Three dimensional configuration of the secretory pathway and segregation of secretion granules in the yeast Saccharomyces cerevisiae.

The structural elements of the secretory pathway in the budding yeast Saccharomyces cerevisiae were analyzed by 3D stereo-electron microscopy using relatively thick sections in which membranes were selectively impregnated. In a wild-type strain, tubular networks of various sizes and staining properties were distributed throughout the cytoplasm. As a rule, wide-meshed, lightly stained polygonal networks were connected to more or less fenestrated sheets of endoplasmic reticulum (ER). Some of these networks were continuous with more intensely stained networks and narrower meshes that displayed at their intersections nodular dilations that progressively increased in size and staining properties to reach those of secretion granules. Such networks presumably corresponded to Golgi elements. Indeed, stacked cisternae typical of the mammalian Golgi apparatus are rarely found in wild-type cells. However, if it is assumed that the Golgi apparatus plays a key role in the segregation and maturation of secretion granules, then tubular networks with nodular dilations should be equivalent to parts of this organelle. In correlation with the increase in size and density of the nodules there was a decrease in diameter and staining intensity of the interconnecting tubules. These results parallel observations on the formation of secretory granules in mammalian cells and suggest that the segregation of secretory material is concomitant with the progressive perforation and tubulization of previously unperforated sheets. When the sec21-3 thermosensitive mutant was examined at the nonpermissive temperature (37 degrees C), the secretory pathway was blocked at exit from the ER, which started to accumulate as clusters of narrow, anastomosed, unperforated ribbon-like elements. When the block was released by shifting down to permissive temperature (24 degrees C), tubular networks of various sizes and caliber, presumably Golgi in nature, formed as soon as 5 minutes after release of the block. At later time intervals, granules of various sizes and densities appeared to be released by rupture of these tubular networks or even to form at the edges of ER fenestrae. These observations support a dynamic maturation process in which the formation of secretion granules occurs by means of an oriented series of membrane transformations starting at the ER and culminating with the liberation of secretion granules from Golgi networks.

Effects of in vivo exposure of Mya arenaria to organic and inorganic mercury on phagocytic activity of hemocytes.

Marine bivalves are aquatic invertebrate organisms which can be used as bioindicators in environmental monitoring. In vivo effects of mercuric chloride (HgCl(2)) and methylmercury (CH(3)HgCl) on phagocytic function of Mya arenaria hemocytes were evaluated in this study. Clams were exposed to single metal in water for up to 28 days at concentrations ranging from 10(-9) to 10(-5) M. Phagocytic activity of hemocytes was determined by uptake of fluorescent microspheres and flow cytometry. All clams exposed to 10(-5) M HgCl(2) died by day 7 of exposure. The viability of hemocytes was decreased only in clams exposed to 10(-6) M HgCl(2) for 28 days. A significant decrease in phagocytic activity of hemocytes was observed in clams exposed to 10(-6) M of HgCl(2) for 28 days. A similar pattern was observed with CH(3)HgCl, but at an earlier time. Chemical analysis performed on the tissues of the animals clearly show a greater uptake of the organic form of mercury by clams. Furthermore, a clear correlation was established between body burden of mercury and effects on phagocytic activity of hemocytes. Overall, the results of this study show that both speciations of mercury inhibited phagocytic function of Mya arenaria hemocytes following in vivo exposures.

Role of endoplasmic reticulum-derived vesicles in the formation of Golgi elements in sec23 and sec18 Saccharomyces Cerevisiae mutants.

