The changing microtubule arrangements in developing hair cells of the chick cochlea.

It has been suggested that microtubules in auditory hair cells might be involved in directing the morphological and hence functional polarisation of the sensory hair bundles. The distribution of microtubules was studied in hair cells of the chick cochlea, during the developmental stages when the stereocilia and cuticular plate were being formed. Cochleae were immunofluorescently labelled with antibodies to tubulin at specific stages in development, and hair cell ultrastructure was observed by electron microscopy. We found that the microtubule array changed from a simple symmetrical apical plate with a central kinocilium before the cuticular plate forms, to a ring with the kinocilium to one side when the cuticular plate begins to form, through to a cup-like arrangement below the cuticular plate once the plate has formed. In the earliest stages, no asymmetries were observed in the distribution of the microtubules, suggesting that structures other than the microtubules set up the functional polarisation of the stereociliary bundle.

Microtubule nucleation and organization in teleost photoreceptors: microtubule recovery after elimination by cold.

Retinal photoreceptors have two separate populations of microtubules: axonemal microtubules of the modified cilium of the outer segment and cytoplasmic microtubules of the cell body. The axonemal microtubules originate from a basal body located at the distal tip of the photoreceptor inner segment and extend in a 9 + 0 configuration into the outer segment of rods and accessory outer segment of cones. The cytoplasmic microtubules of the cell body are axially aligned from the distal tip of the inner segment to proximal synapse, and are oriented with uniform polarity, their minus ends distal toward the outer segment and plus ends proximal toward the synapse (Troutt & Burnside, 1988). To investigate how this regular cytoplasmic microtubule array is generated, we have attempted to identify microtubule nucleation sites in the cones of the tropical teleost fish, Tilapia (Sarotherodon mossambicus) by examining the regrowth of cytoplasmic microtubules after cold disruption in whole retinas or in isolated cone fragments consisting of inner and outer segments (CIS-COS). Incremental stages of microtubule reassembly were examined both by electron microscopy of thin sections and by immunofluorescent localization of microtubules with an antitubulin antibody. Cold treatment completely abolished all cytoplasmic microtubules but did not disrupt axonemal microtubules. Within 2 min after rewarming, cytoplasmic microtubules reappeared in the most distal portion of the inner segment in a small aster-like array associated with the basal body, and subsequently appeared in more proximal parts of the cone. These observations suggest that a favoured microtubule nucleation site is associated with the basal body region of the cone outer segment, and thus that the basal body region could function as a microtubule organizing centre for the photoreceptor. These results are consistent with the findings of our previous investigation of cone microtubule polarity, which showed that the minus ends of the cytoplasmic microtubules of the cone are associated with the basal body region.

Role of microtubules in pigment granule migration in teleost retinal pigment epithelial cells.

In cells of the teleost retinal pigment epithelium (RPE), melanin pigment granules migrate in response to changes in environmental light conditions. Melanin granules disperse into the RPE cell's long apical projections in response to the onset of light, and aggregate towards the base of the RPE cell in response to the onset of darkness. The RPE cells possess numerous microtubules and actin filaments, which in the apical projections are aligned longitudinally. Previous cytochalasin studies have shown that intact actin filaments are required for pigment granule dispersion and maintenance of the dispersed state (Burnside, Adler and O'Connor (1983). Invest. Ophthalmol. Vis. Sci. 24, 1). We report here that pigment granule aggregation is strongly inhibited when the highly stable microtubules of RPE apical projections are disrupted by a combination of cold and nocodazole treatments. Pigment dispersion and maintenance of the dispersed and aggregated states are unaffected by microtubule disruption. These results indicate that microtubules are required for RPE pigment aggregation but not for dispersion.

The unusual microtubule polarity in teleost retinal pigment epithelial cells.

In cells of the teleost retinal pigment epithelium (RPE), melanin granules disperse into the RPE cell's long apical projections in response to light onset, and aggregate toward the base of the RPE cell in response to dark onset. The RPE cells possess numerous microtubules, which in the apical projections are aligned longitudinally. Nocodazole studies have shown that pigment granule aggregation is microtubule-dependent (Troutt, L. L., and B. Burnside, 1988b Exp. Eye Res. In press.). To investigate further the mechanism of microtubule participation in RPE pigment granule aggregation, we have used the tubulin hook method to assess the polarity of microtubules in the apical projections of teleost RPE cells. We report here that virtually all microtubules in the RPE apical projections are uniformly oriented with plus ends toward the cell body and minus ends toward the projection tips. This orientation is opposite that found for microtubules of dermal melanophores, neurons, and most other cell types.

Microtubule polarity and distribution in teleost photoreceptors.

We have characterized the polarity orientation of microtubules in teleost retinal photoreceptors. The highly polarized rods and cones contain large numbers of paraxially aligned microtubules and exhibit dramatic cell shape changes. The myoid portion of the inner segments of both rods and cones undergoes contraction and elongation in response to light or circadian signals. Previous studies in our laboratory have demonstrated that in cones but not rods myoid elongation is microtubule-dependent. To determine polarity orientation, we decorated microtubules in photoreceptors of the green sunfish Lepomis cyanellus, with hooks formed from either exogenous or endogenous tubulin subunits. The direction of curvature of the attached hooks in cross section indicates microtubule polarity orientation by allowing one to determine the relative positions within the cell of the plus (fast-growing) and minus (slow-growing) ends of the microtubules. We found that virtually all cytoplasmic microtubules in photoreceptors are oriented with plus ends directed toward the synapse and minus ends toward the basal body at the base of the outer segment. Axonemal microtubules in photoreceptor outer segments are oriented with minus ends toward the basal body as in cilia and flagella. We have suggested previously that cone myoid elongation is mediated by mechanochemical sliding between microtubules. The polarity observations reported here indicate that if microtubules do slide in cones, sliding would necessarily occur between microtubules of parallel orientation as is observed in cilia and flagella.