Lydia C. Yuan

Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: Evidence for membrane cycling from Golgi to ER

Abstract In cells treated with brefeldin A (BFA), movement of newly synthesized membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus was blocked. Surprisingly, the glycoproteins retained in the ER were rapidly processed by cis/medial Golgi enzymes but not by trans Golgi enzymes. An explanation for these observations was provided from morphological studies at both the light and electron microscopic levels using markers for the cis/medial and trans Golgi. They revealed a rapid and dramatic redistribution to the ER of components of the cis/medial but not the trans Golgi in response to treatment with BFA. Upon removal of BFA, the morphology of the Golgi apparatus was rapidly reestablished and proteins normally transported out of the ER were efficiently and rapidly sorted to their final destinations. These results suggest that BFA disrupts a dynamic membrane-recycling pathway between the ER and cis/medial Golgi, effectively blocking membrane transport out of but not back to the ER.

Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic.

Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFAs effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.

Identification of mammalian sperm surface antigens: II. Characterization of an acrosomal cap protein and a tail protein using monoclonal anti-mouse sperm antibodies.

Monoclonal anti-mouse sperm antibodies have been produced by fusing mouse myeloma cells with spleen cells from rats immunized with epididymal sperm of C3H mice, Immunoprecipitation and immunoperoxidase techniques showed that one such monoclonal antibody, AMS IV-33, recognized a 200 000 dalton protein localized on the acrosomal cap of the sperm cell. Two other monoclonal antibodies AMS IV-54 and -76, reacted with a 68 000 dalton component on the surface of the sperm tail. Both antigenic targets were species specific and were present in about equal amounts on sperm from several different strains of mice. The tail protein was sperm specific, whereas the antibody reacting with the acrosomal cap protein also appeared to react somewhat with antigens present in other mouse tissues.

Lysosomal integral membrane glycoproteins are expressed at high levels in the inclusion bodies of I-cell disease fibroblasts☆

The localization, expression, and transport of two lysosomal integral membrane glycoproteins of human cells, hLAMP-1 and hLAMP-2, have been studied in mucolipidosis II (I-cell disease) fibroblasts. These cells are deficient in N-acetylglucosaminylphosphotransferase, one of the enzymes required for addition of the mannose 6-phosphate recognition signal to newly synthesized lysosomal hydrolases and a prerequisite for the sorting and transport of the hydrolases to lysosomes. I-cells analyzed by immunofluorescence microscopy with monoclonal antibodies against hLAMP-1 and hLAMP-2 showed intense staining of the inclusion bodies covering most of the cytoplasm of the cells. Immunoelectron microscopy confirmed this localization and showed that the hLAMP-positive vesicles commonly contained membrane structures or electron-dense homogeneous material characteristic of secondary lysosomes. Studies of the biosynthesis of hLAMP-2 in I-cells pulse-labeled with [35S]methionine indicated that the molecule is glycosylated in the Golgi system, is transported to vesicles with the high density characteristic of lysosomes, and has chemical properties similar to those of the glycoprotein synthesized in normal cells. The concentration of the hLAMP-2 glycoprotein was three- to fourfold greater than that in normal fibroblasts, in sharp contrast to the reduced levels of lysosomal hydrolases seen in I-cells. These experiments demonstrate that the inclusion bodies in I-cells have properties of secondary lysosomes and that the transport and targeting of the lysosomal membrane glycoproteins to the inclusion bodies of these cells is not coupled to the mannose 6-phosphate system for transporting soluble acid hydrolases.

Association of microperoxisomes with the endoplasmic reticulum in the granulosa lutein cells of the rhesus monkey (Macaca mulatta)

SummaryAldehyde fixed tissue from monkey (Macaca mulatta) corpus luteum was incubated in alkaline 3,3′-diaminobenzidine (DAB), and prepared for electron microscopic histochemical observations. The association of microperoxisomes with the granular (GER) or agranular (AER) endoplasmic reticulum was reconstructed from serially sectioned tissues and by tilting of specimens in the microscope. Out of 107 microperoxisomes, 106 were directly associated with the AER. Two different forms of attachment were found between microperoxisomes and the AER. In the first type of connection, the lumen of the AER and that of the microperoxisome are confluent. In the second, ungulate type of connection, a blunt-end structure either is inserted into an invagination of the AER, or penetrates into the lumen of the AER. The lumen of the lingula is confluent with the microperoxisome, but not with the AER. In addition to these connections, fine thread-like structures were observed extending between AER and adjacent microperoxisomes.

Progesterone production by dispersed monkey (macaca mulatta) luteal cells after exposure to trypsin

In an attempt to justify use of trypsin to achieve more thorough dispersion of luteal cell clumps in vitro, progesterone (P) production by collagenase dispersed monkey luteal cells from the mid-luteal phase corpus luteum (CL) was examined in vitro either after 10 min, or continuous (3h) exposure to trypsin (TR). In the first experiment, cells were pre-incubated in TR, then incubated at 37 degrees C for 3h with human chorionic gonadotropin (hCG) after the addition of soybean-trypsin inhibitor (STI). Pre-incubation of luteal cells with TR had no effect on the level of P production under basal conditions. Cells that were preincubated with TR responded to hCG stimulation with increased progesterone secretion (P less than 0.01) in a fashion similar to untreated cells. P production in response to hCG was independent of TR concentration over the range of 0.05% to 0.2% during the pre-incubation period. However, continuous exposure (3h) of cells to TR significantly depressed (P less than 0.01) basal P secretion and inhibited the response to hCG. We conclude that TR had no effect on the biopotency of hCG per se, but probably the over-exposure to TR had an adverse effect on the LH/hCG receptors. Addition of STI after a 10 min pre-incubation with TR, prevented these deliterious effects, thereby permitting the use of TR to improve the completeness of luteal cell dissociation.

Identification of mammalian sperm surface antigens. I. Production of monoclonal anti-mouse sperm antibodies**Preliminary observations of this study were presented at the Thirty-Seventh Annual Meeting of The American Fertility Society, March 14 to 18, 1981, Atlanta, Georgia, and received the Associate Members’ Award.

Surface antigens of mammalian sperm were studied by use of monoclonal antibodies (MAs). Six hybridoma cell lines were obtained by fusion of mouse myeloma cells with spleen cells from rats immunized with unwashed, epididymal sperm from C3H mice. Quantitative assessment of antibody binding, using a solid phase, antibody-protein A assay, indicated that four MAs bound to integral, sperm surface antigens; two others bound to nonintegral sperm antigens or epididymal fluid components. Immunofluorescence studies showed specific binding of individual MAs to localized regions: acrosome, midpiece, and midpiece and tail. All of these MAs inhibited sperm-egg binding, and those to the midpiece and/or tail immobilized sperm cells. The monoclonal antibodies provide probes for immunochemical characterization of sperm antigens and for elucidation of the role of the antigens in sperm.