Abstract
Purpose: :
Our prior investigations have shown that superficial cortical fibers of rat lenses contain a bipolar structure that is similar to the terminal web of other epithelia. The objective of this study was to further characterize the composition and function of this novel cytoskeletal assembly.
Methods: :
Structural evaluation included immuno–localization of fodrin with respect to the terminal web–like structure in normal rat lenses and assessment of cytoskeletal organization at posterior fiber ends after treatment with cytochalasin D. Functional parameters (focal variability and scatter) were evaluated by laser scan analysis using the ScantoxTM In Vitro Assay System. Right eye (OD) lenses were treated with cytochalasin D, whereas left eye (OS) lenses were utilized as contra–lateral controls. All lenses were scanned at 0°, 60°, and 120° in order to encompass both anterior and posterior (Y) sutures.
Results: :
Posterior polar sections of normal Sprague–dawley rat lenses double–labeled for fodrin and F–actin demonstrated that fodrin was strongly localized both within the web–like actin apparatus at fiber ends and in the membrane skeleton underlying lateral fiber membranes. Fodrin was also confirmed at anterior fiber ends, although the terminal web–like structure was less prominent in this location. Three–hour treatment with 20µM cytochalasin D resulted in perturbation of fiber end organization as assessed by confocal microscopy. Extended (6 hr, 20µM) cytochalasin D treatment did not produce a corresponding increase in fiber end disorder, however, increased concentration of cytochalasin D (100µM, 3 hr) caused fiber degeneration (membrane blebbing). Initial results of laser scan analysis showed that cytochalasin D–treated lenses had greater focal variability (avg. sem = 0.996) as compared to control lenses (avg. sem = 0.481). Additionally, relative scatter was higher in experimental vs. control lenses (281 vs. 188).
Conclusions: :
In conjunction with our prior work localizing F–actin and (non–muscle) myosin to the terminal web–like structure in lens fibers, these results indicate that this cytoskeletal assembly is analogous in organization and composition to other epithelial terminal webs. Measures of lens focal ability and scatter indicate that it probably functions to stabilize fiber end organization during fiber maturation and possibly during accommodative changes.
Keywords: crystalline lens • cytoskeleton • microscopy: confocal/tunneling