Formation of two microtubule-nucleating sites which perform differently during centrosomal reorganization in a mouse cochlear epithelial cell

1995 ◽  
Vol 108 (4) ◽  
pp. 1333-1345 ◽  
Author(s):  
J.B. Tucker ◽  
M.M. Mogensen ◽  
C.C. Paton ◽  
J.B. Mackie ◽  
C.G. Henderson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Apical Cell ◽  
Organ Of Corti ◽  
Longitudinal Axis ◽  
Sequence Of Events ◽  
Pericentriolar Material ◽  
High Concentrations ◽  
Oriented Parallel ◽  
A Site

This report provides evidence for the formation of a cell surface-associated centrosome with two spatially discrete microtubule-nucleating sites that perform differently; the minus ends of microtubules remain anchored to one site but escape from the other. Centrosomal reorganization in the cells in question, outer pillar cells of the organ of Corti, indicates that its pericentriolar material becomes intimately associated with the plasma membrane at the two nucleating sites. Two large microtubules bundles assemble in each cell. A beam which includes about 1,300 microtubules spans most of the cell apex. It is positioned at right angles to a pillar with about 4,500 microtubules which is oriented parallel to the cell's longitudinal axis. The beam's microtubules elongate from, and remain attached to, a centrosomal region with two centrioles which acts as a microtubule-nucleating site. However, the elongating microtubules do not radiate from the immediate vicinity of the centrioles. During beam assembly, the minus ends of the microtubules are concentrated together close to the plasma membrane (less than 0.2 micron away in many cases) at a site which is located to one side of the cell apex. High concentrations of the pillar's microtubules elongating from one particular site have not been detected. Analyses of pillar assembly indicate that the following sequence of events occurs. Pillar microtubules elongate from an apical cell surface-associated nucleating site, which becomes more distantly separated from the centriolar locality as cell morphogenesis progresses. Microtubules do not accumulate at this apical nucleating site because they escape from it. They migrate down to lower levels in the cell where the mature bundle is finally situated and their plus ends are captured at the cell base.

scholarly journals Three microtubule-organizing centres collaborate in a mouse cochlear epithelial cell during supracellularly coordinated control of microtubule positioning

1995 ◽  
Vol 108 (1) ◽  
pp. 37-50 ◽  
Author(s):  
C.G. Henderson ◽  
J.B. Tucker ◽  
M.M. Mogensen ◽  
J.B. Mackie ◽  
M.A. Chaplin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Basement Membrane ◽  
Cell Surface ◽  
Basilar Membrane ◽  
Apical Cell ◽  
Organ Of Corti ◽  
Large Cell ◽  
Cell Junctions ◽  
Microtubule Bundle ◽  
Microtubule Organizing Centres

Large cell surface-associated microtubule bundles that include about 3,000 microtubules assemble in certain epithelial cells called inner pillar cells in the mouse organ of Corti. Microtubule-organizing centres (MTOCs) at both ends and near the middle of each cell act in concert during control of microtubule positioning. In addition, the three cell surface-associated microtubule-organizing centres are involved in coordinating the connection of bundle microtubules to cytoskeletal components in neighbouring cells and to a basement membrane. The precisely defined locations of the three MTOCs specify the cell surface regions where microtubule ends will finally be anchored. The MTOCs are modified as anchorage proceeds. Substantial fibrous meshworks assemble at the surface sites occupied by the MTOCs and link microtubule ends to cell junctions. This procedure also connects the microtubule bundle to cytoskeletal arrays in neighbouring cells at two of the MTOC sites, and to the basilar membrane (a substantial basement membrane) in the case of the third site. A fourth meshwork that is not positioned at a major MTOC site is involved in connecting one side of the microtubule bundle to the cytoskeletons of two other cell neighbours. The term surfoskelosome is suggested for such concentrations of specialized cytoskeletal materials and junctions at cell surface anchorages for cytoskeletal arrays. The large microtubule bundle in each cell is composed of two closely aligned microtubule arrays. Bundle assembly begins with nucleation of microtubules by a centrosomal MTOC that is attached to the apical cell surface. These microtubules elongate downwards and the plus ends of many of them are apparently captured by a basal MTOC that is attached to the plasma membrane at the bottom of the cell. In the lower portion of the cell, the microtubule bundle also includes a basal array of microtubules but these elongate in the opposite direction. This investigation provides evidence that they extend upwards from the basal MTOC to be captured by a medial MTOC which is attached to the plasma membrane and situated near the mid-level of the cell. However, there are substantial indications that the basal array's microtubules are also nucleated by the apically situated centrosomal MTOC, but escape from it, and are translocated downwards for capture of their plus ends by the basal MTOC. If this is the case, then these microtubules continue to elongate after translocation and extend back up to the medial MTOC, which captures their minus ends.

