Cell surface changes during mitosis and cytokinesis of epithelial cells

Summary PtK2 cells were studied with scanning electron microscopy to record changes on the cell surface during mitosis and cytokinesis. During prophase, prometaphase and metaphase, the cells remain very flat with few microvilli on their surfaces. In anaphase cells, there is a marked increase in the number of microvilli, most of which are clumped over the separating chromosomes and polar regions of the mitotic spindle leaving the surface of the interzonal spindle region relatively smooth. Microvilli appear over the interzonal spindle region in telophase and the cells also increase in height. At the beginning of cleavage, the distribution of microvilli is roughly uniform over the surface but it becomes asymmetric at the completion of cleav-age when the daughter cells begin to spread. At this time most microvilli are over the daughter nuclei and the surfaces that border the former cleavage furrow. The regions of the daughter cells distal to the furrow are the first to spread and their surfaces have very few microvilli. When chromosome movement is inhibited by either Nocodazole or Taxol, microvilli formation is inhibited on the arrested cells. Nevertheless cell rounding still takes place in the normal time period. It is concluded from these observations that the signal for the onset of chromosome movement in anaphase is accompanied by a signal for the formation of microvilli. It is suggested that there is also a separate signal for the cell-rounding event in mitosis and that microvilli do not play a role in this contractile process.     

Cell surface proteins during early Xenopus development: analysis of cell surface proteins and total glycoproteins provides evidence for a maternal glycoprotein pool

Summary The populations of cell surface proteins and total glycoproteins were investigated in early Xenopus embryos through lectin staining, affinity binding of glycoproteins to lectins, and use of a succinimide ester to biotinylate cell surface molecules. Lectin staining shows that the egg is endowed with a thick layer of surface glycoprotein, and that glycoprotein is immediately detected on the newly formed membranes of nascent blastomeres. The amount of glycoprotein found in eggs and early embryos remains constant, and electrophoretic analysis reveals no changes in abundant lectin-binding glycoproteins through the neurula stage. In contrast, the amount of cell surface protein increases dramatically from the 2-cell to the gastrula stages. Despite this quantiative increase, only a small number of differences in cell surface proteins were detected during this period. A series of bands was detected which appears to be specific to the outer surface of the embryo. Because the populations of surface proteins and of total glycoproteins overlap to a great extent, the increase in cell surface protein, in the absence of a change in total glycoprotein, indicates the presence of a maternal glycoprotein pool in the Xenopus egg, from which the cell surface proteins of embryonic blastomeres are recruited.     

Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodium-dodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with [3H]D-glucosamine or [3H]L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecules haptenized with trinitrophenol (TNP) groups were followed radiochemically by means of [125I]anti-DNP antibodies. The half-life for internalization of proteins tagged with either [125I]anti-DNP IgG or Fab averaged about 5 min. A similar result was obtained when a monoclonal antibody directed against a single plasma membrane glycoprotein was used, or when the rate of surface loss of TNP groups unoccupied by antibodies was measured. Within 15 min at 37 degrees C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t 1/2 approximately 5 min). Most of the intracellular radioactivity was associated with a membrane fraction of density similar to that of the plasma membrane. Over a 4-h period, there was no significant entry of labeled molecules into lysosomes. By contrast, the fluid-phase marker, horseradish peroxidase, became associated with the lysosomes within 1 h. Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.     

