Cytoskeletal integration in a highly ordered sensory epithelium in the organ of Corti: reponse to loss of cell partners in the Bronx waltzer mouse.

This report is concerned with control of cell shaping, positioning, and cytoskeletal integration in a highly ordered cochlear neuroepithelium. It is largely based on investigations of events that occur during abnormal morphogenesis of the organ of Corti in the Bronx waltzer (bv/bv) mutant mouse. The organ's sensory hair cells and adjacent supporting cells ordinarily construct a spatially elaborate and supracellularly integrated cytoskeletal framework. Large microtubule bundles are connected to cytoskeletal components in neighbouring cells by actin-containing meshworks that link them to substantial arrays of adherens junctions. In bv/bv mice, degeneration and loss of most inner hair cells and outer pillar cells occurs during organ development. These cells flank each side of a row of inner pillar cells that respond by upregulating assembly of their actin-containing meshworks. This only occurs in surface regions where they no longer contact cell types involved in construction of the cytoskeletal framework. The meshworks are larger and exhibit a more extensive sub-surface deployment than is normally the case. Hence, assembly of intercellular cytoskeletal connecting components can proceed without contact with appropriate cell neighbours but termination of assembly is apparently subject to a negative feedback control triggered by successful completion of intercellular connection with the correct cell neighbours. In addition, inner pillar cells compensate for loss of cell neighbours by interdigitating and overlapping each other more extensively than is usually the case to increase opportunities for generating adherens junctions. Certain adherens junctions in the organs of +/+ and bv/bv mice exhibit features that distinguish them from all previously described cell junctions. The dense plaques on their cytoplasmic faces are composed of aligned ridges. We suggest that they are called ribbed adherens junctions. Perturbations of cell shaping and positioning indicate that loss of inner hair cells is the primary consequence of the bv mutation. Most of the other abnormalities can be understood in terms of a secondary sequence of morphogenetic aberrations (precipitated by loss of inner hair cells). These aberrations provide new information about the ways in which supporting cells help to control hair cell positioning.

Keratin filament deployment and cytoskeletal networking in a sensory epithelium that vibrates during hearing.

The intricate and spatially precise ways in which keratin intermediate filaments are deployed in certain cochlear epithelial cells, called supporting cells, suggests that these filaments make a micromechanically important contribution to the functional design of the guinea pig organ of Corti. Filament arrays that include keratins 8, 18, and 19 are confined mainly to regions close to the ends of large transcellular microtubule bundles in supporting cells. These cells and their microtubule bundles link sensory hair cells to a specialized basement membrane that vibrates during hearing. The keratin filament arrays apparently help anchor the ends of the microtubule bundles to cell surfaces. Filaments are concentrated at the apices and bases of most cells that contact hair cells. Substantial arrays of adherens junctions link the apices of these cells. Hence, keratin filaments may contribute to a cytoskeletal network that distributes mechanical forces from cell to cell and that coordinates the displacement of neighboring hair cells. However, high concentrations of keratin filaments have not been detected at the apices of one of the supporting cell types, which apparently has a mechanical role that is different from that of the others. Transmission electron microscopy has revealed previously undescribed filament networks at all the locations where the binding of antibodies to keratins is most marked. There is evidence that intercellular linkage of the keratin networks via their association with actin-containing meshworks and adherens junctions is more extensive than linkage provided by desmosomes.

Centrosomal deployment of gamma-tubulin and pericentrin: evidence for a microtubule-nucleating domain and a minus-end docking domain in certain mouse epithelial cells.

