fragmenting trans-tubular networks.

Animals↗

PubMed1983

Ultrastructural studies of myeloma cells: observations concerning the Golgi apparatus and intermediate-size filaments.

Studies on the ultrastructure of tumor cells of 22 multiple myeloma patients revealed hitherto unreported observations concerning the Golgi apparatus and intermediate-size filaments. Various abnormalities of the Golgi apparatus including displacements of the Golgi stacks, dilatations of cisternae, and massive accumulations of vesicles as well as vacuoles in the Golgi region were found in myeloma cells of 11 patients. Intermediate-size filaments forming a fine intracytoplasmic network were apparent in myeloma cells of all 22 patients. In five patients, in addition to this architecture, intermediate-size filaments were gathered into thick bundles which were primarily but not exclusively located in the perinuclear region.

Cytoskeleton↗

PubMed1988

Contribution of the Golgi apparatus components to the formation of the acrosomic system and chromatoid body in rat spermatids.

The cytochemical distribution of two different acid phosphatases, nicotinamide adenine dinucleotide phosphatase (NADPase) and cytidine monophosphatase (CMPase), was analyzed within the Golgi apparatus, acrosomic system, and chromatoid body of early spermatids. Within the saccules composing the Golgi cortex, the phosphatases were localized in specific and nonoverlapping compartments: the cis-tubular network, or cis-element, and the first underlying saccule were unreactive for both enzymes, the following four to five saccules were reactive for NADPase, and the last three saccules were reactive for CMPase. Vesicles of various sizes and short membranous tubules, present near the edges of saccules and in the medulla of the Golgi apparatus, were NADPase- and/or CMPase-positive. The acrosomic system showed a weak but significant NADPase reactivity and a strong CMPase reactivity. The vesicles associated with the chromatoid body were reactive for NADPase and/or CMPase. The combined observations indicate that the two cytochemically distinct compartments of the Golgi cortex, the NADPase- and CMPase-positive saccules, independently contribute to the formation of the acrosomic system and the vesicular component of the chromatoid body.

Acrosome↗

PubMed1989

Granule formation and polarity of the Golgi apparatus in neutrophil granulocytes of the rat.

The formation of granules in neutrophil (heterophil) progenitor cells was examined with the electron microscope in sections of rat bone marrow fixed in 2% glutaraldehyde and postfixed with reduced osmium (Karnovsky: Proceedings of the 11th Meeting, American Society of Cell Biologists, Abstr. 284, p. 146, 1971). The cells were also osmicated in 2% osmium tetroxide for 36 hours at 37 degrees C to outline the osmiophilic element usually observed on the cis-face of the stacks of saccules of the Golgi apparatus of various cell types. In myeloblasts, which do not produce granules, the cis-osmiophilic element (CE) was found on the concave face of the C-shaped Golgi stacks. In promyelocytes the CE was present on the convex aspect of the C-shaped stacks, while the primary (azurophilic) granules formed in relation to elements on the concave aspects of the stacks. In myelocytes, the situation was reversed: the CE was found on the concave face of the Golgi stacks, while the secondary (specific) granules were seen forming in relation to elements on the convex aspect of the stacks. Finally, in metamyelocytes and mature neutrophils in which no granule formation took place, the appearance on Golgi stacks varied: they were either flat or C-shaped. The CE was indiscriminately found on one face or the other of the flat Golgi stacks of metamyetocytes and on the convex or concave faces of the C-shaped Golgi stacks of mature neutrophils. Using the cis-osmiophilic-element as a marker of the cis-face of the stacked Golgi elements, it thus appeared that despite marked changes in the configuration and orientation of the stacks of the cis-trans polarity of the stacked elements was maintained throughout granulopoiesis. In addition the primary and secondary granules that appeared sequentially in promyelocytes and myelocytes were both seen to form in relation to trans-elements of the Golgi apparatus.

Animals↗

PubMed1991

The Golgi apparatus of rat pachytene spermatocytes during spermatogenesis.