BACKGROUND: In the yeast Saccharomyces cerevisiae, the Golgi apparatus consists of individual networks of membranous tubules interspersed throughout the cytoplasm. When sec23 and sec18 mutants are shifted from the permissive (20 degrees C) to the restrictive (37 degrees C) temperature, the secretory pathway is blocked between endoplasmic reticulum (ER) and Golgi elements. When examined with an electron microscope, sec23 displays an excess of ER membranes, whereas sec18 accumulates small vesicles. The present investigation describes the kinetics of the ultrastructural modifications of the Golgi and vesicular elements when sec23 and sec18 mutants are shifted for 10 min to restrictive temperature and then returned to permissive temperature for various time intervals. METHODS: S. cerevisiae sec23 and sec18 mutants from exponentially growing cultures at 20 degrees C were maintained for 10 min at the restrictive temperature of 37 degrees C and returned to the permissive temperature of 20 degrees C for different time intervals. Following fixation in glutaraldehyde and postfixation in potassium ferrocyanide reduce osmium, 80- to 200-nm-thick sections were prepared from Epon-embedded yeast cells. Using the thicker sections, stereopairs of electron microscopy photographs were prepared and used to visualize the three-dimensional configuration of the organelles. To follow the modifications of cell organelles, cell sections were selected at random in thinner sections and cell organelles were scored. RESULTS: At permissive temperature (20 degrees C), the Golgi apparatus consisted of individual networks of tubules dispersed in the cytoplasm, as in the wild type strain. When both mutants were shifted for 10 min at the restrictive temperature (37 degrees C), the main structural feature was the disappearance of all Golgi networks. In sec23 mutant cells, there was an increase in number of tubular, nonnodular networks corresponding to terminal portions of the endoplasmic reticulum; in sec18 cells, small 20- to 50-nm tubules and vesicles accumulated in the cytoplasm. Within minutes after the return of sec23 cells to permissive temperature (20 degrees C), small vesicles and tubules started to accumulate to reach a number similar or greater than that noted in sec18 cells observed under the same conditions. At later time intervals and in both mutants, the small tubules and vesicles decreased in number. This decrease was concomitant with the reappearance of fine nodular networks, followed later on by the reconstruction of networks of larger caliber and the formation of secretion granules. CONCLUSIONS: It is concluded that a block of the secretory pathway upstream of the Golgi compartment leads to the disappearance of Golgi networks. The small vesicles and tubules that accumulate during the block in sec18 cells and within minutes after the shift at 20 degrees C in sec23 cells appear to fuse together to form new Golgi networks. Thus, the small vesicles would not fuse with a preexisting Golgi apparatus but would rather fuse together to produce new Golgi networks. Such networks appear as transitory structures continuously undergoing renewal.

Impaired fertility in mice deficient for the testicular germ-cell protease PC4.

PC4 is a member of the proprotein convertase family of serine proteases implicated in the processing of a variety of polypeptides including prohormones, proneuropeptides, and cell surface proteins. In rodents, PC4 transcripts have been detected in spermatocytes and round spermatids exclusively, suggesting a reproductive function for this enzyme. In an effort to elucidate this function, we have disrupted its locus (Pcsk4) by homologous recombination in embryonic stem cells and have produced mice carrying the mutation. In intercrosses of heterozygous mutant mice, there was low transmission of the mutant Pcsk4 allele to the progeny, resulting in lower than expected incidence of heterozygosity and null homozygosity. The in vivo fertility of homozygous mutant males was severely impaired in the absence of any evident spermatogenic abnormality. In vitro, the fertilizing ability of Pcsk4 null spermatozoa was also found to be significantly reduced. Moreover, eggs fertilized by these spermatozoa failed to grow to the blastocyst stage. These results suggest that PC4 in the male may be important for achieving fertilization and for supporting early embryonic development in mice.

Modifications of the Golgi apparatus in Saccharomyces cerevisiae lacking microtubules.

BACKGROUND: Disassembly of cytoplasmic microtubules by nocodazole in cultured mammalian cells leads to the disruption of the continuous ribbonlike Golgi apparatus and dispersal of the Golgi elements from their normal juxtanuclear location, close to the microtubule-organizing center (MTOC), toward the cell periphery. Clearing of the drug induces reassembly of the microtubules from the MTOC and reorganization of the Golgi elements into a continuous ribbonlike juxtanuclear structure. In the yeast Saccharomyces cerevisiae, the Golgi apparatus does not form a continuous structure as in mammalian cells but instead constitutes independent units dispersed throughout the cytoplasm. It is the purpose of this article to investigate the role of microtubules in the structure and distribution of the Golgi elements in S. cerevisiae by studying the ultrastructure of cell organelles either in mutant cells deficient in beta-tubulin or in wild-type cells treated with the microtubule-depolymerizing drug nocodazole. METHODS: Two S. cerevisiae yeast strains were used in this study: a control wild-type strain, CUY226 (ade2-101, his3-delta 200, leu2-delta 1, lys2-801, ura3-52 Mat alpha), and a mutant strain, CUY66 (tub2-401, ade2-101, ura3-52, Mat alpha). Nocodazole was added to the wild-type cells cultivated at 30 degrees C, and cells were fixed 5 min, 20 min, and 60 min, respectively, after adding the drug to the culture. Both strains were fixed and examined 5 min, 20 min, and 60 min after shifting the cultures from the permissive temperature of 30 degrees C to the restrictive temperature of 14 degrees C. Cells were fixed in 2% glutaraldehyde, treated for 15 min in 1% sodium metaperiodate, postfixed in reduced osmium, and embedded in Epon. To visualize the three-dimensional configuration of cell organelles, stereopairs were prepared from sections stained with lead citrate and tilted at +/- 15 degrees from the 0 degree position of the goniometric stage of the electron microscope. RESULTS: In mutant cells shifted to restrictive temperature and wild-type cells treated with nocodazole, the main ultrastructural modification was a fragmentation of networks of membranous tubules, which probably correspond to the yeast Golgi apparatus. Secretion granules were still present in growing buds, and they were dispersed in the cytoplasm, which contained in addition numerous small vesicles in the 30-60-nm diameter range. CONCLUSIONS: In normal cells, small vesicles may originate from the endoplasmic reticulum and fuse together to give rise to Golgi networks (Rambourg et al. 1994. Anat. Rec., 240:32-41). If this hypothesis is correct, the observations reported might indicate that intact microtubules orient the flow of small vesicles and favour their fusion into Golgi networks.

