Microinjection of actin antibodies impaired gap junctional intercellular communication in lens epithelial cells in vitro

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Abstract
Purpose. The aim of this study was to check the importance of cytoskeletal actin for gap junction mediated intercellular communication (GJIC) in cultured lens epithelial cells (LEC). Methods. Bovine LEC were cultured until confluency on cover-slides of a collocate-system. In order to study the cytoskeletal influence on cell communication microcinjection of gap junction permeable neurobiotin ® into a single cell was preceded by microinjection of actin antibodies. Confocal laser scanning microscopy of specimens treated with actin antibodies and/or subsequent phalloidin labelling, and electron microscopy, were applied to check for cytoskeleton cell membrane links. Specificity of actin antibodies was proved by immoblotting techniques. Results. Immunohistochemistry and phalloidin-rhodamine staining displayed bundles of actin-filaments extending through the entire LEC. Quantitative analysis of GJIC showed intensive dye-spreading of neurobiotin between adjacent LEC. Injection of actin antibodies thirty minutes prior to microinjection of neurobiotin significantly reduced GJIC. Microinjection of irrelevant antibodies had no effect on GJIC. Conclusion. Integrity of the actin-cytoskeleton is fundamental for unimpaired GJIC in LEC.