A morphological and immunocytochemical study of the Golgi apparatus in pachytene spermatocytes was performed in an effort to correlate the structure and function of this organelle during meiotic prophase. In stages I-III of the cycle, the Golgi complex of pachytene spermatocytes is a flattened discoid, 0.5-1 microns in diameter, composed of vesicles interspersed with classically described Golgi cisternae. During subsequent maturation of pachytene spermatocytes (stages IV-XIII), the size of the Golgi complex increases significantly, attaining a size of 2-3 microns. However, unlike pachytene spermatocytes of stages I-III, the majority of the Golgi complex of more mature spermatocytes is characterized by an abundance of distinct stacks of cisternae interspersed with numerous vesicles and tubules. The composition of the Golgi complex was also studied by using two monoclonal antibodies that recognize either the cis or the trans Golgi cisternae, respectively, and employing biotin-streptavidin-peroxidase immunocytochemistry in 5 micron frozen sections of testes. Immunodetection of the distinct cisternae revealed that the increase in size of the Golgi complex during maturation of pachytene spermatocytes was due predominantly to an accumulation of trans Golgi; the amount of cis Golgi remained unchanged. The morphological data presented in this study are consistent with an heightened secretory activity of pachytene spermatocytes during their maturation. In addition, the increase in size of the Golgi apparatus during the extensive prophase of pachytene spermatocytes may suggest that the mechanism employed by germ cells to partition the Golgi complex during the first division of meiosis varies significantly from that of somatic cells undergoing mitosis.

Animals↗

PubMed1991

Golgi apparatus of epithelial principal cells of the epididymal initial segment of the rat: structure, relationship with endoplasmic reticulum, and role in the formation of secretory vesicles.

The initial segment of the epididymis of rats, fixed with glutaraldehyde, was postfixed with reduced osmium, a technique that clearly delineates the membranes of cisternae of the endoplasmic reticulum (ER) and the various elements of the Golgi apparatus, or with tannic acid to enhance the coats of vesicles and ribosomes on ER cisternae. The material was also treated to demonstrate various phosphatase activities (NADPase, TPPase, CMPase, G-6-Pase) or impregnated with osmium tetroxide. In osmium-impregnated material, the Golgi apparatus of the epithelial principal cells of the initial segment appeared in the light microscope as a branching, anastomosing ribbon forming a large network in the supranuclear region. In the electron microscope, ER were of two types: the heavily granulated, flattened, rough ER seen in the infranuclear and juxtanuclear regions and the distended, tubular, sparsely granulated ER, showing only few ribosomes, seen interlaced with the Golgi ribbon in the supranuclear region and at the apical pole of the cell. Of particular interest in this cell was the fact that the sparsely granulated ER approximated the Golgi stack on both its cis- and trans-faces. On the cis-face of the Golgi stack, the sparsely granulated ER cisternae showed the usual finger- or bud-like protrusions directed toward the cis element of the Golgi stack and around which numerous small 80 nm vesicles or membranous tubules were clustered. The Golgi stack consisted of the following elements in a cis-trans axis: the cis osmiophilic element, a first saccule slightly dilated, saccules two to four (S2-S4), which were NADPase-positive, and saccules five to seven and the eight Golgi element, which were TPPase-positive. On the trans-aspect of the Golgi stacks, several (up to four) CMPase-positive trans-Golgi networks were observed often in close apposition to the sparsely granulated ER cisternae. One of the trans-Golgi networks showed a "peeling-off" configuration, i.e. part of it was closely apposed to the overlying Golgi element of the stack, whereas the remaining part was separated from the stack by a space occupied by a cisterna of sparsely granulated ER. The other trans-Golgi networks were completely separated from the stack and were often seen sandwiched between sparsely granulated ER cisternae. Thus, ER cisternae showed extensive areas of close apposition but no continuity with the trans-Golgi networks. Although the saccules of the Golgi stacks showed NADPase and/or TPPase activity, the trans-Golgi networks displayed CMPase activity, thus facilitating their identification from the closely associated unreactive sparsely granulated ER cisternae.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Changes of cytochemical properties in the Golgi apparatus during in vivo differentiation of the ameloblast in developing rat molar tooth germs.

The cytochemical changes of the Golgi stacks occurring concomitantly with cell differentiation were examined in ameloblasts of developing rat molar tooth germs using osmium impregnation and cytochemistry with nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and acid phosphatase (Acpase). NADPase, TPPase, and Acpase activities were already present in the Golgi stacks of the inner enamel epithelial cells, the undifferentiated form of the ameloblast: NADPase activity existed in the medial Golgi cisternae, TPPase activity in the trans Golgi cisternae, and Acpase activity in almost all cisternae and strongly in the trans-most cisterna of the Golgi stack. At this stage, however, osmium deposits after impregnation were not observed in the cisterna of Golgi stacks but were present in some small vesicles. These vesicles were located throughout the cytoplasm. Osmiophilic cisternae in the Golgi stacks were apparent for the first time at the stage when the Golgi apparatus developed and migrated to the region distal to the nucleus with the progression of cell differentiation. These findings indicate that the cis subcompartment of the Golgi apparatus was incomplete in the inner enamel epithelial cells with regard to appearance of its cytochemical property, as compared with the medial and trans subcompartments. It is suggested that the cis compartment of the Golgi stack may be completed only in the last stage of the compartmentalized Golgi organization during differentiation of the ameloblast.