Evidence for microtubule nucleation at plasma membrane-associated sites in Drosophila

1987 ◽  
Vol 88 (1) ◽  
pp. 95-107 ◽  
Author(s):  
M.M. Mogensen ◽  
J.B. Tucker
Keyword(s):  
Plasma Membrane ◽  
Cell Differentiation ◽  
Cross Section ◽  
Plasma Membranes ◽  
Early Stage ◽  
Apical Cell ◽  
Apical Plasma Membrane ◽  
Cell Surfaces ◽  
Microtubule Organizing Centres ◽  
Oriented Parallel

This report is concerned with the nucleation and organization of microtubule bundles that assemble after ‘conventional’ centrosomal microtubule-organizing centres have been lost. The microtubule bundles in question span the lengths of wing epidermal cells. Bundles extend between hemidesmosomes at the apical cuticle-secreting surfaces of cells and basal attachment desmosomes that unite the dorsal and ventral epidermal layers of developing wing blades. Furthermore, each bundle includes up to 1500 microtubules and most of the microtubules are composed of 15 protofilaments. Individual cells were serially cross-sectioned at an early stage of bundle assembly. The number of microtubule profiles/cell cross-section decreased progressively by up to 59% of the most apical values in section sequences cut from fairly apical to more basal levels in the cells. The apical ends of microtubules were associated with numerous small dense plaque-like sites (diameter 0.1-0.2 micron), which were specialized regions of plasma membranes at the apical surfaces of cells. Many of the microtubules near apical plaques were not well aligned with each other; they ‘radiated away’ from cell apices. This was in contrast to the situation at more basal levels where most microtubules were oriented parallel to the longitudinal axes of cells. These findings indicate that the relatively dispersed arrays of apical plasma membrane-associated plaques act as microtubule-nucleating sites to initiate basally directed elongation of bundle microtubules. Apical cell surfaces and their plaques seem to operate as microtubule-nucleating and -organizing regions that functionally replace the centrosomal microtubule-organizing centres lost earlier in cell differentiation.

scholarly journals Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma cells.

1987 ◽  
Vol 35 (8) ◽  
pp. 809-816 ◽  
Author(s):  
R Pakkanen ◽  
K Hedman ◽  
O Turunen ◽  
T Wahlström ◽  
A Vaheri
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Polyclonal Antibodies ◽  
Apical Cell ◽  
Antigenic Site ◽  
Electron Microscopic ◽  
Permeabilized Cells ◽  
Choriocarcinoma Cells

We have previously purified from cultured JEG-3 choriocarcinoma cells an Mr 75,000 protein, originally detected using antibodies to a retrovirus-related synthetic peptide. Using polyclonal antibodies, we have now localized this protein immunocytochemically in JEG-3 cells at both light and electron microscopic levels. In immunofluorescence microscopy of saponin-permeabilized cells, the antigen appeared as dots and short strands at the apical cell surface. In pre-embedding immunoperoxidase electron microscopy, the Mr 75,000 protein was specifically localized to microvilli on the apical cell surface. Immunoferritin electron microscopy was used to assess more quantitatively the antigen distribution in the plane of the plasma membrane, and to define the position of the antigenic site(s) with respect to the membrane. The immunoferritin results confirmed the microvillus specificity of the Mr 75,000 protein and showed that the antigenic portion of the protein is within a few nanometers from, and on the cytoplasmic side of, the lipid bilayer. In detergent extraction experiments, the Mr 75,000 antigen was highly enriched in the soluble fractions. These results demonstrate that the Mr 75,000 protein is a membrane protein highly specific for microvilli.

scholarly journals Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes.

1991 ◽  
Vol 114 (6) ◽  
pp. 1149-1158 ◽  
Author(s):  
D P Cerneus ◽  
A van der Ende
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Line ◽  
Apical Cell ◽  
Bewo Cell ◽  
Transferrin Receptors ◽  
Membrane Domains ◽  
Bewo Cells ◽  
Early Endosomes ◽  
Plasma Membrane Domains

Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells.

scholarly journals Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane.