Characterization of surface glycoproteins of mouse lymphoid cells

We have labeled exposed surface glycoproteins of mouse lymphoid cells by the galactose oxidase-tritated sodium borohydride technique. The labeled glyco-proteins were separated by polyacrylamide slab gel electrophoresis and visualized by autoradiography (fluorography). The major thymocyte surface proteins have molecular weights of 170,000 and 125,000. Thymocytes from TL antigen-positive mouse strains showed an additional band with a molecular weight of 27,000. Highly purified T lymphocytes contain two major surface glycoproteins with molecular weights of 180,000 and 125,000. Purified B lymphocytes have one major surface glycoprotein with a molecular weight of 210,000. When T lymphocytes are stimulated in vitro by concanavalin A or phytohemag-glutinin, the major proteins characteristic of T cells are relatively weakly labeled, but new components of lower molecular weights appear on the cell surface. A similar change is seen in B lymphocytes stimulated by Escherichia coli lipopolysaccharide. T lymphoblasts isolated from mixed lymphocyte cultures show a slightly different surface glycoprotein pattern. A polypeptide with a molecular weight of 57,000, which was labeled without enzymatic treatment by tritiated sodium borohydride alone, is strongly labeled in proliferating cells.     

Changes in cell surface and intracellular glycoproteins of trophoblastic giant cells during mouse placentation

Summary Changes in lectin bindings of mouse trophoblastic giant cells (TGCs) were examined by light and electron microscopy. Neither Griffonia simplicifolia agglutinin (GS)-II nor succinyl-wheat germ agglutinin (s-WGA) bound to the 1st and 2nd TGCs on day 6.5 post coitum (p.c.), but did so from days 8.5 to 12.5 p.c. Positive reactions with s-WGA were localized in the perinuclear region and cell surface of both 1st and 2nd TGCs; while GS-II bound only to the perinuclear region, where it appeared as network-like deposits. This region was identified as well-developed Golgi lamellae by electron microscopy. Moreover, SDS-PAGE and lectin-blot analysis of the 1st TGCs indicated that the intensity of s-WGA and GS-II bindings increased in the glycoproteins of approximately 43, 40, 37, and 26 kDa and in those of 43 and 38 kDa, respectively, during the 8.5th to 10.5th day p.c. The reaction with GS-I was detected on cell surface of both the 1st and 2nd TGCs on day 6.5 p.c. The reaction in the 1st TGCs was intensely positive throughout their development, whereas the reactivity decreased in the 2nd TGCs on day 10.5 p.c. and completely disappeared on day 12.5 p.c. The GS-I reaction in TGCs was more intense at the maternal side than at the embryonic side. These results suggest that certain Gal and/or GlcNAc glycoproteins on the cell surface and in Golgi lamellae of TGCs dynamically change from the 8.5th to 10.5th day p.c. in association with mouse placentation.     

Changes in cell surface and intracellular glycoproteins of trophoblastic giant cells during mouse placentation

Changes in lectin bindings of mouse trophoblastic giant cells (TGCs) were examined by light and electron microscopy. Neither Griffonia simplicifolia agglutinin (GS)-II nor succinyl-wheat germ agglutinin (s-WGA) bound to the 1st and 2nd TGCs on day 6.5 post coitum (p.c.), but did so from days 8.5 to 12.5 p.c. Positive reactions with s-WGA were localized in the perinuclear region and cell surface of both 1st and 2nd TGCs; while GS-II bound only to the perinuclear region, where it appeared as network-like deposits. This region was identified as well-developed Golgi lamellae by electron microscopy. Moreover, SDS-PAGE and lectin-blot analysis of the 1st TGCs indicated that the intensity of s-WGA and GS-II bindings increased in the glycoproteins of approximately 43, 40, 37, and 26 kDa and in those of 43 and 38 kDa, respectively, during the 8.5th to 10.5th day p.c. The reaction with GS-I was detected on cell surface of both the 1st and 2nd TGCs on day 6.5 p.c. The reaction in the 1st TGCs was intensely positive throughout their development, whereas the reactivity decreased in the 2nd TGCs on day 10.5 p.c. and completely disappeared on day 12.5 p.c. The GS-I reaction in TGCs was more intense at the maternal side than at the embryonic side. These results suggest that certain Gal and/or GlcNAc glycoproteins on the cell surface and in Golgi lamellae of TGCs dynamically change from the 8.5th to 10.5th day p.c. in association with mouse placentation.     

