Faculty Bibliography

Analysis by confocal microscopy has revealed features of the microtubule network of rat hepatocytes in culture, establishing the three-dimensional disposition of the microtubule-based cytoskeleton, its relation to the actin-based cytoskeleton and to ligand-containing endosomes during receptor-mediated endocytosis and the alterations in its structure and disposition by the microtubule pertubant, Taxol. By co-localization studies, we have been able to demonstrate that the microtubules have a significant role in receptor-mediated endocytosis of asialoglycoproteins in this cell. Asialoorosomucoid-containing endosomes attach to widely spaced arrays of microtubules running under the baso-lateral surface of the hepatocytes 5-15 minutes after the initiation of endocytosis and then travel along microtubule paths to become concentrated with microtubules near the centrosome and at bile canaliculi after 30-60 minutes of receptor-mediated endocytosis. Receptor-mediated endocytosis is affected, but not abolished by Taxol, which inhibits the rate of asialoorosomucoid degradation at the same concentrations as those that disrupt microtubule and cytoplasmic dynein distribution, and that prevent the concentration of endosomes centrally. The results support suggestions that asialoorosomucoid-containing endosomes are captured by microtubules just below the actin layer at the cell periphery and these are actively transported centrally along microtubules, possibly by cytoplasmic dynein, so that the concentration of endosomes near the centrosome, and the subsequent efficient lysosomal degradation of ligand, are consequences of the confluence of microtubules in this region.

Detection in combination with localization of low copy gene expression can be difficult to achieve. The use of reverse transcription PCR on tissue sections with a fluorescent marker provides localization of mRNA expression on a cellular level, and when combined with confocal microscopy and image analysis it also allows for an estimate of the relative intensity of fluorescence. A good example of the application of this method is the localization of nerve growth factor (NGF) mRNA expression in the inner ear. NGF through indirect tests appears to be present in this system, yet NGF mRNA could not be localized with in situ hybridization using radiolabeled riboprobes. Using fluorescent in situ RT-PCR, we can easily detect the presence of NGF mRNA and localize it to specific cell types within the maturing inner ear.

The Car-2n/Car-2n mutant mouse is entirely lacking in carbonic anhydrase II (CA), which is normally found in myelin and glial cells in the CNS. CNS tissue from CA-deficient mutant mice was examined to see whether abnormalities could be detected which might help in understanding the function(s) of CA in the normal brain. Analyses of myelin yields and myelin proteins showed no differences between mutants and normal littermates. To visualize the oligodendrocytes and astrocytes in tissue sections, immunocytochemical staining was performed using antibodies against the Pi form of glutathione-S-transferase and glial-fibrillary acidic protein, respectively. In both gray and white matter from the mutants' brains the oligodendrocytes appeared to be shrinking and, possibly, degenerating. Hypertrophy of astrocytes occurred in the white matter, and the astrocytes in gray matter appeared swollen. It is suggested that imbalances in the HCO3-/CO2 ratios in various glial-cell compartments may produce abnormal distributions of water and ions in the brains of the CAII-deficient mice.