Identification and characterization of a 38 kDa glycoprotein functionally associated with mating activity of Paramecium primaurelia

In Paramecium primaurelia mating interactions take place immediately after mixing mating-competent cells of opposite mating types. The cells clump in clusters (mating reaction) and then separate in pairs. Previous results have shown that sialic acid-containing glycoconjugates are present on the cell surface and are involved in mating-cell pairing. In order to identify the sialic acid-containing glycoprotein(s), we first metabolically radiolabelled non-mating-competent cells with D-[6-(3)H]galactose, and then analyzed the radiolabelled proteins by anion exchange chromatography. We characterized a 38 kDa (gp38) sialic acid-containing glycoprotein and raised the corresponding polyclonal antibody by means of which we localized the antigen at the level of the oral region of non-mating-competent cells and on the ciliary surface of mating-competent cells. Immunoblot analysis of the ciliary protein fraction showed that the anti-gp38 serum interacted with a 38 kDa protein in both mating types I and II cells. We also demonstrated the functional activity of gp38 in the mating reaction by means of anti-gp38 antibody competition assays.     

Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: starved versus nonstarved.

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent ("initiated") cells were compared with those from non-starved, mating-incompetent ("non-initiated") cells, by gel electrophoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glycoconjugate as having a potential role in control of pair formation during mating.     

Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: Starved versus nonstarved

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent (“initiated”) cells were compared with those from non-starved, mating-incompetent (“non-initiated”) cells, by gel electro-phoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glyco-conjugate as having a potential role in control of pair formation during mating. © 1992 Wiley-Liss, Inc.     

Inhibition of Pair Formation by Concanavalin A and Concanavalin A-Binding Glycoconjugates in Euplotes octocarinatus

ABSTRACT. Concanavalin A (≥ 50 μg/ml) inhibits pair formation in both of the two complementary mating types of Euplotes octocarinatus studied in this investigation. This effect can be reversed by methyl-α- d -mannose. Concanavalin A is accessible for methyl-α- d -mannose until pairs are formed. Methyl-α- d -mannose as well as methyl-α- d -glucose and 2-acetamido-2-deoxy- d -glucose alone do not inhibit pair formation unless applied in concentrations ≥ 60 mM. The Concanavalin A-sensitive phase of preconjugant interaction starts 2 h after cells are induced to conjugate. Based on these observations we suggest that Concanavalin A might exhibit its action by binding to carbohydrate moieties of preconjugation-specific adhesion molecules and thereby might allosterically block interactions with their counterparts. To identify preconjugation-specific alterations in number or localization of Concanavalin A-binding glycoconjugates, we probed western blots of total cell proteins or fixed cells, respectively, with digoxigenin-labeled Concanavalin A. On Concanavalin A blots 20 different Concanavalin A-binding glycoconjugates were identified in mating-competent cells. Localization of Concanavalin A-binding sites on mating-competent cells by light microscopy resulted in predominant labeling of a comma-shaped structure near the paroral membranelle. During the preconjugation period no changes in number or localization of Con A-binding glycoconjugates were detected. Possible reasons are discussed.     

Isolation of simian virus 40-transformed human mammary epithelial stem cell lines that can differentiate to myoepithelial-like cells in culture and in vivo

Simian Virus 40 (SV40) transformation of primary cultures of human mammary epithelial cells has yielded a cloned epithelial-like cell line and a representative, single-cell subclone. Although apparently homogeneous, both cloned cell lines can also yield small numbers of three other cell types. The more-elongated cell type can be obtained directly by replating cells from the medium of the epithelial-like cell cultures or by picking and culturing single cells to form representative lines. Immunofluorescent and immunocytochemical analysis of these cell lines growing on plastic or as tumor-nodules in nude mice for epithelial membrane antigens, various cytokeratins, various actins, laminin, Type IV collagen, the common acute lymphoblastic leukemia antigen (CALLA), and a 135-kDa glycoprotein confirm the epithelial nature of the epithelial-like cells and suggest a myoepithelial origin for the more-elongated cell type. Ultrastructural analysis largely confirms the results, although the myofilamental bundles can be scanty in the growing myoepithelial-like cells. The other two cell types are possibly related to the keratinizing and casein-secreting cells seen in the epithelial tumor-nodules before and after mating the mice, respectively. The myoepithelial-like cells produce 5- to 17-fold more laminin, Type IV collagen, CALLA, and the 135-kDa glycoprotein than the epithelial cells, and all of these antigens are preferentially found on myoepithelial cells in vivo. It is suggested that the SV40-transformed epithelial cell is an immortalized form of human mammary stem cell which can differentiate in culture and in vivo to myoepithelial-like cells.     

Affinity-Purification of Concanavalin A-Binding Ciliary Glycoconjugates of Starved and Feeding Tetrahymena thermophila

Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.     

Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cells of the 53-kDa precursor with no detectable exposure of this protein on the cell surface. It is concluded that type 1 carbohydrate, which is cotranslationally added in N-glycosidic linkages, is neither required for transport of the protein to the Golgi apparatus nor for type 2 glycosylation or protection of the protein against proteolytic degradation. Incapability of tunicamycin-treated cells of forming EDTA-stable cell contacts suggests a role for type 1 carbohydrate in cell adhesion. Type 2 carbohydrate is added posttranslationally. It is required in the absence of type 1 glycosylation for transport of the protein to the cell surface.     

Electron microscopy of cell surfaces of Paramecium caudatum stained with ruthenium red

The cell surface of Paramecium caudatum, syngen 3, was examined by electron microscopy using ruthenium red (RR) staining. The RR-positive surface coat is of uniform thickness and is found on the entire surface of paramecia, including the gullet area. The surface coat is also observed on the membranes in the tightly united region of conjugating cells. Measurements of the thickness of the surface coat in six stocks of P. caudatum demonstrate a significant difference between complementary mating types: cells of mating type VI have a thicker layer than those of mating type V. No detectable differences in morphology of the surface coat are observed between mating reactive and unreactive cells. Observation in detached cilia indicated that changes in the morphology of the surface coat provoked by the detachment procedure have no effect on mating reactivity. RR stainable substances are detected on both sides of ciliary membranes.     

Fig1 facilitates calcium influx and localizes to membranes destined to undergo fusion during mating in Candida albicans

Few mating-regulated genes have been characterized in Candida albicans. C. albicans FIG1 (CaFIG1) is a fungus-specific and mating-induced gene encoding a putative 4-transmembrane domain protein that shares sequence similarities with members of the claudin superfamily. In Saccharomyces cerevisiae, Fig1 is required for shmoo fusion and is upregulated in response to mating pheromones. Expression of CaFIG1 was also strongly activated in the presence of cells of the opposite mating type. CaFig1-green fluorescent protein (GFP) was visible only during the mating response, when it localized predominantly to the plasma membrane and perinuclear zone in mating projections and daughter cells. At the plasma membrane, CaFig1-GFP was visualized as discontinuous zones, but the distribution of perinuclear CaFig1-GFP was homogeneous. Exposure to pheromone induced a 5-fold increase in Ca(2+) uptake in mating-competent opaque cells. Uptake was reduced substantially in the fig1Δ null mutant. CaFig1 is therefore involved in Ca(2+) influx and localizes to membranes that are destined to undergo fusion during mating.     

A 130-kDa Protein 4.1B Regulates Cell Adhesion,Spreading, and Migration of Mouse Embryo Fibroblasts by Influencing Actin Cytoskeleton Organization

Protein 4.1B is a member of protein 4.1 family, adaptor proteins at the interface of membranes and the cytoskeleton. It is expressed in most mammalian tissues and is known to be required in formation of nervous and cardiac systems; it is also a tumor suppressor with a role in metastasis. Here, we explore functions of 4.1B using primary mouse embryonic fibroblasts (MEF) derived from wild type and 4.1B knock-out mice. MEF cells express two 4.1B isoforms: 130 and 60-kDa. 130-kDa 4.1B was absent from 4.1B knock-out MEF cells, but 60-kDa 4.1B remained, suggesting incomplete knock-out. Although the 130-kDa isoform was predominantly located at the plasma membrane, the 60-kDa isoform was enriched in nuclei. 130-kDa-deficient 4.1B MEF cells exhibited impaired cell adhesion, spreading, and migration; they also failed to form actin stress fibers. Impaired cell spreading and stress fiber formation were rescued by re-expression of the 130-kDa 4.1B but not the 60-kDa 4.1B. Our findings document novel, isoform-selective roles for 130-kDa 4.1B in adhesion, spreading, and migration of MEF cells by affecting actin organization, giving new insight into 4.1B functions in normal tissues as well as its role in cancer.     

Candida albicans MTLalpha tup1Delta mutants can reversibly switch to mating-competent, filamentous growth forms

