Cytolocalization of ion-channel antagonist binding sites in sunflower protoplasts during the early steps of culture

A fluorescently labeled phenylalkylamine (PAA), DM-Bodipy PAA, was used as a probe for in vivo labeling of PAA binding sites in sunflower er hypocotyl protoplasts in culture. Verapamil, a. PAA known as a calcium channel antag onist in plants lowers the division rate of sunflower protoplasts in cultur e. The binding specificity of DM-Bodipy PAA was established at various cult ure times by competition experiments with (-)bepridil. Studies on the cytol ocalization of DM-Bodipy PAA binding sites by confocal imaging showed that in freshly isolated protoplasts PAA receptors were organized into clusters uniformly distributed over the cell surface. During protoplast culture, the fluorescence labeling pattern evolved from peripheral to cytoplasmic. Afte r a few days of culture, PAA binding sites were present inside the cell, al ong cytoplasmic strands, on the membrane of vesicles and vacuoles, and were highly concentrated around the nucleus. After protoplast division, the lab eling was mainly restricted to a zone close to the new cell wall. On symmet rical division, binding sites were uniformly distributed on both sides of t he new cell wall. With asymmetrical division, binding sites were concentrat ed in a ring surrounding the new cell plate.