Cell surface glycoproteins mediate compaction, trophoblast attachment, and endoderm formation during early mouse development

Early mouse embryos undergo several morphogenetic processes, such as compaction, trophoblast attachment, and endoderm formation that can be studied in vitro. Several polyspecific and monospecific antisera have been used to perturb these processes in a nontoxic, reversible fashion. One of the antibody-defined molecules, cell CAM 120/80, promotes epithelial cell adhesion, embryo compaction, and endoderm formation. The results suggest the presence of another such molecule(s) involved in these same processes. Evidence is also presented that another set of antibody-defined molecules, GP 140, involved in attachment of somatic cells to the substrate, mediates trophoblast attachment of the mouse blastocyst. The possible role of these molecules in governing the processes leading to cell lineages in the mouse embryo is discussed.     

Altered expression of glycoproteins on the cell surface of Jurkat cells during etoposide-induced apoptosis: Shedding and intracellular translocation of glycoproteins

Background

The glycoproteins on the cell surface are altered during apoptosis and play an important role in phagocytic clearance of apoptotic cells.

Methods

We classified Jurkat cells treated with etoposide as viable and early apoptotic cells, late apoptotic cells or secondary necrotic cells based on propidium iodide staining and scattered grams and estimated the expression levels of glycoproteins on the cell surface.

Results

The cell surface expression levels of intercellular adhesion molecules (ICAM)-2 and -3 on the apoptotic cells were markedly lower, while those of calnexin, calreticulin, and lysosome-associated membrane proteins (LAMP)-1 and -2 were significantly higher compared to non-apoptotic cells. These decreases in ICAM-2 and -3 on the apoptotic cell surface were reduced in the presence of metalloproteinase inhibitors and caspase inhibitors, respectively. Confocal microscopic analysis revealed that calnexin and calreticulin were assembled around fragmented nuclei of blebbed apoptotic cells.

Conclusions

These results suggest that alteration of glycoproteins on the cell surface during apoptosis is associated with shedding and intracellular translocation of glycoproteins.     

Cell surface changes during preimplantation development in the mouse

Scanning electron microscopy reveals microvilli on all preimplantation stages, indicates that their number and length may be dependent on embryo size, and provides examples of regional alterations in their number. Cellular adherence, as evidence by interactions of microvilli, migration of cellular processes, and junctional complexes, increases during development and is accompanied by changes in the shapes of cells and embryos. Cell surfaces bordering the blastocoel differ markedly from the outer cell surfaces of the embryo.     

Cell surface glycoproteins of hepatocytes and hepatoma cells identified by monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MABs) directed to cell surface components of rat hepatocytes were isolated. The antigens of seven MABs were identified as glycosylated plasma membrane proteins. The presence of these glycoproteins on normal hepatocytes and hepatocellular carcinoma cells was analyzed. A semi-quantitative enzyme-linked immunosorbent assay revealed that only two MABs (Be 8.7, Ne 11.3) recognized proteins which were expressed not only in normal liver but also in chemically induced transplantable Morris hepatomas and hepatoma-derived cell lines. The expression of six antigens was found to be sensitive to transformation. The domain specificity of the MABs was determined by indirect immunofluorescence on sections of liver tissue containing neoplastic nodules. Three MABs (Be 8.4, Ne 11.1, Ne 11.3) specifically bound to the sinusoidal domain and two MABs (Be 9.2, De 13.4) to the bile canalicular domain. These five antigens were transformation-sensitive except for the glycoprotein recognized by the MAB Ne 11.3. Three MABs (Be 8.7, Be 9.1, De 13.2) also showed intracellular immunofluorescence. Two of the antigens (Be 9.1, De 13.2) were not present in hepatomas. The relative molar masses (Mr) of the glycoproteins were determined after protein immunoblotting and immunoprecipitation. Four MABs (Be 8.7, Be 9.1, Be 9.2, De 13.4) recognized antigens with a Mr of 110 000 but did not mutually cross-react. The antigen recognized by MAB De 13.4 was identified as the ectoenzyme dipeptidyl peptidase IV (EC 3.4.14.-).     

