The expression and localization of surface neoantigens in transformed and untransformed cultured cells infected with avian tumor viruses

The presence and localization of neoantigens induced in cultured cells, infected or transformed with avian tumor viruses (ATV), were studied ultrastructurally on carbon platinum replicas of cell surfaces. The use of antibody, labeled with hemocyanin molecules, provided sensitive detection and analysis of cell surface antigen distribution. The subgroup-specific antigens of the viral envelope were found in considerable amount in the plasma membranes of ATV-infected chick embryo fibroblasts. The distribution of these antigens over the cell surface, evaluated on cells which were prefixed with glutaraldehyde, was found to be diffuse with a greater density on the cell processes in some cells. Reaction of antibody to viral envelope antigens with living ATV-infected cells resulted in a number of patterns of redistribution of membrane antigen-antibody complexes (AAC). Redistribution occurred in symmetrical or asymmetrical modes. The former consisted of randomly oriented aggregates (patches) of AAC over the cell surface. The latter included: (a) linear accumulation of AAC at cell margins; and (b) condensation of complexes into one or more centers of coalescence. These observations could be made on chick embryo cells infected (but not transformed) by avian leukosis virus, or on cells oncogenically transformed by avian sarcoma virus. The regions of coalescence were suggestive of the “capping” phenomenon seen in other systems, and their formation was temporally correlated with endocytosis of labeled AAC and the gradual loss of AAC from the surface. The effects of several biologically perturbing substances on the processes of redistribution were investigated in ALV-infected fibroblasts. Sodium azide, puromycin, actinomycin D, and colchicine had no effect on either form of asymmetrical redistribution. Cytochalasin B (CB) and iodoacetic acid (IAA) appeared to have some effect on the marginal redistribution, and to completely prevent the condensation into foci of coalescence (FC). When treated with these compounds, reacted with antibody at low temperature, washed free of unbound antibody, and warmed at 37° C, cells rapidly cleared their surfaces of AAC. This was not accompanied by formation of FC or endocytosis. In some of these cells, a distribution was observed which suggested a possible centrifugal flow of antigenic sites – perhaps an alternate route for disposal of AAC. None of the drugs tested affected symmetrical redistribution. Repeated attempts at detection and topographical analysis of a tumor-specific antigen on the surface of Rous sarcoma virus-transformed chicken and rat cells have provided no evidence for antibody to such an antigen in the serum of immunized animals. Autochthonous, homologous, and heterologous immunizations of chickens and rats did not produce a detectable antibody response to a virus-specific tumor surface antigen. Preliminary results, however, suggest the expression of an individual-specific (unique) tumor antigen on the surface of Rous sarcoma cells.     

Electron microscopic study of measles virus infection: unusual antibody-triggered redistribution of antigens on giant cells.

Vero cells infected with measles virus fuse to form multinucleated cells which incorporated virus-specific antigens in their membrane. The distribution of these antigens was analyzed after a brief treatment with human anti-measles immunoglobulin G, using autoradiography and immunoperoxidase labeling combined with transmission and scanning electron microscopy. Virs-specific antigens were distributed over the entire surface of giant cells treated at 4 degrees C with human anti-measles immunoglobulin G and labeled Protein A. When cells were shifted to 37 degrees C, labeled antigen-antibody complexes were redistributed in two stages. Patch formation occurred in 5 to 15 min. Later, antigen-antibody complexes became concentrated in a paracentral "ring" rather than typical caps. Patch formation occurred in the presence of metabolic inhibitors, whereas ring formation was inhibited by metabolic inhibitors. These rings contained membrane folds, villi, and viral buds, whereas the rest of the membrane was smooth. In addition, shedding, endocytosis of antigen-antibody complexes, and reexpression of antigens were observed. Antibodies to nonviral membrane antigens induced the same pattern of redistribution. Infected cells treated with anti-measles Fab' fragments maintained a homogenous distribution of label throughout the experiments. In conclusion, intact immunoglobulins, but not Fab' fragments, were able to induce a dramatic redistribution of viral antigen on the membrane of giant cells infected with measles virus.     