Acid Phosphatase↗

PubMed1993

Transport of casein submicelles and formation of secretion granules in the Golgi apparatus of epithelial cells of the lactating mammary gland of the rat.

Lactating mammary glands fixed by perfusion with 5% glutaraldehyde subsequently were postfixed with potassium ferrocyanide reduced osmium or were treated with tannic acid. Stained thin sections were examined with the electron microscope and stereopairs were prepared. The distribution of casein submicelles was analyzed in the various components of the Golgi apparatus. The Golgi stacks were composed of five or six elements, all of which contained casein submicelles 20 nm in diameter. The cis-tubular network or cis-element, as well as the underlying three or four midsaccules, showed these casein submicelles either attached to their membrane or free in the lumen. The trans-most element of the stacks formed distended prosecretory granules in which both isolated or clustered casein submicelles were suspended in an electron-lucent fluid. These micellar aggregates increased in size and became progressively more compact to form spherical dense bodies or casein micelles, in which the individual 20 nm particles could easily be resolved. Casein micelles were seen in secretory granules in addition to a wispy material of low density. The numerous small spherical vesicles (80 nm or larger) seen on the cis, lateral, or trans aspects of the stacks did not appear to contain free casein submicelles. This raises questions regarding the role of these vesicles in the transport of casein macromolecules through the Golgi stacks. It was noticeable that in this Golgi apparatus a trans-Golgi network was limited to a few small residual tubules free from casein submicelles. It thus appears that the greater part of the trans-most Golgi element gives rise to the large prosecretory granules. After leaving the Golgi region and prior to exocytosis, the secretory granules often fuse to form larger granules before exocytosis.

Animals↗

PubMed1993

Structure of the Golgi apparatus in stimulated and nonstimulated acinar cells of mammary glands of the rat.

The structural features of the Golgi apparatus of acinar cells of mammary glands were examined with the electron microscope in 3 groups of rats: (1) in lactating female animals at 8 days postpartum, which served as controls; (2) in female rats sacrificed at various intervals from 2 to 30 hours following separation from their 8-day old pups; and (3) in females separated from their 8-day-old pups for a period of 12 hours and returned to their litters for durations of 1, 2, 4, and 8 hours. In animals of group 2, the Golgi stacks remained identical to that of controls between 2 and 8 hours. At 12 hours and later, the Golgi stacks decreased progressively in size, but the number of elements composing the stacks remained similar to that of lactating females and all contained casein submicelles. At 24 and 30 hours, typical secretory granules containing casein micelles disappeared from the trans aspect of the stacks. The earliest and most striking changes observed in the Golgi apparatus of the rats of group 2 took place at 12 hours. At this time, the prosecretory and secretory granules decreased considerably in volume and lost most of their electron-lucent content. This indicated that the delivery of small molecules, i.e., lactose and H2O, to these structures was soon altered following arrest of the sucking stimulus. In animals of group 3, the size of prosecretory and secretory granules and the amount of their electron-lucent content reverted to normal at 4 hours. Thus the influx of lactose and H2O into these structures appears to be rapidly restored after returning the pups to their mothers. The decrease in size of the Golgi stacks noted at 12, 18, and 24 hours following arrest of lactation (group 2), was accompanied by an increase in number of small vesicles that formed clusters next to the Golgi stacks and in "wells." Thus in these regressing Golgi stacks, many of the associated small vesicles appear to arise by vesiculation of the saccules.

Animals↗

PubMed1996

Two major autoantigen-antibody systems of the mitotic spindle apparatus.