Deficient uridine diphosphate-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity in a clone of Chinese hamster ovary cells with altered surface glycoproteins.

We have reported the isolation of a clone (termed 15B) of Chinese hamster ovary (CHO) cells which are deficient in certain plant lectin-binding sites and have decreased amounts of sialic acid, galactose, and N-acetylglucosamine in its membranes (Gottlieb et al. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1078-1082). This study demonstrates that extracts of 15B cells, in contrast to the parent cell line, do not transfer N-acetylglucosamine residues from UDP-GlcNAc to certain glycopeptide and glycoprotein acceptors containing terminal nonreducing alpha-linked mannose residues. The decreased enzyme activity could not be accounted for by the presence of inhibitors, altered pH, or Mn2+ requirements of the glycosyltransferase or increased N-acetylglucosaminidase activity in the extracts. The finding that the 15B cell extracts have significant but reduced N-acetylglucosaminyltransferase activity toward a degraded orosomucoid acceptor suggests that these cells have a selective loss of one of several specific N-acetylglucosaminyltransferases which are present in the parent CHO cells. The sialyl- and galactosyltransferase activities of 15B and parent CHO cells are comparable. Parent CHO and 15B cells were grown in radioactive glucosamine to label the membrane glycoproteins. Solubilization of these glycoproteins and passage over a Ricinus communis agglutinin I (RCA I) Sepharose affinity column revealed that no labeled 15B glycoprotein material bound, whereas 50 percent of the CHO membrane glycoproteins bound and could be eluted with the haptene lactose, demonstrating that 15B cells are virtually devoid of membrane oligosacharides capable of binding to the RCA I lectin. The 15B membrane glycoproteins exhibited a marked shift toward glycoprotein species of lower molecular weight when examined by gel electrophoresis in sodium dodecyl sulfate. It is proposed that this shift in the mobility of the 15B membrane glycoproteins results from a decreased glycosylation of a number of membrane glycoproteins relative to their counterparts in CHO cells. The deficient N-acetylglucosaminyltransferase activity in 15B cells can account for the decreased glycosylation of the 15B cell membrane glycoproteins.     

Altered Cell surface glycoproteins in phytohemagglutinin-resistant mutants of Chinese hamster ovary cells

Purified membranes from surface-labelled phytohemagglutinin-resistant (Pha^R) and wild-type chinese hamster ovary cells have been analysed by sodium dodecyl sulphate gel electrophoresis. Gel patterns were compared for cells labelled via galactose oxidase and B³H₄ or lactoperoxidase and radioactive iodide. The results suggest that Pha^R cells are altered in the carbohydrate portion of a number of their membrane glycoproteins.     

Cell surface N-glycans-mediated isolation of mouse neural stem cells

The isolation of neural stem cells (NSCs) has been hampered by the lack of valid cell-surface antigens on NSCs, and novel valuable markers have been proposed. Glycan (oligosaccharide chain) is a potential candidate as a marker to isolate NSCs, because the species and the combination order of saccharides in glycan generate remarkable structural diversity and specificity. At present, the expression of hundreds of glycoconjugates with glycans have been found in the NSCs; however, just a few glycan-epitopes have been identified as valuable cell-surface markers. This review focused on the isolation of NSC using glycoprotein, especially complex type N-glycans. The cell-surface N-glycan-mediated isolation of NSCs is therefore expected to provide a comprehensive understanding of the biologic characteristics of NSCs in the brain, and thereby help to develop novel strategies in the field of regenerative medicine.     

WGA-binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells.

