Abstract
Abstract: :
Purpose: To investigate whether differentiation-dependent changes in the expression patterns of fiber cell adhesion proteins, MIP and MP20 affects the diffusion of molecules into the lens via the extracellular space. Methods: Lenses were organ cultured for varying times (up to 18 hours) in a medium that contained fluorescent extracellular space markers Lucifer yellow (MW 456 Da) or Texas Red-dextran (MW 10 kDa). Lenses were fixed, cryoprotected, and equatorial and axial sections cut. Sections were labelled with antibodies to either MP20 or MIP and images of antibody labelling patterns and the extracellular space markers collected at high-resolution using a confocal microscope. Adjacent image stacks were collected and tiled together to give flat images over large radii in the lens. Results: While the expression patterns of MIP and MP20 were distinctively different they both changed as a function of fiber cell differentiation. MIP labelling was always associated with the membrane, but, changes in labelling intensity with distance into the lens were observed. The initial signal increased in a region some 100µm in from the capsule then dropped in intensity (~350µm) before sharply increasing again. This secondary increase in MIP labelling was presumably not due to an increased expression of MIP, since it coincided with the loss of fiber cell nuclei. In contrast MP20 was found in the cytoplasm of peripheral differentiating fiber cells, but when cell nuclei were lost, MP20 was inserted into fiber cell membranes. Interestingly, the diffusion of both extracellular space markers into the lens was halted when fiber cell nuclei were lost. This implies that MIP and MP20, which are both thought to play a role in cellular adhesion, change their distribution patterns at a position in the lens where diffusion via the extracellular space becomes restricted. Conclusions: Based on these results we propose the following working hypothesis. The differentiation-dependent insertion of MP20 into fiber cell membranes increases the interaction between the cell membranes of anucleate lens fiber cells as witnessed by the exclusion of tracer dyes from the extracellular space. This in turn causes a change in the distribution of MIP already in the membrane that results in an increase in the intensity of MIP antibody labelling. The functional consequences of the restriction of the extracellular space, in terms of operation of the lens circulation system, need to be further investigated.
Keywords: cell adhesions/cell junctions • microscopy: light/fluorescence/immunohistochem • protein structure/function