Visualization of assembled and disassembled microtubule protein by double label fluorescence microscopy

Indirect immunofluorescence with rhodamine labelled antibodies and fluoresceinated colchicine (FC) are used to simultaneously localize microtubules and soluble tubulin in cultured ovarian granulosa cells. FC labelled tubulin is most concentrated in regions of the cell occupied by antitubulin stained microtubule bundles. Pretreatment of granulosa cells with colchicine results in a central accumulation of FC and antibody labelled tubulin that coincides with the disposition of 10-nm filament cables. In contrast, the microtubule disrupting agent nocodazole produces a diffuse tubulin distribution as detected with both FC and antibody probes. Taxol treatment, which enhances microtubule assembly, results in a striking concentration of microtubule bundles associated with the nucleus that avidly bind FC. These results suggest that disassembled tubulin is preferentially associated with cytoplasmic microtubules and possibly other formed elements of the cytoskeleton.     

Spontaneous redistribution of cell-surface glycoproteins in lymphoid cells during cytokinesis.

The 'unperturbed' distribution of plasma membrane glycoproteins during cytokinesis has been examined by immunofluorescence and electron microscopy on dividing mouse and rat lymphoid cells fixed before being labelled with the appropriate reagents. Two groups of molecules which cap 'spontaneously' to the uropod of non-dividing cells, i.e., the common receptors for Helix pomatia (HPA) and peanut agglutinin (PNA) (and in particular the thymocyte glycophorin-like glycoprotein) and membrane immunoglobulins, redistribute spontaneously to the cleavage furrow during cytokinesis. By electron microscopy, the redistributed molecules (HPA receptors) appear to be aggregated in clusters. Other glycoproteins, such as Concanavalin A receptors and Thy.1 antigens, which do not cap spontaneously on interphase cells, remain uniformly distributed or are somewhat depleted over the cleavage furrow. The results suggest that a spontaneous 'transport' of certain membrane molecules from the nuclear pole to the cleavage furrow occurs normally during cytokinesis by a mechanism analogous to that of uropod formation and spontaneous capping in interphase cells. The existence of redistribution phenomena in dividing cells imposes some restrictions on the possible mechanisms of redistribution and on certain aspects of the cleavage process.     

ULTRASTRUCTURAL DISTRIBUTION OF CELL SURFACE ANTIGENS IN AVIAN TUMOR VIRUS-INFECTED CHICK EMBRYO FIBROBLASTS

The distribution of neoantigens in the surface membrane of avian tumor virus-infected chicken embryo fibroblasts was examined on carbon replicas of cell cultures using hemocyanin-labeled antibody. New determinants appearing on the cell surface of virally infected but not transformed cells are thought to be common with components of the viral envelope. These antigens were found to exist in a diffuse, random array on the dorsal cell surface, with a denser accumulation along the cell processes. In living cells, surface antigens are capable of several types of redistribution when activated by reaction with antibody. Leukosis virus-infected (non-transformed) cells showed two apparently independent modes of redistribution: a relocation of some antibody-related sites to the cell margin; or an involvement of essentially all sites in randomly dispersed aggregates. Viral antigenic sites on sarcoma virus-infected (transformed) cells, reacted with antibody, were able to produce weak marginal relocation; but revealed a more striking tendency to migrate to some central location. The centripetal coalescence thus formed resembles the "cap" noted in other systems. Prior aggregation into "patches" may not be a prerequisite for such cap formation. Tumor-specific surface antigen detection and mapping was attempted by this technique, but results were equivocal. An antigen possibly characteristic of rapidly dividing cells occurred in a sparse, diffuse fashion over the surface of morphologically distinct "round" cells.     

The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing.