Transformations of membrane-bound organelles in sec 14 mutants of the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica.

BACKGROUND: In early descriptions of ultrastructural alterations of secretory (sec) mutants of the yeast Saccharomyces cerevisiae, two mutants, sec7 and sec14, were shown to produce cell structures, the so-called Berkeley bodies thought at first to correspond to Golgi structures. In sec7 mutants grown at restrictive temperature, secretion granules soon dis-appeared, whereas networks of Golgi tubules increased in size and transformed into stacks of seven to eight flattened elements. At these time intervals, structures resembling Berkeley bodies appeared to be extensions of the endoplasmic reticulum (Rambourg et al., 1993). It is the purpose of the present study to examine by electron microscopy S. cerevisiae sec14 mutants and to compare the modifications along their secretory pathway with those occurring in a homologous mutant of Yarrowia lipolytica. METHODS: S. cerevisiae sec 14 mutant cells coming from exponentially growing cultures were examined either at 24 degrees C or after shifting at 37 degrees C for 0, 2, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min. Y. lipolytica mutant cells were first cultured in YNB in 5000 medium and then transferred for 0, 6, 8, 12, 20, and 24 hr, in a phosphate-buffered YPD medium, which allows wild cells to differentiate from yeast to mycelian form. In both cases, cells were fixed in 2% glutaraldehyde, treated for 15 min in 1% sodium metaperiodate, post-fixed in reduced osmium, and embedded in Epon. To visualize the three-dimensional configuration of cell organelles, stereopairs were prepared from section stained with lead citrate and tilted at +/- 15 degrees from the 0 degree position of the goniometric stage of the electron microscope. RESULTS: In S. cerevisiae mutant cells shifted for 2 min at the restrictive temperature, faintly stained networks of thin anastomosed tubules were located at close proximity and often continuous with faintly stained ER cisternae. More intensely stained tubular networks with nodular dilations having the size of secretion granules were dispersed throughout the cytoplasm. Later on, the faintly stained ER elements and related tubular networks decreased in number, whereas the intensely stained nodular tubular networks increased in frequency. The incidence of secretion granules also increased and were distributed at random throughout the cytoplasm. Widemeshed, intensely stained fenestrated spheres were often encountered and increased in number, in parallel to the increase in the number of nodular tubular networks. At late time intervals, the fenestrated spheres decreased in number as they seemingly transformed into spherical bodies identical to vacuoles. In contrast to what occurred in S. cerevisiae sec14 mutant, the main ultrastructural modification observed in Y. lipolytica transferred to the YPD medium was the formation of deep plasma membrane invaginations. CONCLUSIONS: It appears that two functionally homologous PI/PC transfer proteins (Sec14psc and Sec14pyl) control distinct physiological processes in the two sec14 mutants examined. Such differences are perhaps related to the regulatory role of these proteins in different target organelles, i.e., the Golgi apparatus in S. cerevisiae or the plasma membrane in Y. lipolytica.

Three-dimensional structure of tubular networks, presumably Golgi in nature, in various yeast strains: a comparative study.

