Caffeine inhibits cell plate formation by disrupting membrane reorganization just after the vesicle fusion step

Abstract

We have re-examined the effects of caffeine on cell plate formation in synchronized tobacco BY-2 cells by means of cryofixation, immunocytochemistry, and calcium staining techniques. Because cryofixation preserves structural intermediates of cell plates that are not seen in chemically fixed cells, this methodology has enabled us to define not only when caffeine acts but also which assembly steps are inhibited. Caffeine acts at an early stage of cytokinesis, just after the Golgi-derived vesicles have arrived at the cell equator and begun to fuse with each other via thin (20 nm) membrane tubules. This initial round of fusions produces a delicate membrane network which in control cells is rapidly converted in a more substantial tubulo-vesicular network covered by a thick, fuzzy coat on its cytoplasmic surface. Caffeine disrupts the conversion of the fragile, thin, fusion tube-generated membrane network into the more stable tubulo-vesicular network, the assembly of its fuzzy coat, and the budding of clathrin-coated vesicles from its surface. Normally, the tubulo-vesicular network also provides the structural framework for calcium-dependent callose synthases that deposit a callose layer over the lumenal surface of the cell plate membranes. In the presence of caffeine, no stabilizing callose layer is formed, and the thin tubule membrane network fragments into vesicles of variable sizes. Cell plates in caffeine-treated cells stained with chlortetracycline, a fluorescent stain of membrane-associated calcium, also display a significant reduction in fluorescence at the cell plate, suggesting a major decrease in cell plate membrane-associated calcium. However, this latter finding needs to be confirmed by more sophisticated calcium measuring techniques. Current theories of the mechanism of action of caffeine, including its ability to disrupt local calcium gradients, are discussed within the new ultrastructural context that this study provides. Our findings, finally, suggest a new method for isolating just fused but not further matured cell plate forming vesicles for biochemical studies.