Herpes simplex virus type 1 infection results in a reorganization of antigens associated with the small nuclear ribonucleoprotein particles (snRNPs), resulting in the formation of prominent clusters near the nuclear periphery. In this study, we show that the immediate-early protein ICP27, which is involved in the impairment of host cell splicing and in the changes in the distribution of snRNPs, is also required for reassorting the SR domain splicing factor SC35. Other viral processes, such as adsorption and penetration, shutoff of host protein synthesis, early and late gene expression, and DNA replication, do not appear to play a role in changing the staining pattern of splicing antigens. Furthermore, the C-terminal repressor region of ICP27, which is required for the inhibitory effects on splicing, also is involved in redistributing the snRNPs and SC35. During infection or transfection with five different repressor mutants, the speckled staining pattern characteristic of uninfected cells was seen and the level of a spliced target mRNA was not reduced. Infections in the presence of activator mutants showed a redistributed snRNP pattern and a decreased accumulation of spliced target mRNA. Moreover, two arginine-rich regions in the N-terminal half of ICP27 were not required for the redistribution of snRNPs or SC35. Substitution of these regions with a lysine-rich sequence from simian virus 40 large-T antigen resulted in a redistribution of splicing antigens. Unexpectedly, a repressor mutant with a ts phenotype showed a redistributed staining pattern like that seen with wild-type infected cells. During infections with this ts mutant, splicing was not inhibited, as shown in this and previous studies, confirming its repressor phenotype. Furthermore, both the mutant and the wild-type protein colocalized with snRNPs. Therefore, the redistribution of snRNPs and SC35 correlates with ICP27-mediated impairment of host cell splicing, but these alterations are not sufficient to fully inhibit splicing. This indicates that active splicing complexes are still present even after dramatic changes in the organization of the snRNPs.     

Cell surface glycoproteins of CHO cells. II. Surface distribution and pathway of internalization

The surface distribution and pathway for internalization of 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. Molecules, haptenized with trinitrophenol groups, the majority of which are in a group of high molecular weight acidic glycoproteins (HMWAG), were labelled at 4 degrees C with anti-dinitrophenol immunoglobulins coupled to fluorescein isothiocyanate (FITC), horseradish peroxidase, or colloidal gold and either immediately fixed for mapping their distribution or followed intracellularly after warming to allow endocytosis to proceed. The distribution of label on the CHO cell surface was non-random with a large proportion arranged in clusters from 100 to 300 nm in diameter. Antibody label was concentrated heavily on microvilli, and about 10% of the molecules were always associated with clathrin-coated pits. Upon warming the cells to 37 degrees C, HMWAG were internalized immediately into smooth-membraned tubules (less than 80 nm luminal diameter) that appeared to connect with vesicles (less than 300 nm luminal diameter) located in the cortical cytoplasm. By 60 min, labelled antibody was located within larger vesicles (greater than 300 nm luminal diameter) that had a morphology characteristic of multivesicular bodies and not lysosomes. There was no evidence for entry of labelled molecules into either electron-dense, secondary lysosomes or into the Golgi cisternae, suggesting that neither compartment is involved in the major pathway of cell surface endocytosis. Our results are consistent with the view that the majority of plasma membrane protein are internalized as small discrete domains by a pathway very similar to that described by others for adsorptive endocytosis.     

CD1 and major histocompatibility complex II molecules follow a different course during dendritic cell maturation

The maturation of dendritic cells is accompanied by the redistribution of major histocompatibility complex (MHC) class II molecules from the lysosomal MHC class II compartment to the plasma membrane to mediate presentation of peptide antigens. Besides MHC molecules, dendritic cells also express CD1 molecules that mediate presentation of lipid antigens. Herein, we show that in human monocyte-derived dendritic cells, unlike MHC class II, the steady-state distribution of lysosomal CD1b and CD1c isoforms was unperturbed in response to lipopolysaccharide-induced maturation. However, the lysosomes in these cells underwent a dramatic reorganization into electron dense tubules with altered lysosomal protein composition. These structures matured into novel and morphologically unique compartments, here termed mature dendritic cell lysosomes (MDL). Furthermore, we show that upon activation mature dendritic cells do not lose their ability of efficient clathrin-mediated endocytosis as demonstrated for CD1b and transferrin receptor molecules. Thus, the constitutive endocytosis of CD1b molecules and the differential sorting of MHC class II from lysosomes separate peptide- and lipid antigen-presenting molecules during dendritic cell maturation.     

