Abstract
Abstract: :
Purpose: To precisely characterise the location of several recently observed changes in the distribution of the membrane proteins, AQP0, MP20, Cx50 and SGLT2, that occur as a function of fibre cell differentiation; and to develop methods to investigate the underlying mechanisms responsible for such changes. Methods: Lenses were fixed, cryoprotected, and sectioned using protocols developed in our laboratory. Sections were labelled with a membrane marker, a nuclear stain, and antibodies to one of a number of lens membrane proteins (AQP0, MP20, Cx50 and SGLT2). Image stacks of labelling patterns were collected at high resolution using a confocal microscope and tiled together to yield data sets that reflected the distribution of membrane proteins over large radial distances. The resultant map of membrane protein distribution changes through fibre cell differentiation was then used to guide the isolation of specific regions of the lens by a laser dissection microscope. Results: While all four proteins are members of distinctly different families and perform different functions in the lens, all staining patterns changed at a discrete zone that coincided with the loss of fibre cell nuclei. Both MP20 and SGLT2 labelling exhibited a redistribution of signal from cytoplasmic to membranous, indicating that insertion of the proteins into the membrane occurs when nuclei are lost. In peripheral fibre cells Cx50 was initially in large broad side ‘plaques’, but then underwent a redistribution around the entire cell membrane, before being cleaved when nuclei degraded. In contrast AQP0 labelling was initially dispersed around peripheral fibre cell membranes, but shortly before nuclei were lost AQP0 signal coalesced into large broad side ‘aggregates’ before sharply redistributing around the cell membrane again as cell nuclei were lost. Since abrupt changes in all antibody labelling patterns were observed when cell nuclei degraded the use of a laser dissection microscope to isolate tissue from this zone was investigated. Results on the yield of protein obtained and its suitability for the identification of possible targeting signals and/or post–translational modifications of the different membrane proteins will be discussed. Conclusions: As lens fibre cells lose their nuclei, a large number of changes in membrane protein distribution take place. These transitions are abrupt, necessitating the development of a highly accurate dissection system to extract membrane proteins with changing expression patterns and subject them to biochemical analysis.
Keywords: cell adhesions/cell junctions • protein structure/function • microscopy: light/fluorescence/immunohistochemistry