The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-beta on pericentromeric heterochromatin in S-phase and G(2), decreased pericentromeric targeting of Aurora B kinase, resulting in decreased premitotic phosphorylation of pericentromeric histone H3(S10) in G(2), followed by assembly of deficient kinetochores in M-phase. HP1-beta, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.

A Chinese hamster ovary cell mutant DTG 1-5-4, was selected for pleiotropic defects in receptor-mediated endocytosis by methods previously described (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071. DTG 1-5-4 exhibited increased resistance to modeccin, Pseudomonas toxin, diphtheria toxin, Sindbis virus, and vesicular stomatitis virus, as well as decreased uptake via the mannose 6-phosphate receptor. Fluorescein-dextran-labeled endosomes isolated from DTG 1-5-4 were deficient in ATP-dependent acidification in vitro. Endocytosis and endosome acidification were both restored in revertants of DTG 1-5-4 and in hybrids of DTG 1-5-4 with DTF 1-5-1, another endocytosis mutant exhibiting decreased ATP-dependent endosome acidification.Both DTG 1-5-4 and DTF 1-5-1 were blocked at two stages of infection with Sindbis virus: at low multiplicities of infecting virus, resistance reflected a block in viral penetration into the cytoplasm, but at higher multiplicities of infection the block was in virus release. Like endocytosis, release of Sindbis virus was increased in revertants of DTG 1-5-4 and in DTG 1-5-4 x DTF 1-5-1 hybrids. Decreased release of virus from DTG 1-5-4 correlated with defects in some of the Golgi apparatus-associated steps of Sindbis glycoprotein maturation: proteolytic processing of the precursor pE2, galactosylation, and transport to the cell surface all were inhibited. In contrast, mannosylation, fucosylation, and acylation of the Sindbis glycoproteins, and galactosylation of vesicular stomatitis virus and cellular glycoproteins occurred to similar respective extents in mutant and parent. Electron microscopic examination of Sindbis-infected DTG 1-5-4 showed a remarkable accumulation of nucleocapsids bound to cisternae adjacent to the Golgi apparatus; virions were observed in the lumina of some of these cisternae.That the alterations in both endocytosis and Golgi-associated steps of viral maturation result from a single genetic lesion indicates that these processes are dependent on a common biochemical mechanism. We suggest that endocytic and secretory pathways may share a common component involved in ion transport.

Populations of Chinese hamster ovary cells selected for resistance to diphtheria toxin were found to be highly enriched for mutants deficient in the uptake of lysosomal hydrolases via the mannose 6-phosphate receptor. One doubly defective mutant, DTF 1-5-1, exhibited increased resistance to Sindbis virus, although it was able to bind and internalize virus normally. Normal production of virus was obtained when, subsequent to virus binding, the mutant was exposed for 2 rain to acidic pH. Similarly, a shift to acidic pH increased the sensitivity of DTF 1-5-1 to diphtheria toxin 12-fold. Decreased uptake of lysosomal hydrolases by the mutant correlated with decreased mannose 6-phosphate receptor activity at the cell surface; results of lactoperoxidase-catalyzed iodination indicated that the surface-associated receptor was present but inactive on DTF 1-5-1. Total mannose 6-phosphate receptor activity was also decreased in the mutant and this decrease was reflected by increased secretion of lysosomal hydrolases. The phenotype of DTF 1-5-1 resembles in many ways that of cells treated with ammonia. We suggest that the defect in DTF 1-5-1 stems from an inability to deliver virus, diphtheria toxin, and lysosomal hydrolases to an acidic compartment. Other ligands may be endocytosed through a different pathway since the defect of DTF 1-5-1 did not decrease the endocytosis of ricin, modeccin, or Pseudomonas toxin and had minimal effects on uptake and degradation of low density lipoprotein.