Candida albicans strains that are homozygous at the mating type locus (MTLa or MTLalpha) can spontaneously switch from the normal round-to-oval yeast cell morphology to an elongated, so-called opaque cell form that can mate with opaque cells of the opposite mating type. In response to environmental signals, C. albicans also undergoes a transition from yeast to filamentous growth, which is negatively regulated by the general repressor Tup1p. Therefore, C. albicans mutants in which the TUP1 gene is inactivated grow constitutively in the filamentous form. We found that tup1Delta mutants of the MTLalpha strain WO-1 are still able to undergo phenotypic switching. Although the mutants had lost the capacity to grow in the normal yeast (white) or opaque forms, they could still reversibly switch between four different cell and colony phenotypes (designated as fuzzy, frizzy, wrinkled and smooth) at a frequency of about 10(-3) to 10(-4). Deletion of TUP1 resulted in deregulated expression of phase-specific genes. While the white-specific WH11 gene was constitutively expressed in all four cell types, the opaque-specific SAP1 gene remained repressed and the opaque-specific OP4 gene was weakly induced in all phase variants. In spite of the loss of white- and opaque-specific cell morphology and gene expression, the tup1Delta mutants retained an important characteristic of their wild-type parent, the ability to switch to a mating-competent form. The three filamentous phase variants (fuzzy, frizzy and wrinkled) all were able to mate and produce recombinant progeny with opaque cells of an MTLa strain at frequencies that were somewhat lower than those of normal opaque cells, whereas the smooth phase variant was unable to do so. Therefore, although deletion of TUP1 in C. albicans MTLalpha cells affects cellular morphology and gene expression patterns, the mutants can still reversibly switch between mating-competent and -incompetent cell types and mate with a partner of the opposite mating type.     

The role of plant villin in the organization of the actin cytoskeleton, cytoplasmic streaming and the architecture of the transvacuolar strand in root hair cells of Hydrocharis

In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the sub-cortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells. Received: 16 September 1999 / Accepted: 3 December 1999     

Motor protein Myo5p is required to maintain the regulatory circuit controlling WOR1 expression in Candida albicans

The Candida albicans MYO5 gene encodes myosin I, a protein required for the formation of germ tubes and true hyphae. Because the polarized growth of opaque-phase cells in response to pheromone results in mating projections that can resemble germ tubes, we examined the role of Myo5p in this process. We localized green fluorescent protein (GFP)-tagged Myo5p in opaque-phase cells of C. albicans during both bud and shmoo formation. In vegetatively growing opaque cells, Myo5p is found at sites of bud emergence and bud growth, while in pheromone-stimulated cells, Myo5p localizes at the growing tips of shmoos. Intriguingly, cells homozygous for MTLa in which the MYO5 gene was deleted failed to switch efficiently from the white phase to the opaque phase, although ectopic expression of WOR1 from the MET3 promoter can convert myo5 mutants into mating-competent opaque cells. However, when WOR1 expression was shut off, the myo5-defective cells rapidly lost both their opaque phenotype and mating competence, suggesting that Myo5p is involved in the maintenance of the opaque state. When MYO5 is expressed conditionally in opaque cells, the opaque phenotype, as well as the mating ability of the cells, becomes unstable under repressive conditions, and quantitative real-time PCR demonstrated that the shutoff of MYO5 expression correlates with a dramatic reduction in WOR1 expression. It appears that while myosin I is not directly required for mating in C. albicans, it is involved in WOR1 expression and the white-opaque transition and thus is indirectly implicated in mating.     

A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-src lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p60v-src and related oncogene products.     

A 130-kDa protein on endothelial cells binds to amino acids 15-42 of the B beta chain of fibrinogen.

Factors which stimulate the release of von Willebrand factor (vWf) from endothelial cell Weibel-Palade bodies and which induce the expression of the leukocyte-binding adhesion molecule P-selectin (PADGEM, GMP-140, CD62) on the endothelial cell surface remain incompletely characterized. Fibrin but not fibrinogen is a potent stimulus for the release of stored von Willebrand factor from endothelial cells. Removal of fibrinopeptides A and B from fibrinogen occurs during the formation of fibrin, and the removal of fibrinopeptide B is a requirement for fibrin to induce vWf secretion. The cleavage of fibrinopeptide A by reptilase enzyme forms a fibrin gel yet it is incapable of stimulating Weibel-Palade body degranulation. As a consequence of removing fibrinopeptide B, B beta 15-42 becomes the new NH2 terminus of the beta chain of fibrin. We have shown that the peptide B beta 15-42 in solution inhibits the release of vWf stimulated by fibrin. In addition, B beta 15-42 coupled to ovalbumin supports the binding and spreading of endothelial cells, while a scrambled form of this peptide coupled to the same carrier does not. We investigated whether these determinants near the amino terminus of the beta chain of fibrin bind to a specific protein on the surface of endothelial cells. A 130-kDa protein was isolated from surface-labeled human umbilical vein endothelial cells by specific binding to B beta 15-42 immobilized on Sepharose. This glycoprotein was eluted with the B beta 15-42 peptide in solution but not with the scrambled form of this peptide. The fibrin-derived peptides B beta 19-26 and B beta 37-56-cysteine were also incapable of eluting the 130-kDa protein bound to immobilized B beta 15-42 as were the arginine-glycine-aspartic acid-serine RGDS tetrapeptide and EDTA. The 130-kDa protein is recognized neither by antibodies to the known integrins found on endothelial cells nor by antibodies to CD31 (endoCAM, PECAM-1), a member of the immunoglobulin family of receptors found on endothelial cells. The beta chain of fibrin thus contains a sequence near its amino terminus which specifically binds to what is likely a novel endothelial cell surface protein. This glycoprotein may promote endothelial cell adhesion to fibrin during the wound healing process and is a candidate for a receptor involved in fibrin-mediated release of Weibel-Palade bodies from endothelial cells.     