BACKGROUND: In the yeast Saccharomyces cerevisiae, the Golgi apparatus consists of discrete units distributed throughout the cytoplasm. When such units are examined in three dimensions, in relatively thick sections prepared for the electron microscope, they usually appear as small tubular networks with a stained material accumulating in dilations located at the junctions of membranous tubules. To see whether such tubular networks are observed in other yeast species, the three-dimensional structure of organelles in eight additional yeast strains, endowed with diverse biological properties, are examined. METHODS: Yeast strains were grown at 24 degrees C in YPD medium (2% Bactopeptone, 1% Bactoyeast extract, and 2% glucose). Cells that were examined by electron microscopy came from exponentially growing cultures grown in a shaking water bath and maintained at a OD 600 (optical density at 600 nm) of 0.5. Cells were fixed in a fixative containing 2% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 and 0.8 M sorbitol. They were then treated for 15 min in 1% sodium metaperiodate and postfixed for 1 hr in potassium ferrocyanide-osmic acid. They were preembedded in agarose prior to dehydration and finally embedded in Epon. In these conditions, the preservation of cell organelles was improved and the cytoplasmic retraction from the cell wall was minimized. Photographs of sections tilted at +/- 15 degrees from the 0 degrees position of the goniometric stage were used to prepare stereopairs from which the three-dimensional configuration of the organelles was visualized. RESULTS: In all yeast strains, tubular networks appeared as separate elements or units disperse throughout the cytoplasm. Each unit consisted of anastomosed membranous tubules. In some strains such as Saccharomyces cerevisiae, Zygosaccharomyces rouxii, or Saccharomyces pombe, such units appeared mainly as polygonal networks of intensely stained membranous tubules. Along these networks, distensions filled with stained material were similar in size to nearby secretory granules, suggesting that the latter formed by fragmentation of the tubular networks. In Hansenula polymorpha, Pichia pastoris, and Debaryomyces hansenii, networks of anastomosed tubules were closely superposed to each other and formed parallel arrays reminiscent of the stacks of Golgi saccules seen in mammalian cells. However, in contrast to what is usually found in the latter, the layers making up the parallel arrays in yeasts, were clearly continuous to each other. In other strains, i.e., Kluyveromyces lactis, Candida albicans, and Candida parapsilosis, the situation was intermediate and their cytoplasm contained only arrays of small size with two or at most three superposed layers of membranous tubules. Small vesicles in the 30-50 nm range were rarely encountered in most yeast strains. CONCLUSIONS: It is therefore concluded that tubular networks, presumably Golgi in nature, are present in all yeasts examined so far. Yet, in some strains, these tubular networks may be arranged in parallel arrays or stacks.

Trans-Golgi network (TGN) of different cell types: three-dimensional structural characteristics and variability.

BACKGROUND: The trans-Golgi network (TGN) is generally considered as a distinct and permanent structural compartment of the Golgi apparatus of various cell types. To verify this postulate we examined and compared the three-dimensional characteristics of the TGNs of 14 different mammalian cell types as presented in our various publications since 1979 when we initially described the trans-tubular network of Sertoli cells. METHODS: In all these studies we used low and high voltage electron microscopes on thin or thick sections of tissues fixed with glutaraldehyde and postfixed with reduced osmium. The sections were stained with uranyl acetate and lead citrate. Stereopairs, prepared from photographs of tilted specimens, permitted a direct observation of the three-dimensional structure of the various elements of the Golgi apparatus. RESULTS: The TGNs are multilayered and extensive in cells which do not form large typical secretory granules (Sertoli cells, nonciliated cells of ductuli efferentes, spinal ganglion cells) but have an extensive lysosomal system. The TGN is absent in cells forming very large secretory granules (secretory cells of seminal vesicles and lactating mammary glands). The TGNs are small in cells producing small to medium-size secretory granules and/or appear as residual fragments on the trans aspect of the Golgi stacks (e.g., mucous cells of Brunner's gland, pancreatic acinar cells, etc.). In cells with multiple and extensive TGNs, a continuity of these tubular networks with the two or three transmost saccules of the stack is observed but there are seemingly no connections between the TGNs. Whenever the TGNs are present, they do not form a continuous structure along the Golgi ribbon. However, they do present, in all cases, configurations suggestive of desquamation and renewal. CONCLUSIONS: The structure of the TGN varies considerably from one cell type to another, being extensive in cells not showing typical secretory granules but having an extensive lysosomal system, while in secretory cells showing small or large secretory granules the TGN is either small or even entirely absent.

Effects of brefeldin A on the three-dimensional structure of the Golgi apparatus in a sensitive strain of Saccharomyces cerevisiae.

