Abstract
Purpose: :
Connexin 43 (Cx43) is a major lens epithelial gap junction protein. Its assembly and disassembly depends on phosphorylation by PKC–gamma and interaction with ZO1. Disruption of this interaction leads to retention of Cx43 in the cytoplasmic fraction and decreases the ability of cells to communicate. The purpose of our study is to identify a specific function for ZO–1 in signal transduction between phosphorylation of Cx43 by PKC–gamma, assembly/disassembly of Cx43 gap junction plaques and function of dye transfer.
Methods: :
N/N 1003A lens epithelial cells were transiently transfected by anti–ZO–1–siRNA and subsequently subjected to immunoprecipitation, western blots, confocal image analysis and scrape loading/dye transfer assay in the absence and presence of TPA–induced phosphorylation of Cx43.
Results: :
Here we show by immunoprecipitation and western blot analysis after transfection with anti–ZO–1–siRNA: 1) shut down of ZO–1 protein synthesis by approximately 40% after 24–36 hours, 2) decreased amount of ZO–1 in immunoprecipitates with anti–Cx43 antibodies, 3) no change in levels of Cx43 phosphorylation. Confocal microscopy demonstrates: 1). Increased size of Cx43 plaques, 2) redistribution of the Cx43 from membrane into cytoplasmic fraction. 3) Retention of Cx43 in the cytoplasm and decreased total number of Cx43 plaques in the membrane fraction. 4). Blocked ability of membrane and cytoplasmic Cx43 to respond to TPA–induced phosphorylation by PKC–gamma, 5) loss of ability of Cx43 to disassemble membrane plaques after phosphorylation.
Conclusions: :
Our results demonstrate that interaction between ZO–1 and Cx43 and phosphorylation of Cx43 are both necessary not only to maintain structural integrity, size and location of gap junctions, but also to provide an adequate transduction pathway for regulatory response to phosphorylation signals via PKC–gamma. We propose that direct interaction between ZO–1 and Cx43 is absolutely necessary for converting phosphorylation signals into structural and functional events. The lack of ZO–1 removes the ability of Cx43 to respond to PKC–gamma activation.
Grant: :
NEI/RO1 EY13421 to DJT Nord Funding to DL
Keywords: gap junctions/coupling • microscopy: confocal/tunneling • immunohistochemistry