The role of Src-kinase in the regulation of endocytosis of EGF-receptor complexes. Distribution of clathrin after stimulation of EGR endocytosis in various cell lines during inhibition of Src-kinase activity

A distribution of EGF receptor and clathrin during EGF endocytosis in A431, HER14, WT and PURO cell lines was studied by indirect immunofluorescence. Though the initial distribution of EGF-receptors on A431 and HER14 cells was somewhat different, the late stages of endocytosis proceeded equally and were marked by formation of bright spots in the juxtanuclear region characteristic of the late endosomes. The Src-family kinase inhibitor CGP77675 had no influence on the dynamics of receptor endocytosis at the immunofluorescent level in both cell lines. Stimulation of EGF-receptor endocytosis in A431 cells did not also result in any redistribution of clathrin in the areas where the majority of EGF-receptors are localized, i.e. in the lateral plasma membrane both in the control cells and under CGP77675 treatment. Clathrin in A431, WT and PURO cells demonstrated even a punctuated pattern throughout the cytoplasm with some accumulation in the juxtanuclear region. This distribution depended neither on the absence or presence of Src activity nor on EGF addition. The data obtained indicate that 1) EGF-receptors do not serve as the initiation sites during clathrin coated pit assembly; 2) Src-kinase activation does not result in significant clathrin redistribution in the plasma membrane, and its influence on EGF endocytosis can be considered as a secondary effect.     

Surface distribution and partition during freeze-fracture of CD8 antigens on human lymphocytes and on epithelial transfected cells

Freeze-fracture immunocytochemistry was used to analyse the surface distribution, redistribution induced by antibodies, and partition during freeze-fracture, of CD8 molecules on human T lymphocytes and rat epithelial transfected (FRT-U10) cells. Immunogold labelling of CD8 antigens was uniform over the unfractured cell surfaces of both lymphocytes and epithelial transfected cells. After freeze-fracture, the gold particles were associated with the exoplasmic outer leaflets of the plasma membranes in both cell types. In lymphocytes, incubation with antibodies at 37° C up to 20 min induced patching and capping of the antigens on the unfractured cell surface. After fracture, the patched molecules appeared associated with the protoplasmic inner leaflet of the plasma membranes. Parallel antibody-treatment at 37° C of FRT-U10 cells induced clustering of CD8 molecules but failed to cause further aggregation in larger patches or in caps. After freeze-fracture, the immunola-belling was clustered, but associated with the exoplasmic outer leaflet of the plasma membranes as in untreated cells. The different redistribution induced by antibodies and the different behaviour on fracture of the redistributed molecules in the two cell types may be regulated by CD8 interaction with the cytoskeleton.     

Role of coated vesicles, microfilaments, and calmodulin in receptor- mediated endocytosis by cultured B lymphoblastoid cells

Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) binding of the multivalent ligand in a diffuse cell surface distribution, (b) clustering of the ligand-receptor complexes, (c) recruitment of clathrin coats to the cytoplasmic surface of the cell membrane opposite ligand-receptor clusters, (d) assembly and (e) internalization of coated vesicles, and (f) delivery of label into a large vesicular compartment, presumably partly lysosomal. Most of the labeled ligand enters this pathway. The recruitment of clathrin coats to the membrane opposite ligand-receptor clusters is sensitive to the calmodulin-directed drug Stelazine (trifluoperazine dihydrochloride). In addition, Stelazine inhibits an alternate pathway of endocytosis that does not involve coated vesicle formation. The actin-directed drug dihydrocytochalasin B has no effect on the recruitment of clathrin to the ligand-receptor clusters and the formation of coated pits and little effect on the alternate pathway, but this drug does interfere with subsequent coated vesicle formation and it inhibits capping. Cortical microfilaments that decorate with heavy meromyosin with constant polarity are observed in association with the coated regions of the plasma membrane and with coated vesicles. SDS-polyacrylamide gel electrophoresis analysis of a coated vesicle preparation isolated from WiL2 cells demonstrates that the major polypeptides in the fraction are a 175-kdalton component that comigrates with calf brain clathrin, a 42- kdalton component that comigrates with rabbit muscle actin and a 18.5- kdalton minor component that comigrates with calmodulin as well as 110- , 70-, 55-, 36-, 30-, and 17-kdalton components. These results clarify the pathways of endocytosis in this cell and suggest functional roles for calmodulin, especially in the formation of clathrin-coated pits, and for actin microfilaments in coated vesicle formation and in capping.     

Regulation of cell wall synthesis by auxin and fusicoccin in different tissues of pea stem segments

Cell wall synthesis was studied by determining the incorporation of [¹⁴C]-glucose into epidermal and cortical cell walls of etiolated Pisum sativum L. cv. Alaska stem segments. Walls were fractionated into the matrix and cellulose components, and incorporation into these components assessed in terms of the total uptake of label into that tissue. When segments were allowed to elongate, the stimulation of total glucose uptake by indole-3-acetic acid (IAA) and fusicoccin (FC) was greater than their stimulation of incorporation. IAA and FC thus did not stimulate precursor incorporation in elongating segments. When elongation was inhibited by calcium, however, IAA and FC significantly promoted wall synthesis in the cortex and vasular tissue (which shows almost no growth or acidification response to auxin). In these tissues incorporation into matrix and cellulose was promoted approximately equally. In the epidermis (thought to be the tissue responsive to auxin in the control of growth), FC promoted a significant increase in wall synthesis, although less than that in the cortex, while there was some evidence of a similar promotion by IAA. Both IAA and FC had a greater effect on incorporation into the matrix component of the wall than into cellulose. The results that FC caused a substantial promotion of cell wall synthesis which was not due solely to elongation, and that the inner non-growth responsive cortical tissues can respond to IAA. Moreover, a comparison of the effects of IAA and FC on the different components of the wall suggests that the response in the epidermis differs from that in the other tissues.     

Comparison of the effect of indoleacetic Acid and fusicoccin on the breakdown of phosphatidylinositol in maize coleoptiles

The effect of indoleacetic acid (IAA) and fusicoccin (FC) on the breakdown of phosphatidylinositol in maize (Zea mays L.) coleoptiles has been studied. Coleoptiles were able to incorporate [³H] myo-inositol into the phospholipid fraction almost linearly for 8 hours. Thin layer chromatography analysis of total phospholipids showed that [³H]myo-inositol was incorporated only into phosphatidylinositol. Prelabeled coleoptiles treated with IAA showed a loss of the radioactivity incorporated in the phospholipid fraction, whose level decreased by 34% after 1 hour. Treatment with FC, on the contrary, did not modify the content of labelled phosphatidylinositol with respect to the control. The different effects of IAA and FC and a possible mechanism of IAA action on growth are discussed.     

Antibody-induced capping of measles virus antigens on plasma membrane studied by electron microscopy.

Antibodies specific for measles virus could redistribute ("cap") virus antigens on infected HeLa cells as shown by transmission and scanning electron microscopy. Using an indirect immunoperoxidase technique, infected cells showed diffuse, circumferential distribution of virus antigens over the cell surface when mixed with antibody at 4 C. At 37 C, virus-coated microvilli concentrated on one pole of the cell, leaving the remainder of the plasma membrane devoid of both viral antigens and microvillus projections. Whereas extreme polar displacement of virus-antibody complexes frequently occurred, endocytosis was rarely seen. The findings indicate that antiviral antibodies can move and cluster virus on plasma membranes and suggest that virus-antibody complexes are stripped and shed from the cell surface.     