Expression of apical cell surface proteins and glycoproteins was examined in polarized primary cultures of mouse uterine epithelial cells (UEC). Lectin-gold cytochemistry revealed that wheat germ agglutinin (WGA) bound specifically to the components of the apical glycocalyx as well as intracellular vesicles. Double labeling with the pH sensitive dye 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP) demonstrated the acidic nature of the WGA-staining intracellular vesicles. The enzymatic and chemical sensitivities of the WGA binding sites on the apical cell surface were monitored both by WGA-gold staining as well as by 125I-WGA binding assays. In thin sections, a large fraction of these sites were removed by pronase; however, application of a wide variety of proteases, glycosidases, or chemical treatments to the apical surface of intact UEC failed to reduce WGA binding. In no case did treatments designed to remove sialic acids reduce 125I-WGA binding more than 12%. In contrast, endo-beta-galactosidase as well as a combination of beta-galactosidase with beta-hexosaminidase succeeded in removing 28% and 77% of these sites, respectively. These studies suggested that the majority of the apically disposed WGA binding sites involved N-acetylglucosamine residues rather than sialic acids and included lactosaminoglycans. Many of the proteins detected at the apical cell surface by lactoperoxidase-catalyzed radioiodination were WGA-binding glycoproteins. A major class of these glycoproteins displayed Mr > 200 kDa by SDS-PAGE and was heavily labeled metabolically by 3H-glucosamine or by vectorial labeling at the apical cell surface with galactosyl transferase and UDP-3H-galactose. Analyses of the 3H-labeled oligosaccharides labeled by either procedure indicated that a large fraction of the apically disposed WGA-binding oligosaccharides consisted of neutral, O-linked mucin-type structures with median MW of approximately 1,500. Oligosaccharides in this fraction were partially (15%) sensitive to endo-beta-galactosidase digestion and bound to Datura stramonium agglutinin (68%), demonstrating the presence of lactosaminoglycan sequences. UEC were an extremely effective barrier to attachment or invasion by either a highly invasive melanoma cell line, B16-BL6, or implantation-competent mouse blastocysts. In contrast, neither uterine stromal cells nor a non-polarizing UEC cell line, RL95, prevented B16-BL6 attachment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell surface antigens reacting with antiretrovirus sera on normal mouse spleen cells: a flow cytometric study

The development of erythroleukemia in Balb/c mice injected with Rauscher leukemia virus has been followed by indirect immunofluorescence technique and flow cytometry, using antiserum against disrupted of virions. The progression of the disease was accompanied by a great increase in the number of large, immunofluorescence positive cells. A subpopulation of normal spleen cells was also highly positive. The expression of the antigens in normal cells was examined in relation to the cell cycle. The majority of the S-G2/M phase cells were found in the antigen positive compartment of larger cells. A two-color analysis of immunofluorescence and DNA content revealed that the distribution of antigen expression in G1 phase was broad, gradually decreasing from a low-intensity mode. The cell with double DNA content distributed evenly around a modus of five-fold higher intensity. In experiments with stimulated bone-marrow cells, superiority of S-phase cells in anti-Rauscher serum binding was found. Cell-surface gp70 antigen is suggested to be involved in this cell-cycle dependent binding of antibodies by normal cells.     

Trypanosoma cruzi: monoclonal antibodies to the surface glycoprotein superfamily differentiate subsets of the 85-kDa surface glycoproteins and confirm simultaneous expression of variant 85-kDa surface glycoproteins.

Most surface glycoproteins expressed by mammalian-stage forms of Trypanosoma cruzi are homologous to the parasite's trans-sialidase and therefore are members of the parasite's trans-sialidase superfamily. Few members of this superfamily have trans-sialidase activity. The SA85-1 family is a subfamily of the trans-sialidase superfamily whose members lack trans-sialidase activity. The function of these non-trans-sialidase members remains unknown. In this report a series of monoclonal and polyclonal antibodies to the SA85-1 glycoproteins is presented. The mAbs define distinct subgroups of SA85-1 glycoproteins, and these distinct subgroups are simultaneously expressed by individual trypomastigotes, supporting previous studies indicating that multiple SA85-1 glycoproteins and trans-sialidase superfamily glycoproteins are simultaneously expressed by each trypomastigote. In addition, the antibodies define two major subsets of the SA85-1 family (subset 1 and subset 2) based on differences in migration in SDS-PAGE; the subsets do not appear to be created by differences in glycosylation. Subset 1 migrates slower and is spontaneously released or shed preferentially from the parasite surface compared to subset 2. In addition, subset 1 is attached to the trypomastigote surface by a GPI linkage. Since these glycoprotein subsets are differentially expressed, they may have different functions.     