BACKGROUND: Brefeldin A (BFA), when added to the medium of cultured mammalian cells, induces a reversible block of secretion and disrupts the Golgi apparatus whereas Golgi enzyme markers appear to redistribute into the endoplasmic reticulum (ER). It has been shown in addition that in mammalian cells, BFA would prevent the assembly of coatomer proteins (COP) onto membranes by inhibiting the GTP-dependent interaction of the ADP-ribosylation factor (ARF) with such membranes. The purpose of the present study is to analyze, by stereoelectron microscopy, the structural modifications of Golgi elements and of the ER-Golgi relationship in a BFA-sensitive yeast mutant, S. cerevisiae erg6. METHODS: S. cerevisiae erg6 cells were placed in a medium containing 100 micrograms/ml BFA dissolved in 1% alcohol and collected after exposures of 0.5, 1.5, 5, 10, 15, 20, 30, and 70 min to the drug. Yeasts placed in a BFA-free medium but containing 1% alcohol served as controls. After fixation in 2% glutaraldehyde, the cells were postfixed in reduced osmium and embedded in Epon. Then 0.08-0.2 microns thick sections stained with lead citrate were examined with the electron microscope. Photographs of the thicker sections, tilted at +/- 15 degrees from the 0 degree position of the goniometric stage, were used to prepare stereopairs from which the three-dimensional configuration of the organelles was visualized. Since BFA is known to prevent the interaction of ARF with membranes, the phenotype of the arf1 mutant deficient in this protein was also examined for comparative purposes. RESULTS: In control cells, as in wild-type strains, two types of Golgi elements were observed: small networks of fine tubules seen close and occasionally connected to ER cisternae and coarser tubular networks showing nodular distensions having a size comparable to that of secretion granules. The latter networks were considered as trans-Golgi elements and the former as cis-Golgi elements. Several networks of both types were distributed throughout the cytoplasm. At short time intervals (0.5-5 min) of BFA treatment, the trans-Golgi elements disappeared from the cytoplasm, while the ER-connected cis-Golgi elements developed and formed large spheroidal masses frequently showing concentrically arranged fine tubular networks. Such spheroidal, cage-like structures later disappeared, and after 30 min Golgi elements were no longer identifiable, while ER cisternae assumed pleomorphic configurations as the cells showed signs of degeneration. S. cerevisiae arf1 mutants presented a phenotype similar to that of BFA-treated S. cerevisiae erg6. CONCLUSIONS: It is therefore concluded that soon after exposure to BFA there is, in this sensitive yeast mutant, a transitory hypertrophy of the ER-connected cis-Golgi network presumably resulting from a block at the exit end of this compartment. At longer time intervals (i.e., after 30 min) the Golgi elements are no longer formed, and the cells present signs of cell degeneration.

Connections between the various elements of the cis- and mid-compartments of the Golgi apparatus of early rat spermatids.

BACKGROUND: The exact structural relationships of the saccules, membranous tubules, and vesicles that compose the cis- and mid-compartments of the Golgi cortex of rat spermatids was investigated to determine the relationship of these elements to each other. METHODS: Tissues fixed with glutaraldehyde and buffered in sodium cacodylate were examined with the electron microscope. Electron micrographs, including stereopairs, were analyzed to determine the three-dimensional organization of the Golgi elements. RESULTS: The deeper layer of the Golgi cortex was composed of stacks of saccules connected to each other either by saccules or membranous tubules. The peripheral region of the Golgi cortex, located between the cis-side of the stacks and a network of overlying ER cisternae contained numerous membranous tubules and vesicles of two class sizes: 50-100 nm vesicles and microvesicles 5-10 nm in diameter. The tubules formed tight networks, known as cis-elements or cis-Golgi networks (CGN), which were strictly parallel and next to the first or cis-saccule of the stack. The cis-elements were continuous with more loosely arranged peripheral tubules which formed elaborate, intertwined and interconnected networks. These peripheral tubules closely approximated the overlying ER cisternae in areas often showing fuzz-coated finger-like projections. Occasionally such peripheral tubules were continuous with ER cisternae. The saccules forming the stacks were continuous with membranous tubules which not only connected saccules of adjacent stacks, but also saccules of the same stack. These tubules were also connected with the tight tubular networks forming the cis-elements and the broad networks formed by the peripheral membranous tubules. Vesicles (50-100 nm) and microvesicles (5-10 nm) frequently formed aggregates in the peripheral Golgi region next to areas of ER membrane free of fuzz-coated projections. The microvesicles, embedded in a denser cytoplasmic matrix, had a more or less distinct delimiting membrane suggestive of their disintegration in this juxta-ER location. The 50-100 nm vesicles that were seen at the periphery of the vesicular aggregates appeared to form mainly from the membranous tubules of the Golgi cortex. CONCLUSIONS: Thus the saccules and membranous tubules of the spermatid's Golgi cortex formed a single continuous membraneous system connected to ER cisternae. The vesicles, seemingly arising from the membranous tubules, appear to follow a retrograde pathway and undergo dissolution next to ER cisternae.

Ultrastructural modifications of vesicular and Golgi elements in the Saccharomyces cerevisiae sec21 mutant at permissive and non-permissive temperatures.