Abstract. We have isolated three independent Chinese hamster ovary cell mutants (B3853, I223, and M311) with temperature-sensitive, pleiotropic defects in receptor-mediated endocytosis. Activities affected at 41°C include uptake via the D-mannose 6-phosphate receptor, accumulation of Fe from diferric transferrin, uptake of ctz-macroglobulin, compartmentalization of newly synthesized acid hydrolases, resistance to ricin, and sensitivity to diphtheria and Pseudomonas toxins and modeccin. The three mutants also displayed decreased sialylation of some secreted glycoproteins at 41°C, reminiscent of the nonconditional mutant DTG1-5-4 that showed both endocytic and Golgi-associated defects (Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman, 1984, J. Cell Biol., 99:1296-1308. Phenotypic changes were detectable within 30 min after transfer of the mutants to 41°C; maximal alteration of most susceptible functions was obtained 4 h after temperature shift. At 39°C, the mutants exhibited many but not all of the changes manifested at 41°C; resistance to diphtheria and Pseudomonas toxins required the higher temperature.Analysis of cell hybrids showed that B3853 and DTG1-5-4 are in one complementation group ("Endl"); M311 and I223 are in another CEnd2"). In the Endl mutants, loss of endocytosis correlated with complete loss of ATP-dependent endosomal acidification in vitro; in the End 2 mutants partial loss of acidification was observed. At the nonpermissive temperature, residual levels of endocytic activity in B3853 and M311 were nearly identical; thus, we conclude that the differences measured in endosomal acidification in vitro reflect the different genetic loci affected, rather than the relative severity of the genetic lesions. The mutations in M311 and I223 appear to have different effects on the same protein; in 1223 (but not in M311) the full spectrum of phenotypic changes could be produced at the permissive temperature by inhibition of protein synthesis. S EVERAL laboratories have described mutant Chinese hamster ovary (CHO) cells that are pleiotropically defective in receptor-mediated endocytosis (17,21,22,28,29). Endosomes isolated from some of these mutants were shown to be defective in ATP-dependent acidification (20,29). Although decreased endosomal acidification has yet to be demonstrated in vivo, many aspects of the mutants' phenotypes are consistent with such a defect; e.g., increased resistance to diphtheria toxin and enveloped RNA viruses (21,22,28,29) and decreased accumulation of Fe from transferrin (13).However, other facets of the mutants' phenotypes do not appear to follow directly from loss of endosomal acidification; decreased release of Sindbis virus and decreased galactosylation of Sindbis glycoproteins (29) suggest a defect in Golgi function. Other phenotypic changes, e.g., oversecretion of newly synthesized acid hydrolases (28,29,36), are ambiguous in that they could result from either Golgiassociated or endosomal defects.Through analysis of revertants and cell-cel...

B4-2-1 is a Chinese hamster ovary cell mutant previously isolated and characterized as deficient in mannose 6-phosphate receptor activity. We show here that B4-2-1 is a pleiotropic mutant, defective in biosynthesis of asparagine-linked oligosaccharides. B4-2-1 is unable to synthesize mannosylphosphoryldolichol; the consequences of this defect on glycosylation are (i) biosynthesis of one major lipid-linked oligosaccharide, characterized by its resistance to endoglycosidase H and decreased size; this oligosaccharide is similar to a minor species of lipid-linked oligosaccharide found in parental cells; (ii) transfer of this oligosaccharide to newly synthesized proteins; and (iii) absence of normal "high-mannose" oligosaccharides on mature glycoproteins isolated from B4-2-1; glycoproteins from the mutant contain complex oligosaccharides as well as endoglycosidase H-resistant, amannosidase-sensitive species. While the glycosylation defect may alter adversely the function ofseveral glycoproteins in the mutant, including that of the mannose 6-phosphate receptor, it appears to have no effect on the formation or function ofthe mannose 6-phosphate recognition marker on acid hydrolases of B4-2-1.The uptake of acid hydrolases into human fibroblasts and Chinese hamster ovary (CHO) cells involves binding of a mannose 6-phosphate recognition marker on the enzymes to a receptor for mannose 6-phosphate on the cell surface (1)(2)(3). This receptor appears to be similar in the two cell types with respect to both structure (3) and function (4).The mannose 6-phosphate recognition marker and mannose 6-phosphate receptor appear to be involved not only in enzyme uptake but also in translocation of newly synthesized endogenous acid hydrolases to the lysosomes (2, 5). Most of the receptor has been shown to exist intracellularly (2). Human fibroblasts defective in mannose phosphorylation (6-9) fail to compartmentalize acid hydrolases but instead secrete those enzymes. Aberrant compartmentalization was also observed in CHO cell mutants defective in mannose 6-phosphate receptor activity (10).One ofthe receptor-defective CHO cell mutants, B4-2-1, was found to synthesize acid hydrolases ofincreased electrophoretic mobility (10). Since the receptor and the hydrolases are all glycoproteins, we speculated that this mutant might have a pleiotropic defect in glycosylation. We show here that B4-2-1 cells synthesize altered asparagine-linked oligosaccharides due to a defect in the biosynthesis of mannosylphosphoryldolichol. MATERIALS AND METHODSCells. Isolation and culture conditions for the CHO cell lines N211-1-8 and B4-2-1 (4) and WTB (11) have been described. All media contained 5% fetal bovine serum (Armour, Kankakee, IL); (North American Biologicals, Miami, FL). The cultures were checked routinely for mycoplasma (12) and were found to be negative. (-100 mCi/mmol) was prepared as described (13). Reagents. endo-p-N-acetylglucosaminidase H (endo H)Incubation of Cells with [2-3H]Mannose. Cells were labeled with 1 ml of a-modified minima...