Membrane shuttle between plasma membrane, phagosomes, and pinosomes in Dictyostelium discoideum amoeboid cells

The intracellular redistribution of membrane internalized during endocytosis was studied quantitatively by a biochemical approach and by a morphometric analysis of autoradiographs in electron microscopy. Plasma membrane glycoconjugates, enzymatically labelled with radioactive galactose, were used as a membrane marker. In cells labelled at their surface either before or after the phagocytotic uptake of latex beads, subsequent endocytosis led to a redistribution of label between the plasma membrane and endosomal membranes until a steady-state was reached after about 1 h with 43% of the label on the plasma membrane. The steady-state resulted when all participating membranes carried the same surface density of label. During phagocytosis or pinocytosis the equivalent of the plasma membrane was internalized and recycled once every 20 min or 40 min, respectively. Compared to this rate a very rapid and complete mixing of membranes was observed between newly formed phagosomes and preexisting digestive vacuoles or between newly formed pinosomes and preexisting phagosomes. Due to this rapid mixing, the membranes enclosing undigestible latex beads remained fully linked to the shuttle of membrane to and from the cell surface.     

Hormonal and gravitropic specificity in the regulation of growth and cell wall synthesis in pulvini and internodes from shoots of Avena sativa L. (oat)

Segments can be cut from the peduncular-1 internode of oat (Avena sativa L.) shoots so as to contain the graviresponsive leaf-sheath pulvinus and gibberellin-sensitive internodal tissue. Incorporation of [14C]glucose was used to monitor cell wall synthesis in these two tissues as affected by gravistimulus, indoleacetic acid (IAA), gibberellic acid (GA3), and fusicoccin (FC). Pulvinar cell wall synthesis was promoted by IAA and FC (both within about 1 h), as well as by gravistimulus (starting between 3 and 6 h), whereas GA3 had no effect on nongravistimulated pulvini. In contrast, GA3 and FC promoted internodal cell wall synthesis (initiated between 1 and 2 h), whereas IAA and gravistimulus caused a decrease in internodal uptake. FC preferentially promoted incorporation into the matrix component of the wall in both tissues. Gravistimulus failed to increase responsiveness of pulvinar tissue to IAA, whereas GA3 partially overcame gravistimulus-promoted incorporation into pulvinar cell wall, probably because of preferential movement of label into the rapidly elongating internode. The results demonstrate that these eight stimulus/tissue combinations can be examined easily in an isolated 10-mm stem segment, providing new opportunities for the comparative study of tissue- and stimulus-specific events in gene regulation and signal transduction in agronomically important cereals.     

Accumulation of WGA Receptors in the Cleavage Furrow during Cytokinesis of Sea Urchin Eggs

Sea urchin eggs stained with fluorescein-conjugated wheat germ agglutinin (F-WGA) before or after fixation showed a marked accumulation of fluorescence at the cleavage furrow in the first and the second cell divisions. WGA receptors (WGA-binding membrane glycoproteins) were redistributed to the equatorial region through several steps in compressed eggs. Accumulated WGA receptors showed a distribution similar to that of contractile-ring microfilaments throughout most of the steps. Therefore, the former is probably associated with the latter directly or indirectly. Labeling with F-WGA provides a simple method to detect contractile-ring microfilaments in living eggs. Treatment of eggs with colcemid shortly before cytokinesis dispersed the ring-like accumulation of WGA receptors together with contractile-ring microfilaments. This result suggests that microtubule structures, probably asters, are involved in the redistribution of WGA receptors. Cytochalasin B prevented furrowing when it was applied shortly before cytokinesis. While contractile-ring microfilaments showed a spotty distribution in the expected furrow region, WGA receptors were normally redistributed. Furthermore, a higher concentration of the drug allowed the appearance of accumulated WGA receptors in compressed eggs although the development into a ring-like configuration was inhibited. These observations suggest the possibility that the redistribution of WGA receptors is involved in the formation of contractile ring.     

