Dynamic expression patterns of major lens membrane proteins

Abstract

Differentiation-dependent expression of membrane proteins is emerging as a common theme in a variety of protein expression studies conducted in the lens. Often however, the physiological relevance of, and cellular signalling mechanisms responsible for these expression patterns is not well understood. The lens microcirculation system, proposed to maintain lens homeostasis, is generated by spatial differences in lens membrane transport and structural proteins. To investigate the roles of major membrane transport and structural proteins in lens homeostasis, high resolution microscopy of lens tissue over large distances is required. This thesis presents the expression patterns of the two most abundant lens membrane proteins, Aquaporin-0 and MP20, which were both shown to change with lens fibre cell differentiation. While changes in the expression pattern of both membrane proteins has been shown previously, the application of a new lens sectioning and imaging protocol has enabled, for the first time, the changing expression patterns of both proteins to be placed in the context of the intrinsic differentiation and age gradient in the lens. Additionally, correlations between protein expression pattern and lens function have been made through immunolabelling of lens sections containing extracellular space markers. Finally, novel techniques have been developed to investigate the post-translational modifications responsible for the changing expression patterns. MP20 was initially found in a cytoplasmic pool which inserted into the lens fibre cell membrane upon degradation of cell nuclei. This membrane insertion event correlated to the formation of a barrier to extracellular diffusion. To investigate the cellular signals involved in MP20 membrane insertion, a biochemical protocol was developed to isolate a pool of MP20 derived from cytoplasmic vesicles, which was compared to membrane-bound MP20 using mass spectrometry. While MP20 was detected in both samples, differential post-translational modifications indicative of a membrane insertion signal were not observed. Initially localised to peripheral lens fibre cell membranes, Aquaporin-0 was found to aggregate predominantly on the broad sides of fibre cells, often in dome-like structures deeper into the lens. As cell nuclei degraded, the signal from an Aquaporin-0 C-terminal antibody was lost briefly, before returning in non-nucleated cells to be strong and membranous. In the lens core, signal was lost, which was shown by Western blotting and mass spectrometry to be due to cleavage of the Aquaporin-0 C-terminus. Conventional dissection techniques were unable to separate the fine zones of Aquaporin-0 labelling, necessitating the development of a novel laser microdissection/mass spectrometry approach to correlate Aquaporin-0 labelling to post-translational modifications. Additionally, MALDI tissue profiling has also been developed to investigate regional differences in the post-translational modification state of Aquaporin-0. Results from these experiments confirmed that loss of Aquaporin-0 C-terminal labelling was due to cleavage. These exciting techniques will be useful tools in the continuing investigation of changes in post-translational modifications of major lens membrane proteins. Knowledge of the high resolution expression patterns of the two most abundant lens membrane proteins has enhanced our understanding of their roles in lens homeostasis. The development of tools to investigate the cellular signals involved in the generation of lens membrane protein expression patterns is crucial if we are to advance our understanding of lens development and function.