. The mAb AA4 binds to novel derivatives of the ganglioside GD1b on rat basophilic leukemia (RBL-2H3) cells . Some of the gangliosides are located close to the high affinity IgE receptor (FCcRI), and binding of mAb AA4 inhibits FCERI-mediated histamine release. In the present study, mAb AA4 was found to bind exclusively to mast cells in all rat tissues examined. In vitro, within 1 min of mAb AA4 binding, the cells underwent striking morphologic changes . They lost their normal spindle shaped appearance, increased their ruffling, and spread over the surface of the culture dish. These changes were accompanied by a redistribution of the cytoskeletal elements, actin, tubulin, and vimentin, but only the actin was associated with the membrane ruffles . Binding of mAb AA4 also induces a rise in intracellular calcium, stimulates phosphatidyl inositol breakdown, and activates PKC . However, the extent of these changes was less than that observed when the cells were stimulated with antigen or antibody directed against the FcERI. None of these changes associated with mAb AA4 binding were seen when the cells were exposed to nonspecific IgG, IgE, or four other anti-cell surface antibodies, nor were the changes induced by binding mAb AA4 at 4°C or in the absence of extracellular calcium . Although mAb AA4 does not stimulate histamine release, it enhances the effect of the calcium ionophore A23187 mediated release. The morphological and biochemical effects produced by mAb AA4 are similar to those seen following activation of the cell through the IgE receptor. Therefore, the surface gangliosides which bind mAb AA4 may function in modulating secretory events .phate by mobilizing intracellular calcium pools . Receptor activation also results in calcium influx, activation of phospholipase A2, the phosphorylation of a number of intracellular proteins (7), and the rearrangement of the cytoskeleton before mediator release (35,37,48) .The rat basophilic leukemia cell line, RBL-2113, has been widely used as a model to study 48,49) . A number ofmAbs have been raised against surface molecules on these cells (3,5,29,50) . These mAb were selected for their ability to either stimulate or inhibit histamine release in the RBL-2113 cells. The majority of the antibodies were directed against the Fcc-RI ; however, two antibodies, mAb ADl and AA4, bind other cell surface components . The mAb ADl binds to a novel 50-60-kD protein on the surface of the RBL-2113 cells and modulates histamine release (31). The other antibody, mAb AA4, binds to unique a-galactosyl derivatives of the ganglioside GD, b (18). These gangliosides are closely related to the FceRl, both spatially and functionally (5) . Binding ofboth the intact mAb AA4 and its Fab fragments to cells inhibits the binding

Abstract. The CHO cell mutant FD1.3.25 exhibits both increased accumulation and altered distribution of endocytosed fluid phase tracers. Neither the rate of tracer internalization nor the kinetics of recycling from early endosomes was affected, but exocytosis from late endocytic compartments appeared to be decreased in the mutant. Endocytosed tracer moved more rapidly to the cell poles in FD1.3.25 than in wild type cells. An abundant 36-kD polypeptide was found associated with taxol-polymerized microtubules in preparations from wild type and mutant; in the former but not the latter this polypeptide could be dissociated by incubation of the microtubules in ATP or high salt. The 36-kD polypeptide co-electrophoresed in two dimensions with the monomer of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Analysis of cDNA clones showed that the mutant is heterozygous for this enzyme, with ~25 % of the GAPDH RNA containing a single nucleotide change resulting in substitution of Ser for Pro234, a residue that is conserved throughout evolution. Stable transfectants of wild type cells expressing the mutant monomer at ~15% of the total enzyme exhibited the various changes in endocytosis observed in FD1.3.25.

A Chinese hamster ovary cell mutant defective in the receptor-mediated endocytosis of several unrelated ligands (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071) failed to accumulate iron provided in the form of diferric transferrin. Analysis of the steps of the transferrin cycle indicated that binding and internalization of transferrin proceeded normally in mutant cells. However, the mutant appeared unable to dissociate iron from transferrin, as evidenced by release of diferric transferrin from the mutant versus apotransferrin from the parent. Uptake of ferric ions from the growth medium was enhanced in the mutant.