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

Dominik Harant and Ingeborg Lang - Department of Functional and Evolutionary Ecology, Faculty of Life Sciences, University of Vienna,

Cell-to-cell contact is essential for communication and development of multicellular organisms. A prerequisite is the passage through membranes. That way, molecular exchange and information flow is regulated via hormones, membrane proteins and pores.
In plants, the rigid cell walls prevent large membrane contact areas between protoplasts. Only plasmodesmata, minute channels between adjacent cells, form direct connections. Often, molecular data of the proteins involved are manifold but there is a lack of knowledge on functional and structural information.

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.

How Amira-Avizo Software is used

High-resolution 3D-models of the ER, PM and chloroplasts of plasmolyzed chloronema and caulonema cells, were obtained by setting the step size in z-direction to “system optimized” (0.13 µm) in order to match with the resolution in the x–y direction. Z-stacks were then transferred to the 3D visualization program Amira Software. Subsequently, the detected cell structures were segmented with the threshold tool to Int. J. Mol. Sci. 2021, 22, 158 10 of 12
generate a pseudo-colored and smoothed surface. This way, we could analyze the labeled membranes and chloroplasts even further. To highlight the attachments of the protoplasts, we marked the membrane in this area by hand using the brush tool. At the adjoining cell wall, the ER is shown in green and the plasma membrane as transparent yellow with its surface generated by a threshold-tool. Twenty to thirty cell pairs were segmented and
reconstructed in this way.