BACKGROUND: The secretory protein transit between cisternae of endoplasmic reticulum (ER) and Golgi elements is blocked when the yeast Saccharomyces cerevisiae sec21 mutant is shifted from the permissive (24 degrees C) to a non-permissive (37 degrees C) temperature, but 30-50 nm vesicles accumulate in the cytoplasm. At the semi-permissive temperature of 33 degrees C there is no complete block but rather a slowdown of the protein transport between ER and Golgi. The purpose of the present investigation is to analyze the structural expression of these events. METHODS: S. cerevisiae sec21 mutants were maintained for 90 min at semi-restrictive (33 degrees C) or restrictive (37 degrees C) temperatures and then progressively returned to 24 degrees C. Following fixation in glutaraldehyde and a postfixation in potassium ferrocyanide reduced osmium, 0.08 to 0.2 microns thick sections were cut from Epon embedded yeasts. Using the thicker sections, stereopairs of electron microscope photographs were prepared and used to visualize the three-dimensional configuration of the organelles. RESULTS: At permissive temperature, the Golgi elements appeared as isolated networks of membranous tubules dispersed throughout the cytoplasm. The diameter of these membranous tubules varied considerably from one Golgi element to another. Larger tubules showed at their intersections distensions with size and staining intensity comparable with that of the secretory granules seen at proximity of the Golgi networks or at the cell periphery. Small vesicles in the 30-50 nm size range were rarely if ever observed in cells grown at permissive temperature. Golgi networks and secretion granules were less conspicuous in mutant cells maintained at 33 degrees C and completely disappeared at 37 degrees C. In both cases, the main structural feature was the presence in the cytoplasm of numerous small vesicles and of short membranous tubules with a diameter identical to that of the small vesicles. As soon as 5 minutes after shifting mutants from 33 degrees C to 24 degrees C, the small vesicles disappeared from the cytoplasm, while secretory granules were actively produced in extensively developed Golgi network. When mutants were returned from 37 degrees C to 24 degrees C, the disappearance of small vesicles was more progressive and concomitant with the progressive reconstruction of Golgi networks. CONCLUSIONS: It is thus postulated that, in the above mentioned conditions, the small vesicles of the sec21 mutant did not act as intermediate carriers between the endoplasmic reticulum and a pre-existing Golgi apparatus, but rather fused together to produce newly formed Golgi networks.

Molecular cloning and developmental expression of an mRNA encoding the 27 kDa outer dense fiber protein of rat spermatozoa.

We have isolated a cDNA (ODF27), encoding the major 27 kDa protein of rat sperm outer dense fibers (ODF), by screening a testicular lambda-gt11 phage cDNA library with an affinity-purified anti-27 kDa ODF polyclonal antibody. A cyanogen bromide derived internal amino acid (a.a.) sequence of the 27 kDa ODF protein was identical to an internal region of the deduced a.a. sequence of this cDNA. The cDNA encodes a protein with a high proportion of a repetitive motif, Cys-Gly-Pro, at the carboxy-terminal end, reminiscent of the testis-specific Mst(3)CGP proteins of Drosophila melanogaster (Schäfer et al., 1993. Mol Cell Biol 13:1708-1718). Nick translation probes of the ODF27 cDNA recognized two complementary mRNAs of 1.2 and 1.5 kb in the rat testis. Developmental Northern blot analysis revealed that these mRNAs are first transcribed in round spermatids. In situ hybridization confirmed the haploid expression of these transcripts and demonstrated that they are found in the cytoplasm of spermatids throughout most of the duration of spermiogenesis. They reach a peak in steps 8-10 of spermiogenesis at the time transcription ceases, remain at high levels from steps 11 to 15, and diminish in steps 16-18 at the time ODF protein synthesis and assembly are shown to be maximum. The translation of these transcripts, therefore, appears to be post-transcriptionally controlled. A literature and NCBl database search revealed that the nucleotide sequence of the 027 cDNA is homologous to the rat gene RT7 (Van Der Hoorn et al., 1990. Dev Biol 142:147-154) and to the rat testis-specific cDNA rts 5/1 (Burfeind and Hoyer-Fender, 1991. Dev Biol 148:195-204), which encodes a 27 kDa polypeptide.

Modulation of the Golgi apparatus in Saccharomyces cerevisiae sec7 mutants as seen by three-dimensional electron microscopy.

