3D Dissection of Structural Membrane-Wall Contacts in Filamentous Moss Protonemata.

In conventional light microscopy, the adjacent cell walls of filamentous moss protonemata are seen from its narrow side thereby obscuring the major area of cell-cell connection. Optical sectioning, segmentation and 3D reconstructions allow the tilting and rotation of intracellular structures thereby greatly improving our understanding of interaction between organelles, membranes and the cell wall. Often, the findings also allow for conclusions on the respective functions. The moss Physcomitrium (Physcomitrella) patens is a model organism for growth, development and morphogenesis. Its filamentous protonemata are ideal objects for microscopy. Here, we investigated the cell wall between two neighboring cells and the connection of membranes towards this wall after plasmolysis in 0.8 M mannitol. An m-green fluorescent protein (GFP)-HDEL cell line was used to visualize the endoplasmatic reticulum (ER), the plasma membrane (PM) was stained with FM4-64. Our studies clearly show the importance of cell-cell contacts in P. patens protonemata. In 86% of the investigated cell pairs, at least one of the protoplasts remained fully attached to the adjacent cell wall. By tilting of z-stacks, volume renderings and 3D reconstructions, we visualized the amount of attached/detached PM and ER components after plasmolysis and membrane piercings through the wall of cell neighbors.