Resistance of human cells to the adenovirus E3 effect on class I MHC antigen expression. Implications for antiviral immunity

Group C human adenovirus (Ad) serotypes (e.g., Ad2 and Ad5) cause persistent infections in man. One proposed mechanisms to explain human adenovirus persistence is an ineffective CTL response due to reduced cell surface expression of class I MHC Ag on virally infected cells, an effect mediated by the 19-kDa glycoprotein encoded by Ad early region 3 (E3). In the present study, the generality of this phenomenon was tested by analyzing E3 19-kDa glycoprotein down-regulation of cell surface class 1 MHC Ag on a variety of human cell types. With the exception of the Ad5 early region 1 (E1) transformed cell line, 293, Ad2/5 infection of fibroblastic, epithelial, and lymphoid cells did not cause major decreases in surface class I Ag until the terminal stages of infection when cell death is imminent. Furthermore, newly synthesized class I Ag continued to be surface expressed on most cell types at times when infected cells contained large amounts of Ad E3 19-kDa glycoprotein. These data indicate that most types of human cells are resistant to the E3 19-kDa glycoprotein effect, suggesting that virus-specific CTL recognition and lysis of most Ad2/5-infected human cells should not be limited by E3 19-kDa-mediated reduction in class I MHC Ag expression.     

In vivo and in vitro anaerobic mating in Candida albicans

Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37 degrees C, block production of farnesol, and permit in vitro mating at 37 degrees C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains.     

White Cells Facilitate Opposite- and Same-Sex Mating of Opaque Cells in Candida albicans

Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTL a) in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.     

Gametic differentiation in Chlamydomonas reinhardi: cell cycle dependency and rates in attainment of mating competency

Withdrawal of a utilizable nitrogen source during mid G₁ of the cell cycle induces gametic differentiation in synchronously grown vegetative cultures of Chlamydomonas reinhardi. Cell division accompanies gametic differentiation in such cultures, and the ability of mid G₁ vegetative cells to form gametes is matched by their ability to undergo a round of cell division after nitrogen withdrawal. Synchronously grown cultures require up to 19 hr in nitrogen-free medium to complete a round of division and to form mating-competent cells. Asynchronously grown liquid cultures require less time after nitrogen withdrawal (generally 5–8 hr) to achieve mating competency. In these cultures cell division did not necessarily accompany gametic differentiation since gametic differentiation took place in induced cultures at high cell concentrations which prevented cell division. Maximum mating competency was achieved in less than 2 hr after induction of vegetative cells grown on agar plates. Little cell division was observed during that short induction interval. The relationship between the attainment of mating competency (gametogenesis) and other physiological events resulting from nitrogen withdrawal is discussed.     

CD43 functions as a ligand for E-Selectin on activated T cells

E-selectin, an inducible cell adhesion molecule expressed on endothelial cells, mediates the rolling on endothelium of leukocytes expressing E-selectin ligands, such as neutrophils and activated T cells. Although previous studies using mice lacking P-selectin glycoprotein ligand-1 (PSGL-1) have indicated that PSGL-1 on Th1 cells functions as an E-selectin ligand, the molecular nature of E-selectin ligands other than PSGL-1 remains unknown. In this study, we show that a 130-kDa glycoprotein was precipitated by an E-selectin-IgG chimera from mouse Th1 cells. This protein was cleaved by O-sialoglycoprotein endopeptidase and required sialic acid for E-selectin binding. The mAb 1B11, which recognizes the 130-kDa glycoform of CD43, recognized the 130-kDa band in the E-selectin-IgG precipitate. In addition, immunoprecipitation of the E-selectin-IgG precipitate with 1B11 depleted the 130-kDa protein, further confirming its identity as CD43. CD43 was also precipitated with E-selectin-IgG from cultured human T cells. E-selectin-dependent cell rolling on CD43 was observed under flow conditions using a CD43-IgG chimera generated in Chinese hamster ovary cells expressing alpha-1,3-fucosyltransferase VII and a core 2 beta-1,6-N-acetylglucosaminyltransferase. These results suggest that CD43, when modified by a specific set of glycosyltranferases, can function as an E-selectin ligand and therefore potentially mediate activated T cell migration into inflamed sites.