It is not easy being green: the technical path for the ultrastructural study of the locomotory apparatus of the moss Physcomitrium patens

Abstract text (incl. figure legends and references)

Electron microscopy (EM) as a whole technique enables the ultrastructural studies of many samples from individual cells to full organisms. For the visualization of a single image under the electron beam, extensive time investment is made in technical preparation and several EM techniques often need to be applied until an image or model can be presented. The path is not always easy, and optimizations are commonly needed to avoid unintentional artifacts that can impact the final ultrastructural observation. This problem is even more pronounced when it comes to processing plant samples, such as the moss Physcomitrium patens, where there is a need to consider the existence of the cell wall, that provides rigidity and strength to cells, but also functions as a barrier for the chemicals needed to observe the ultrastructure using room temperature EM.

In this study, the challenge presented to our facility was the characterization of the locomotory apparatus of the moss P.patens, namely the de novo centriole assembly that is involved in the motility of the sperm cells during spermatogenesis. These sperm cells develop inside an antheridium located at the tip of the gametophore, a complex structure that contains leaves, rhizoids and both sexual organs (the antheridia and the archegonia). We addressed this question by combining the observations from Chemical Fixation, High-Pressure Freezing–Freeze Substitution, Correlative Light-Electron Microscopy and Electron Tomography.

Each of the different EM techniques helped to address different parts of the biological question. Chemical Fixation allowed to carefully identify the main stages of development and to do the initial ultrastructural characterization. High-Pressure Freezing – Freeze Substitution was used to confirm that the identified structures were not artifacts induced by chemical fixation and allowed a more accurate and detailed ultrastructural characterization of each structure. Electron Tomography gave three-dimensional models of the structures. Correlative Light-Electron Microscopy (CLEM) helped to elucidate the protein composition of the sperm cells" locomotory apparatus and associate the light microscopy results with the structures observed by electron microscopy.

Overall, this work shows how complex the technical path in an EM study of a larger specimen can be and how crucial it is to keep optimizing and developing EM techniques that supports the answering of biological questions even when using bigger and more intricate specimens.

Figure 1: 3D models of different stages in centriole assembly and maturation in Physcomitrium patens. a) Bicentriole stage; b) Sister centrioles associated with the multilayered structure (MLS); c) Asymmetrical sister centrioles docked to the cell membrane, serving as basal bodies for ciliogenesis. Cyan – astral microtubules; Light green – centriolar microtubules; Red – Centriolar cartwheel; Dark green – microtubular spline of the MLS; Yellow – lamellar strip of the MLS; Magenta – ciliary transition zone. Scale-bars = 200nm. Adapted from: The 3D architecture and molecular foundations of de novo centriole assembly via bicentrioles (2021).