Abstract
Purpose:
Undulating aquaporin junctions are formed by square-array aquaporin 0 proteins located preferentially in deep cortical and nuclear regions of the lens. They belong to one type of interlocking complexes in the lens to play an adhesion role between fiber cells. The cytoskeletal regulation for the formation and maintenance of these junctions with a unique ridge-and-valley configuration is not known. Here, we tested the possible involvement of tropomodulin1-actin complexes and CP49 beaded filaments using genetically modified mouse lenses.
Methods:
SEM, freeze-fracture TEM, immunoconfocal microscopy and western blotting were applied to examine the mouse lenses with the following genetically modified combinations: (1) Tmod1-/-;CP49+/+, (2) Tmod1-/-;CP49-/- and (3) Tmod1+/+;CP49-/-. The Tmod1+/+; CP49+/+ lenses were used for controls.
Results:
Normal distribution of undulating aquaporin junctions was seen in the deep cortex (~500 μm deep from the surface) in Tmod1+/+; CP49+/+ lenses. These junctions with ridge-and-valley surface patterns disappeared almost completely in the same regions in all Tmod1-/-; CP49+/+ and Tmod1-/-; CP49-/- lenses studied, suggesting that Tmod1-actin complexes are essential for the formation and maintenance of undulating aquaporin junctions. In contrast, there were significant differences in the Tmod1+/+ lenses whether or not the CP49 was present. An enormous amount of aquaporin junctions with extensive ridge-and-valley patterns were found only in the Tmod1+/+; CP49-/- lenses. It is suggested that CP49 beaded filaments may play a role in maintaining a flat membrane surface in various lens regions. Freeze-fracture studies confirmed the presence of a unique arrangement of square-array AQP0 particles along the undulating aquaporin junctions. Immunofluorescence labeling showed that the staining of AQP0 was increased in the deeper cortex only in the Tmod1+/+; CP49-/- lenses, but without changes in AQP0 protein levels measured by western blotting.
Conclusions:
This result indicates that the actin-Tmod1 network is essential for extensive assembly of aquaporin junctions, and is regulated by the CP49 beaded filaments in the lens. In the presence of Tmod1 and the absence of CP49 beaded filaments, the formation of undulating aquaporin junctions reaches the highest level in the deep cortical regions of the lens.
Keywords: 448 cell membrane/membrane specializations •
493 cytoskeleton •
597 microscopy: electron microscopy