1995 ◽  
Vol 128 (6) ◽  
pp. 1043-1053 ◽  
Author(s):  
K Fiedler ◽  
F Lafont ◽  
R G Parton ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Cell ◽  
Protein Composition ◽  
Apical Plasma Membrane ◽  
Vesicular Traffic ◽  
Transport Vesicles ◽  
Apical Vesicles

The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.

scholarly journals Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes.

1994 ◽  
Vol 125 (1) ◽  
pp. 67-86 ◽  
Author(s):  
G Apodaca ◽  
L A Katz ◽  
K E Mostov
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Cell ◽  
Fluid Phase ◽  
Secretory Component ◽  
Apical Plasma Membrane ◽  
Recycling Endosome ◽  
Endosomal Compartment ◽  
Early Endosomes

Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non-transcytosing molecules.

The three cortical membranes of the gregarines. I. Ultrastructural organization of Gregarina blaberae

1983 ◽  
Vol 61 (1) ◽  
pp. 151-174
Author(s):  
J. Schrevel ◽  
E. Caigneaux ◽  
D. Gros ◽  
M. Philippe
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Freeze Fracture ◽  
Longitudinal Axis ◽  
Ultrastructural Organization ◽  
Transmission Electron ◽  
Movement Mechanism ◽  
Cortical Membranes ◽  
Integral Proteins ◽  
Gliding Movement

Gregarines, parasitic protozoa of invertebrates, possess a highly differentiated cell surface, with three cortical membranes and associated structures. Transmission electron microscopy and freeze-fracture reveal the presence of two cytomembranes lying uniformly under the plasma membrane. The density and the distribution of the intramembraneous particles (IMPs) in the plasma membrane of Gregarina blaberae are similar to those reported for other eukaryotic cells. The IMP density is lower in the cytomembranes than in the plasma membrane. The distribution of IMPs in the different fracture faces of the two cytomembranes suggests that they are in topological continuity, forming either side of a flattened vesicle or cisterna. The sizes of the cytomembrane IMPs show a high variability. The nature of the IMPs, both for the plasma membrane and the cytomembrane, is discussed with regard to the integral proteins and glycoproteins of the ghost. The cell surface of G. blaberae exhibits numerous longitudinal folds with three types of cortical membrane-associated structures: 12 nm filaments, an internal lamina, and homogeneous structures described as ‘rippled dense structures’. The 12 nm filaments, running under the cytomembranes along the longitudinal axis of each fold, exhibit the properties of intermediate filaments. Their distribution in mature cells and during the growth process suggests a participation in cell surface morphogenesis. The internal lamina, also localized under the cytomembranes, would stabilize each fold and assure a scaffolding function between the numerous folds. The rippled dense structures, settled on the external cytomembrane, show a regular distribution at the top of each fold. The membrane-associated structures are discussed with regard to the gliding movement mechanism.

Vasopressin-induced changes in the three-dimensional structure of toad bladder apical surface

1987 ◽  
Vol 253 (5) ◽  
pp. C707-C720 ◽  
Author(s):  
J. H. Hartwig ◽  
D. A. Ausiello ◽  
D. Brown
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Apical Cell ◽  
Three Dimensional ◽  
Granular Cell ◽  
Cytochalasin D ◽  
Toad Bladder ◽  
Dimensional Structure ◽  
Apical Plasma Membrane ◽  
Apical Surface

The apical plasma membrane of toad bladder granular cells undergoes a rapid and dramatic increase in water permeability in response to vasopressin stimulation. Previous studies have shown that this permeability increase is accompanied by characteristic changes in the morphology of this membrane and that these changes may be involved in the hormonal response. In this report, we have used the technique of rapid freezing and freeze drying to obtain high resolution stereo images of the surface of the granular cell apical plasma membrane before and during vasopressin stimulation. Using this approach, we confirmed that vasopressin induces a ridge-to-villus transformation of the cell surface even in the absence of osmotic water flow, but now show that this transformation occurs at least in part via a retraction of segments of preexisting ridges, rather than by the growth of new microvilli from the apical cell surface. This is also demonstrated by the finding that vasopressin induces the ridge-to-villus transformation of the cell surface even in the presence of cytochalasin D. In addition, the rapid-freeze, freeze-dry technique reveals that the surface glycocalyx of the epithelial cells consists of a complex, three-dimensional network of filaments that is heterogeneous among different cells. Finally, vasopressin-induced tubular invaginations of the apical plasma membrane were visualized in stereomicrographs, and the number and size of such invaginations were altered in the presence of cytochalasin D. These may represent surface images of vasopressin-induced exo- and endocytotic events that are related to membrane permeability changes.

