Electron tomography of plant organelles and the outlook for correlative microscopic approaches

Abstract

Structural analyses of organelles and localization of proteins in their confines are essential to investigate inner workings of eukaryotic cells. Electron tomography (ET) is a threedimensional electron microscopy method with which we can extract sliced views of organelles from any direction and quantify their structural parameters at nanometer-level resolution. This advanced electron microscopy tool is suited for characterization of convoluted membrane compartments and of cellular constituents of dimensions smaller than 100 nm. ET studies of plant cells fixed by rapid freezing have expanded our understanding of the biogenesis and functions of plant organelles. Here we describe how the molecular imaging capacity of correlative light and electron microscopy can be integrated with ET in studies of plant organelles.

A Diverse Membrane Interaction Network for Plant Multivesicular Bodies: Roles in Proteins Vacuolar Delivery and Unconventional Secretion

“…Compared to the tremendously diverse roles of the MVBs in mammals and yeast, the reported functions of the PVC/MVBs in plants remain somewhat limited. State-of-the-art microscopy technology, such as super-resolution fluorescence microscopy with 3D structures in living cells, light-sheet microscopy for 4D imaging, as well as 3D TEM and correlative light-electron microscopy (CLEM) approaches (Feeney et al, 2018;Wang et al, 2019), can be expected to be useful tools for the analysis of endosomal structure and trafficking when plants are placed under multiple environments. This will undoubtedly bring new insights into our understanding of the PVC/MVB membrane interaction network and their associated functions.…”

Electron Microscopy Views of Dimorphic Chloroplasts in C4 Plants

“…Novel membrane elements in the chloroplast will be uncovered in cryo-fixed plant cell samples ( Kirchhoff, 2018 ). Intermediates of membrane assembly that were not previously documented are discerned in high-pressure frozen plant cell samples because these structures are not lost during chemical fixation that takes minutes to hours ( Staehelin and Kang, 2008 ; Wang et al., 2017 ; Karahara and Kang, 2013 ; Wang et al., 2019 ). For example, the kinked thylakoid membranes at the midplane of dividing chloroplasts have not been reported until Mai et al.…”

It Started With a Kiss: Monitoring Organelle Interactions and Identifying Membrane Contact Site Components in Plants

“…Electron microscopy (EM) can offer higher resolution, and FPs can be detected using immunogold (Pérez-Sancho et al, 2015;Cui et al, 2016;Wang et al, 2016) or Correlative light and electron microscopy (CLEM), although these approaches are low-throughput. While EM requires sample fixation and therefore abolishes system dynamics, it allows much more accurate quantification of organelle proximity (Caplan et al, 2015) and MCS area, especially if tomography approaches are used to allow 3D reconstruction (Wang et al, 2019). FIGURE 1 | Temporal and spatial characterization of organelle interactions.…”