Abstract
Purpose::
To study the structure of beaded filaments, the principal components of the lens fiber cells cytoskeleton, and their interaction with the plasma membrane, with additional emphasis on their relation to gap junctions.
Methods::
We have used conical electron tomography to examine rat and mouse lenses in three experimental conditions: wild type animals, wild type animals that have had the soluble material extracted, and PKC gamma knockout animals. The tilt series was aligned using fiduciary markers, reconstructed using the weighted back projection algorithm and refined using projection matching. The resolution of the maps was approx. 3nm. Visualization included 3D volume rendering and density segmentation methods.
Results::
In reconstructions of wild type animals, beaded filaments could not clearly be observed since they were predominantly localized in regions of high density that obstructed their visualization. In reconstructions of tissue where soluble proteins had been extracted, however, regions of high density no longer obfuscated visualization and beaded filaments could be visualized in 3D. The structures appeared as meandering filaments of 5 to 6 nm in diameter with attached spheres of approx. 23nm clustered in polarized pairs spaced approx. 40 nm apart. These filaments were dispersed throughout the cytoplasm running parallel to each other, and in some cases sharing beads among two or more filaments. Association to the plasma membrane and the gap junctions occurred at regions where the beads were in direct apposition with the plasma membrane. In the PKC gamma knockout animals, the number of beaded filaments decreased, though they could still be observed in the cytoplasm. However, the frequency with which beads associated to the plasma membrane and to gap junctions significantly decreased, causing the membrane to curve into an approximately sinusoidal wave that likely induced the shedding of vesicles into the extracellular space.
Conclusions::
Our results indicate that conical electron tomography and density segmentation are exemplary methods for determining the structure of organelles as well as their interaction in the cellular environments. The resolution makes this method ideally suited to study the changes introduced by ablation of proteins in tissues of genetically altered animals.
Keywords: cytoskeleton • gap junctions/coupling • cataract