A cell surface-associated centrosomal layer of microtubule-organizing material in the inner pillar cell of the mouse cochlea

1992 ◽  
Vol 102 (2) ◽  
pp. 215-226 ◽  
Author(s):  
J.B. Tucker ◽  
C.C. Paton ◽  
G.P. Richardson ◽  
M.M. Mogensen ◽  
I.J. Russell
Keyword(s):  
Cell Surface ◽  
Cultured Cells ◽  
Organ Of Corti ◽  
Microtubule Assembly ◽  
Apical Surface ◽  
Large Numbers ◽  
Pericentriolar Material ◽  
A Cell ◽  
Mouse Organ

This investigation provides evidence that pericentriolar material is divorced from the immediate vicinities of centrioles and becomes functionally associated with the plasmalemma during the differentiation of a mammalian cell type. Such events occur prior to the assembly of large transcellular microtubule bundles in columnar epithelial cells called inner pillar cells in the mouse organ of Corti. The microtubules do not radiate from a typical centrosome and its centrioles. They elongate from a microtubule-organizing centre (MTOC), which is deployed as a subapical cell surface-associated layer in each cell. Most of the dense material of this layer, and the tops of most of the microtubules, are initially concentrated around the sides of a cell about 1 microns below its apical surface. In addition, a pair of centrioles is located above the layer, which acts as if it is a pericellular concentration of the pericentriolar material of a modified centrosome. Although microtubule nucleation takes place in a centrosome-like region, 13 protofilament fidelity is not exercised. Most of the microtubules have 15 protofilaments. Microtubule assembly progresses in these cells after the organ of Corti has been isolated for in vitro culture. However, large numbers of microtubules elongate from pericentriolar material juxtaposed against the centrioles. Hence, there is some reversion by the centrosomes of cultured cells to the operational configuration regarded as typical for animal tissue cells in general.

scholarly journals Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells.

1986 ◽  
Vol 102 (4) ◽  
pp. 1242-1255 ◽  
Author(s):  
T A Gottlieb ◽  
A Gonzalez ◽  
L Rizzolo ◽  
M J Rindler ◽  
M Adesnik ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
G Protein ◽  
Apical Cell ◽  
Viral Envelope ◽  
Apical Plasma Membrane ◽  
Protein Synthesis Inhibitor ◽  
Transfected Cells ◽  
Cell Biol ◽  
Viral Glycoproteins

Previous studies (Rindler, M. J., I. E., Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100: 136-151; Rindler, M. J., I. E. Ivanov, H. Plesken, E. J. Rodriguez-Boulan, and D. D. Sabatini, 1984, J. Cell Biol., 98: 1304-1319) have demonstrated that in polarized Madin-Darby canine kidney cells infected with vesicular stomatitis virus (VSV) or influenza virus the viral envelope glycoproteins G and HA are segregated to the basolateral and apical plasma membrane domains, respectively, where budding of the corresponding viruses takes place. Furthermore, it has been shown that this segregation of the glycoproteins reflects the polarized delivery of the newly synthesized polypeptides to each surface domain. In transfection experiments using eukaryotic expression plasmids that contain cDNAs encoding the viral glycoproteins, it is now shown that even in the absence of other viral components, both proteins are effectively segregated to the appropriate cell surface domain. In transfected cells, the HA glycoprotein was almost exclusively localized in the apical cell surface, whereas the G protein, although preferentially localized in the basolateral domains, was also present in lower amounts, in the apical surfaces of many cells. Using transfected and infected cells, it was demonstrated that, after reaching the cell surface, the G protein, but not the HA protein, undergoes interiorization by endocytosis. Thus, in the presence of chloroquine, a drug that blocks return of interiorized plasma membrane proteins to the cell surface, the G protein was quantitatively trapped in endosome- or lysosome-like vesicles. The sequestration of G was a rapid process that was completed in many cells by 1-2 h after chloroquine treatment. The fact that in transfected cells the surface content of G protein was not noticeably reduced during a 5-h incubation with cycloheximide, a protein synthesis inhibitor that did not prevent the effect of chloroquine, implies that normally, G protein molecules are not only interiorized but are also recycled to the cell surface.

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