Actin, myosin and alpha-actinin containing filament bundles in hyaline cells of the caiman cochlea.

Hyaline cells of the auditory organ of the spectacled caiman contain smooth muscle-like filament bundles within their basal cell pole. These bundles were heavily labeled with antibodies to actin, myosin and alpha-actinin (muscular Z-line protein). Since hyaline cells are firmly attached to the basilar membrane these cells may actively modify the stiffness of the basilar membrane. A contractile mechanism in hyaline cells might affect frequency tuning of primary auditory afferents. This frequency tuning has been shown to be a temperature-dependent process in caimans and other submammalian species. The presence of synaptic contacts between efferent nerve fibres and hyaline cells suggests neural control of hyaline cell activity.

Three different actin filament assemblies occur in every hair cell: each contains a specific actin crosslinking protein.

The apex of hair cells of the chicken auditory organ contains three different kinds of assemblies of actin filaments in close spatial proximity. These are (a) paracrystals of actin filaments with identical polarity in stereocilia, (b) a dense gellike meshwork of actin filaments forming the cuticular plate, and (c) a bundle of parallel actin filaments with mixed polarities that constitute the circumferential filament belt attached to the cytoplasmic aspect of the zonula adhaerens (ZA). Each different supramolecular assembly of actin filaments contains a specific actin filament cross-linking protein which is unique to that particular assembly. Thus fimbrin appears to be responsible for paracrystallin packing of actin filaments in stereocillia; an isoform of spectrin resides in the cuticular plate where it forms the whisker-like crossbridges, and alpha actinin is the actin crosslinking protein of the circumferential ZA bundle. Tropomyosin, which stabilizes actin filaments, is present in all the actin filament assemblies except for the stereocilia. Another striking finding was that myosin appears to be absent from the ZA ring and cuticular plate of hair cells although present in the ZA ring of supporting cells. The abundance of myosin in the ZA ring of the surrounding supporting cells means that it may be important in forming a supporting tensile cellular framework in which the hair cells are inserted.

Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea.

The sensory epithelium of the chick cochlea contains only two cell types, hair cells and supporting cells. We developed methods to rapidly dissect out the sensory epithelium and to prepare a detergent-extracted cytoskeleton. High salt treatment of the cytoskeleton leaves a "hair border", containing actin filament bundles of the stereocilia still attached to the cuticular plate. On SDS-PAGE stained with silver the intact epithelium is seen to contain a large number of bands, the most prominent of which are calbindin and actin. Detergent extraction solubilizes most of the proteins including calbindin. On immunoblots antibodies prepared against fimbrin from chicken intestinal epithelial cells cross react with the 57- and 65-kD bands present in the sensory epithelium and the cytoskeleton. It is probable that the 57-kD is a proteolytic fragment of the 65-kD protein. Preparations of stereocilia attached to the overlying tectorial membrane contain the 57- and 65-kD bands. A 400-kD band is present in the cuticular plate. By immunofluorescence, fimbrin is detected in stereocilia but not in the hair borders after salt extraction. The prominent 125 A transverse stripping pattern characteristic of the actin cross-bridges in a bundle is also absent in hair borders suggesting fimbrin as the component that gives rise to the transverse stripes. Because the actin filaments in the stereocilia of hair borders still remain as compact bundles, albeit very disordered, there must be an additional uncharacterized protein besides fimbrin that cross-links the actin filaments together.

Role of microtubules in polarized delivery of apical membrane proteins to the brush border of the intestinal epithelium.

Colchicine- and vinblastine-induced depolymerization of microtubules (MTs) in the intestinal epithelium of rats and mice resulted in significant delivery of three apical membrane proteins (alkaline phosphatase, sucrase-isomaltase, and aminopeptidase N) to the basolateral membrane domain. In addition, typical brush borders (BBs) occurred at the basolateral cell surface, consisting of numerous microvilli that contained the four major components of the cytoskeleton of apical microvilli (actin, villin, fimbrin, and the 110-kD protein). Formation of basolateral microvilli required polymerization of actin and proceeded at glycocalyx-studded plaques that resembled the dense plaques located at the tips of apical microvilli. BBs from the basolateral membrane became internalized into BB-containing vacuoles which served as recipient organelles for newly synthesized apical membrane proteins. The BB vacuoles fused with each other and finally were inserted into the apical BB. Polarized distribution of Na+,K+-ATPase, a basolateral membrane protein, was not affected by drug-induced depolymerization of MTs. These observations indicate that Golgi-derived carrier vesicles (CVs) containing apical membrane proteins are vectorially guided to the apical cell surface by a retrograde transport along MTs. MTs are uniformly oriented towards a narrow space underneath the apical terminal web (termed subterminal space) that contains MT-organizing properties and controls polarized alignment of MTs. In contrast to apical CVs, targeting of basolateral CVs appears to be independent of MTs but demands a barrier at the apical membrane domain that prevents basolateral CVs from apical fusion (transport barrier hypothesis).

Restriction of the human kidney band 3-like anion exchanger to specialized subdomains of the basolateral plasma membrane of intercalated cells.

In this study, the kidney analog of the erythrocyte anion exchanger, band 3, served as the first example of an anion translocating membrane protein in a nucleated cell type to be localized at the ultrastructural level. Kidney band 3 was found to be confined to the basolateral membrane of the intercalated cells in the human collecting duct. The immunogold label displayed a striking non-uniform distribution along the basolateral plasma membrane with a preferential concentration at pleated areas of the membrane surface. The pleated portions are suggested to represent specialized subdomains to which the band 3 analog might be restricted by linkage via ankyrin to the spectrin-based membrane cytoskeleton. The immunolabel did not extend apically to the level of the zonula adherens and zonula occludens indicating that tight junctions might not be important for maintaining the polarized distribution of this integral membrane protein. Association of antibody label with the rough endoplasmic reticulum and other types of cytoplasmic membranes indicate pathways in the biosynthesis and degradation of this anion exchanger.