Dynamics of phragmoplastin in living cells during cell plate formation and uncoupling of cell elongation from the plane of cell division.

The cell plate is formed by the fusion of Golgi apparatus-derived vesicles in the center of the phragmoplast during cytokinesis in plant cells. A dynamin-like protein, phragmoplastin, has been isolated and shown to be associated with cell plate formation in soybean by using immunocytochemistry. In this article, we demonstrate that similar to dynamin, phragmoplastin polymerizes to form oligomers. We fused soybean phragmoplastin with the green fluorescence protein (GFP) and introduced it into tobacco BY-2 cells to monitor the dynamics of early events in cell plate formation. We demonstrate that the chimeric protein is functional and targeted to the cell plate during cytokinesis in transgenic cells. GFP-phragmoplastin was found to appear first in the center of the forming cell plate, and as the cell plate grew outward, it redistributed to the growing margins of the cell plate. The redistribution of phragmoplastin may require microtubule reorganization because the microtubule-stabilizing drug taxol inhibited phragmoplastin redistribution. Our data show that throughout the entire process of cytokinesis, phragmoplastin is concentrated in the area in which membrane fusion is active, suggesting that phragmoplastin participates in an early membrane fusion event during cell plate formation. Based on the dynamics of GFP-phragmoplastin, it appears that the process of cell plate formation is completed in two phases. The first phase is confined to the cylinder of the phragmoplast proper and is followed by a second phase that deposits phragmoplast vesicles in a concentric fashion, resulting in a ring of fluorescence, with the concentration of vesicles being higher at the periphery. In addition, overexpression of GFP-phragmoplastin appears to act as a dominant negative, slowing down the completion of cell plate formation, and often results in an oblique cell plate. The latter appears to uncouple cell elongation from the plane of cell division, forming twisted and elongated cells with longitudinal cell divisions.     

Nucleophilic Distribution of Metallothionein in Human Tumor Cells

Metallothionein (MT), a major zinc-binding intracellular protein thiol, has been associated with cytoprotection from heavy metals, antineoplastic drugs, mutagens, and cellular oxidants. Despite its small mass (7 kDa), nuclear partitioning of MT has been observed in both normal and malignant tissues. The factors controlling MT sequestration are unknown. Thus, we examined the regulation of MT subcellular distribution in human cancer cell lines that exhibit prominent nuclear MT. The nuclear disposition of MT was unaltered during cell cycle passage in synchronized cells. MT redistributed to the cytoplasm when cells were exposed to reduced temperature. Cytoplasmic redistribution was also seen in DU-145 and HPC36M prostatic cancer cells after ATP depletion, but not in PC3-MA2 and SCC25/CP cells. Pretreatment with 10 μMCdCl₂did not significantly alter MT distribution but did render all cells sensitive to cytoplasmic redistribution after either reduced temperature or ATP depletion. Thus, nuclear retention of MT is energy requiring and this ability of MT to accumulate in subcellular compartments against its concentration gradient may be important in the capacity of MT to supply Zn or other metals to target sites within the cell.     

Gangliosides and β1‐Integrin Are Required for Caveolae and Membrane Domains

Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin‐mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo‐glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol (‘lipid rafts'), reduced caveolae and caveolin‐1 at the plasma membrane by approximately 80%, and blunted activation of β1‐integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo‐gangliosides or other sphingolipids) at 10°C, suggesting that sialo‐lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Iγ away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin‐1 could be recapitulated by β1‐integrin knockdown. These results suggest that both gangliosides and β1‐integrin are required for maintenance of caveolae and plasma membrane domains.