The three-dimensional configuration of the Golgi apparatus has been examined with the electron microscope in thick Golgi sections of Saccharomyces cerevisiae prepared from a wild-type strain and from sec7 mutants maintained for various periods of time at the nonpermissive temperature of 37 degrees C and then returned to the permissive temperature of 24 degrees C. Reduced osmium postfixation of glutaraldehyde fixed specimens stained intensely the content of Golgi elements and thus facilitated their three-dimensional characterization. In wild-type S. cerevisiae, the Golgi elements usually appeared as isolated networks of membranous tubules dispersed throughout the cytoplasm. Along such networks, distensions filled with stained material were similar in size to nearby secretory granules, suggesting that the latter formed by fragmentation of the Golgi elements. In sec7 mutants maintained at 37 degrees C in low (0.1%) glucose medium, secretion granules progressively decreased in number and soon disappeared. Concomitantly the networks of Golgi tubules increased in size and complexity, lost their distensions, and then transformed into flattened saccules forming stacks of up to seven or eight saccules that were similar to the Golgi stacks seen in mammalian cells. However in contrast to the latter, connections between the saccules were evident and Golgi-associated small vesicles were generally absent. Following return to the permissive temperature (24 degrees C), secretion granules reappeared, the Golgi stacks progressively decreased in size, and resumed their initial state of separated small tubular networks. Thus in sec7 mutant, grown at 37 degrees C in low glucose medium, segregation of secretory granules is blocked. As a result, Golgi membranes accumulate to form a continuous system of stacked and interconnected saccules.

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.

Striated anchoring fibrils-anchoring plaque complexes and their relation to hemidesmosomes of myoepithelial and secretory cells in mammary glands of lactating rats.

Striated anchoring fibrils (SAF) are associated with the basement membrane underlying myoepithelial and acinar cells of mammary glands. Their proximal extremities are inserted in electron-dense areas of the lamina densa, the anchoring plaques seen facing the hemidesmosomes of both myoepithelial and acinar cells. In the case of myoepithelial cells, the hemidesmosomes show a thick cytoplasmic plaque applied to the basal plasma membrane in which cytoplasmic filaments are inserted. Facing this plaque but on the extracellular aspect and at a short distance of 5-10 nm, there is a thin layer of electron-dense nodular material called the subcell membrane plate, which is connected to the plasma membrane by short filamentous bridges. Between this subcell membrane plate and the anchoring plaque, there is an abundance of fine anchoring filaments crossing the lamina lucida. Such anchoring filaments are less abundant in the lamina lucida outside the hemidesmosomal areas. In the case of acinar cells, the cytoplasmic plaques of the hemidesmosomes are thin and the associated cytoplasmic filaments less conspicuous. No distinct subcell membrane plate is seen on the extracellular aspect of the plasma membrane facing the cytoplasmic plaque of the hemidesmosomes. However, in this area numerous anchoring filaments cross the lamina lucida between the plasma membrane and the SAF-anchoring plaque complex. The abundance, in these cells, of hemidesmosomes and their association with SAF-anchoring plaque complexes seen in the basement membrane must constitute a strong attachment for both myoepithelial and acinar cells and bind them to the underlying collagen fibrils, thus preventing their detachment from the connective tissue during the contractions of myoepithelial cells during milk ejection.

Modulation of the Golgi apparatus in stimulated and nonstimulated prolactin cells of female rats.

The three-dimensional structure of the Golgi apparatus and its compartments in prolactin cells has been examined in lactating rats in which secretion of prolactin was suppressed by removing the litter or stimulated by allowing the pups to suckle again. As soon as 2 hr after removal of the litter, large irregular progranules and numerous large pale vesicles accumulated in the trans-Golgi area together with vesicular or tubular fragments. The cis-tubular network was no longer recognizable on the cis-face of the Golgi ribbon; the saccules of the midcompartment were partitioned by narrow fissures and also became perforated in register by numerous fenestrations of various sizes and irregular contours. The concomitant appearance of numerous vesicles in the cavities thus formed as well as in the surrounding cytoplasm indicated that they probably arose by the progressive cavitation and fragmentation of saccules of the mid compartment. Such a process, which reached a maximum between 4 and 6 hr after removal of the litter from the mother, was no longer observed at 8 and 12 hr, at which time intervals the Golgi apparatus was reduced in size with no cis-tubular elements and progranules on its trans-aspect and few vesicles in its surroundings. When mothers, separated from their litters for a period of 12 hr, were returned to their pups for 20 min, the cis-tubular network reappeared on the cis-aspect of the Golgi stacks and presumably formed by fusion of vesicles and anastomosed tubules located next to the cisternae of the rough endoplasmic reticulum. In addition, the structure of the midsaccules returned to the stimulated condition, and early progranules were again segregated within the trans-most saccules of the Golgi stack. Hence, the Golgi apparatus of prolactin cells was rapidly and deeply modified in the presence or absence of stimulation.