Cell cycle dependent exposure of a high molecular weight protein on the surface of mouse L cells

The non-penetrating lactoperoxidase iodination probe has been employed in conjunction with synchronously dividing populations of mouse L cells to identify a high molecular weight protein which is preferentially exposed on the L cell surface during G1 phase of the cell cycle. Progression of cells from G1 to S is accompanied by a marked decrease in the availability of this structure, called band 1, to lactoperoxidase-catalyzed iodination and it remains unavailable until cells re-enter G1. It is suggested that the band 1 polypeptide may be functionally involved in the regulation of L cell growth.     

Cell surface N-glycans mediated isolation of mouse neural stem cells

The isolation of neural stem cells (NSCs) from the brain has been hampered by the lack of valid cell surface markers and the requirement for long-term in vitro cultivation that may lead to phenotype deterioration. However, few suitable specific cell surface antigens are available on NSCs that could be used for their prospective isolation. The present study demonstrated that the expression of complex type asparagine-linked oligosaccharide ( N- glycans) was detected on brain cells dissociated from embryonic and adult brain using Phaseolus vulgaris erythroagglutinating lectin (E-PHA) which binds to biantennary complex type N- glycans, and demonstrated that E-PHA bound preferentially to purified NSCs, but not to neurons, microglia, or oligodendrocyte precursor cells. The labeling of dissociated mouse embryonic brain cells or adult brain cells with E-PHA enabled the enrichment of NSCs by 25-fold or 9-fold of the number of neurosphere-forming cells in comparison to that of unsorted cells, respectively. Furthermore, a lectin blot analysis revealed the presence of several glycoproteins which were recognized by E-PHA in the membrane fraction of the proliferating NSCs, but not in the differentiated cells. These results indicate that complex type N- glycans is a valuable cell surface marker for living mouse NSCs from both the embryonic and adult brain.     

Identification of exposed surface glycoproteins in undifferentiated and differentiated mouse N-18 neuroblastoma cells

A simple method is described that permitted rapid isolation of plasma membranes from mouse N-18 neuroblastoma cells. The purified plasma membranes gave a 10-fold increase in the specific activity of incorporated [³H]fucose over that of the cell homogenate. The specific activities of two other membrane markers, 5′-nucleotidase and alkaline phosphatase, increased 11-fold and 15-fold, respectively. Metabolic labeling with [³H]fucose identified a major fucosyl glycoprotein with apparent molecular weight of 92 000. Three surface labeling methods together with SDS-polyacrylamide gel electrophoresis and fluorography were used to characterize and compare the surface glycoproteins of undifferentiated and differentiated N-18 cells. The galactose oxidase/NaB³H₄ method labeled two major galactoproteins (M_r = 52 000, 42 000) in both undifferentiated and differentiated cells. The neuraminidase/galactose oxidase/NaB³H₄ method revealed many sialylgalactoproteins. Among them, the 220-kdalton, 150-kdalton and 130-kdalton bands were at least 100% more prominently labeled in the differentiated calls whereas the 76-kdalton and 72-kdalton bands were less prominently labeled in the differentiated cells when compared to their undifferentiated counterparts. The prominently iodinated protein bands in the undifferentiated cells had apparent molecular weights of 130 000, 92 000, 76 000 and 72 000 as compared to 150-, 130-, 92- and 76-kdalton bands in the differentiated cells. The labeling data obtained will enable us to further study the changes of these identified surface glycoproteins, both quantitatively and topologically, during the differentiation of neuroblastoma cells.