This report provides evidence for two functionally and spatially distinct centrosomal domains in certain mouse cochlear epithelial cells. The vast majority of microtubules elongate from sites associated with the apical cell surface in these cells rather than from pericentriolar material surrounding the immediate environs of their apically situate centrioles. The distribution of gamma-tubulin and pericentrin at cell apices has been examined while microtubule nucleation is progressing because these centrosomal proteins are believed to be essential for microtubule nucleation. Antibodies to both proteins bind to pericentriolar regions but no binding has been detected at the apical cell surface-associated sites where the ends of thousands of recently nucleated microtubules are concentrated. Sparse transient microtubule populations can be detected between pericentriolar regions and surface sites while microtubule assembly advances. A procedure apparently operates in which the pericentriolar region functions as a microtubule-nucleating domain and the cell surface-associated sites operate as docking domains which capture the minus ends of microtubules that migrate to them shortly after nucleation. Docking domains may include some components of the pericentriolar material that have been relocated at the cell apex. A docking element hypothesis for centrosomal control of minus end positioning and dynamics in animal cells generally is proposed. This investigation has also shown that the concentration of gamma-tubulin and pericentrin around centrioles differs spatially and quantitatively in ways that are characteristic for the four cell types studied. Some of these characteristics can be related to differences in control of microtubule number and positioning.

Remodeling adult nursing.

This article describes a nursing education experience in which a critical thinking approach was planned and implemented. Background discussion on critical thinking concepts and related research provides a foundation for presentation of the Mackie teaching model. The model uses a community-based, family-centered scenario as the basis for developing problem-focused nursing intervention skills from a holistic viewpoint. Role expectations of students and faculty are outlined, and related implementation difficulties, together with their resolution strategies, are described. Evaluation methods and outcomes are reviewed in the conclusion.

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

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.

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

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.

Reorganization of the centrosome and associated microtubules during the morphogenesis of a mouse cochlear epithelial cell.

Reorganization of centrosomal microtubule-organizing centres and the minus ends of microtubules occurs as the centrosomal ends of large microtubule bundles are repositioned and anchored to cell junctions in certain epithelial cells called inner pillar cells in the mouse organ of Corti. The microtubule bundle that assembles in each cell consists of two distinct microtubule arrays that run closely alongside each other. Both arrays are attached to the cell surface at their upper and lower ends. One of the arrays spans the entire length of a cell but the other is confined to its lower portion. Initially, about 3,000 microtubules elongate downwards from an apically situated centrosome in each cell. Subsequently, the minus ends of these microtubules, and the centrosome and its two centrioles, migrate for about 12 microns to the tip of a laterally directed projection. Then, a meshwork of dense material accumulates to link microtubule minus ends and the centrosome to cell junctions at the tip of the projection. Pericentriolar satellite bodies, which form after the initial burst of microtubule nucleation, may represent a condensed and inactive concentration of microtubule-nucleating elements. Surprisingly, as a cell matures, about 2,000 microtubules are eliminated from the centrosomal end of the microtubule bundle. However, about 2,000 microtubules are added to the basal portion of each bundle at levels that are remote with respect to the location of the centrosome. Possibly, these microtubules have escaped from the centrosome. If this is the case, then both the plus and minus ends of most of the errant microtubules are captured by sites at the cell surface where the ends are finally anchored. Alternatively, each cell possesses at least one other major microtubule-nucleating site (which does not possess centrioles) in addition to its centrosome.

Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium.

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.

Configurational changes in helical microtubule frameworks in feeding tentacles of the suctorian ciliate Tokophyra.

Microtubules at the tip of a resting (non-feeding) tentacle are arranged helically in two concentric tube-shaped arrays. The pitches of the helical paths followed by tubules in the two arrays differ. At the start of feeding these microtubules bend along their longitudinal axes and splay outwards and downwards away from the tentacle tip as it 'everts'. Tubules in the two arrays slide across each other as this occurs. Comparison of the fine structure of the tips of feeding and resting tentacles with a dynamic model of the microtubular framework indicates that movement of the tubules is not brought about by active sliding of the tubules against each other or by the action of contractile elements attached along the lengths of the tubules. The tips of microtubules forming the inner tube may be pulled downwards by contractile elements in the tentacular pellicle; these tubules apparently push those in the outer tube to their new position. The pattern of configurational changes in a tentacle tip at the start of feeding appears to be largely defined by the elastic resistance of the microtubules to bending, and the ways in which tubules are packed and linked together and attached to the pellicle.