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.

Segregation of secretory material in all elements of the Golgi apparatus in principal epithelial cells of the rat seminal vesicle.

At the apex of the epithelial principal cells of the seminal vesicle, there appears to be two types of mature secretory granules, i.e., large and small. Both types of secretory granules showed an eccentric electron-dense spherical body with one pole attached to the delimiting membrane. The remainder of the large granule surrounding the eccentric body showed a granulofilamentous texture, whereas that of the small granule was electron lucent. The formation of these two types of granules was traced back to the various elements of the Golgi stacks. In the case of the large granules, the earliest stage of segregation of the precursor of the eccentric dense body was observed in distensions of the cis-element. Within distensions of all subjacent saccules, the dense bodies continued to be present but progressively increased in size while remaining attached to the saccular membrane. Following separation from the trans-face of the stack, the large prosecretory granules continued to increase in size by fusing with each other. The very large prosecretory granules, as they migrated toward the cell apex to become mature secretory granules, reduced in size prior to exocytosis. The small granules formed exclusively on the trans-aspect of the Golgi stacks and did not appear to fuse with each other. Observations on the formation of the large prosecretory granules within the Golgi apparatus and of the eccentric body in particular, which may be taken as a marker of the saccular membrane, were suggestive of a cis-trans migration and renewal of Golgi saccules.

Formation of secretory granules in the Golgi apparatus of prolactin cells in the rat pituitary gland: a stereoscopic study.

The mode of secretory granule formation in prolactin cells was analyzed in thin or thick sections of pituitary glands from non-lactating or lactating female as well as from male rats. In all these animals, the Golgi apparatus of prolacting cells consists of a continuous twisted ribbon-like structure that branches and anastomoses to form a hollow sphere located in the juxtanuclear area. The early signs of secretory granule formation are observed along the trans-aspect of the Golgi ribbon where progranules appear as focal distensions simultaneously occurring anywhere in the last trans thiamine pyrophosphatase (TPPase)-containing Golgi element. In the transmost Golgi saccule, such dilatations usually contain several nodular masses of electron opaque material which are separated from each other and from the saccular membrane by a less intensely stained material. While this transmost saccule becomes more fenestrated, its focal polynodular distensions seemingly yield polynodular tubular progranules which are initially closely apposed and usually parallel to the trans face of the Golgi ribbon. Subsequently, these progranules, which frequently show small membranous tubules or tubular networks attached to them, are seen some distance from the Golgi stacks and progressively transform into the more compact polymorphous granules characteristic of prolactin cells. These observations suggest that the polynodular tubular progranules arise by fragmentation of portions of the trans-Golgi elements rather than by fusion of small uninodular granules budding from the edges of a trans-Golgi saccule. Once the progranules have been liberated, the rest of the transmost Golgi element appears to break down into small residual networks, tubules, and vesicles. Thus, in prolactin cells as in other glandular cells, the whole transmost Golgi element would fragment during formation of prosecretory granules.

Three-dimensional structure of cytidine monophosphatase reactive trans-Golgi elements in spinal ganglion cells of the rat.

In order to analyse, at the electron microscope level, the three-dimensional configuration of the trans compartment of the Golgi apparatus rat dorsal root ganglia were treated to demonstrate cytidine monophosphatase (CMPase) activity. The localization of enzymatic activity in the Golgi apparatus varied according to cell types. In type A and C cells, CMPase was exclusively located in the transmost sacculotubular element, whereas in type B cells all the saccules of the stacks forming the Golgi ribbon and the trans-Golgi networks were impregnated. Numerous dense bodies seen at proximity were also CMPase positive. In 3 microns thick sections of type A cells examined at low magnification, the impregnated element was scattered throughout the cytoplasm and never formed a continuous structure. In type B cells, the strongly reactive trans-Golgi networks did not follow the entire length of the impregnated Golgi ribbon but were preferentially located in the concavity of its arched portions. At higher magnification and in all cell types some tubular portions of the trans-Golgi networks took the appearance of spheroidal cage-like structures, the CMPase positive anastomotic tubules forming the bars of the cage. Anastomotic tubules separated from the trans-Golgi networks formed fenestrated spheres, while nearby CMPase-reactive dense bodies exhibited a paler hilus. These observations were taken to indicate that in ganglion cells, some CMPase positive dense bodies, presumably lysosomes, formed by fragmentation of the trans-Golgi networks.