OBJECTIVE: To characterize human autoantigen-antibody systems related to the mitotic poles and spindles. METHODS: Thirty-seven human sera with autoantibodies staining mitotic poles and spindles in indirect immunofluorescence (IIF) studies were further characterized by immunofluorescence on mitotic cells and by immunoblotting and immunoprecipitation. Clinical diagnoses meeting the American College of Rheumatology criteria were based on chart review and interview with the corresponding physicians. RESULTS: Two autoantibody systems reactive with mitotic poles and spindles were defined. Type 1 nuclear mitotic apparatus (NuMA-1) antibodies were identified in the serum of 30 patients. Interphase cells showed a fine, speckled, nuclear staining, while mitotic cells had bright staining of the rim of the centrosomes and light staining of the spindles proximal to the centrosomes. In telophase, the staining shifted from the centrosomes to the reforming nuclei. On immunoblotting, anti-NuMA-1 sera reacted with a 210-kd protein. The reactivity of these sera was identified (with the aid of reference antibodies) as the previously described NuMA antigen-antibody system. Clinical information was available for only 17 of the 30 patients with anti-NuMA-1; of these, 17 (53%) had clinical and lip biopsy findings that met the criteria for Sjögren's syndrome. NuMA-2 antibodies were found in the sera of 7 patients. Interphase cells showed no nuclear or cytoplasmic staining, but mitotic cells had brightly stained poles and spindles. At anaphase/telophase, staining shifted to the midbody and the intercellular bridge. Anti-NuMA-2 sera immunoprecipitated a protein of 116 kd. This group of patients was more heterogeneous and had both systemic and organ-specific autoimmune diseases. CONCLUSIONS: NuMA protein (here called NuMA-1) and a 116-kd protein (here called NuMA-2) are the major targets of the autoimmune response in the mitotic apparatus, since most of the selected sera (based on IIF staining of the mitotic spindles and poles) recognized 1 of these 2 antigens.

Adult↗

PubMed1985

Reorientation of the Golgi apparatus and the microtubule-organizing center inside macrophages subjected to a chemotactic gradient.

Mouse peritoneal macrophages subjected to gradients of activated mouse serum were found by immunofluorescence observations to have their Golgi apparatus and their microtubule-organizing center largely oriented in the direction of the gradient. By analogy with similar results obtained with motile fibroblasts, it is proposed that these two organelles are rapidly and coordinately reoriented inside the macrophages in order to direct the insertion of new membrane mass, via vesicles derived from the Golgi apparatus, into the leading edge of the cell. Consistent with the importance of such membrane insertion to cell migration, we found that the ionophore monensin, an inhibitor of Golgi functions, inhibited cell motility in the chemostactic gradient. It was further shown that several inhibitors of chemotaxis (monensin, cytochalasin D, cycloheximide) did not inhibit the reorientation of the Golgi apparatus/microtubule-organizing center in cells exposed to a chemotactic gradient, and that the reorientation required extracellular Ca+2.

Animals↗

PubMed1995

A 62-kDa mitotic apparatus protein required for mitotic progression is sequestered to the interphase nucleus by associating with the chromosomes during anaphase.

A protein component of 62-kDa (p62) in the mitotic apparatus of the sea urchin embryo has been shown to be important for the proper progression of mitosis [Dinsmore and Sloboda, 1989: Cell 57:127-134]. To study the subcellular distribution of p62 during the cell cycle of sea urchin embryos, indirect immunofluorescence microscopy was used coupled to a modified detergent extraction procedure. The improved fluorescent images obtained by this procedure provide new information concerning the subcellular localization of p62 during the cell cycle that could not be obtained with previous conventional staining procedures [Johnston and Sloboda, 1992: J. Cell Biol. 119:843-854]. Using affinity purified antibodies to p62, we observed a cell cycle-dependent localization of p62 to the chromosomes/chromatin. Prior to nuclear envelope breakdown of the first or second cell cycle, p62 localizes to chromatin in the nucleus. During mitosis, p62 associates with the region of the spindle occupied by the microtubules of the mitotic apparatus. As anaphase proceeds, but before the nuclear envelope reforms, p62 becomes progressively associated with the chromosomes. Thus, p62 is incorporated into the forming interphase nucleus due to its association with chromosomes during late anaphase, rather than by active translocation into the newly formed daughter nuclei through the nuclear pores. The protein is not unique to marine embryos, as demonstrated by immunofluorescence of Y-1 cells, a mouse adrenal tumor cell line. In these cells, the localization of p62 is similar to the localization of the protein in echinoderm embryos, suggesting its possible function in mitotic progression in mammalian somatic cells as well.

Anaphase↗