Abstract:Thylakoid membranes scaffold an assortment of large protein complexes that work together to harness the energy of light. It has been a longstanding challenge to visualize how the intricate thylakoid network organizes these protein complexes to finely tune the photosynthetic reactions. Previously, we used in situ cryo-electron tomography to reveal the native architecture of thylakoid membranes (Engel et al., 2015). Here, we leverage technical advances to resolve the individual protein complexes within these mem… Show more
“…This technique has already been applied successfully to provide 3D models of eukaryotic cells (Decelle et al, 2019;Flori et al, 2017;Gavelis et al, 2019). Although the spatial resolution of FIB-SEM (4-8 nm) is lower than the resolution of transmission electron microscopy (Engel et al, 2015;Wietrzynski et al, 2020), it has the advantage of providing contextual 3D images of whole cells at a specific time. The FIB-SEM technique does not require a priori knowledge on proteins and genomes to observe cellular structures, making it applicable where other label-dependent methods (e.g.…”
Phytoplankton is a minor fraction of the global biomass playing a major role in primary production and climate. Despite improved understanding of phytoplankton diversity and genomics, we lack nanoscale subcellular imaging approaches to understand their physiology and cell biology. Here, we present a complete Focused Ion Beam -Scanning Electron Microscopy (FIB-SEM) workflow (from sample preparation to image processing) to generate nanometric 3D phytoplankton models. Tomograms of entire cells, representatives of six ecologically-successful phytoplankton unicellular eukaryotes, were used for quantitative morphometric analysis. Besides lineage-specific cellular architectures, we observed common features related to cellular energy management: i) conserved cell-volume fractions occupied by the different organelles; ii) consistent plastid-mitochondria interactions, iii) constant volumetric ratios in these energy-producing organelles. We revealed detailed subcellular features related to chromatin organization and to biomineralization. Overall, this approach opens new perspectives to study phytoplankton acclimation responses to abiotic and biotic factors at a relevant biological scale.
“…This technique has already been applied successfully to provide 3D models of eukaryotic cells (Decelle et al, 2019;Flori et al, 2017;Gavelis et al, 2019). Although the spatial resolution of FIB-SEM (4-8 nm) is lower than the resolution of transmission electron microscopy (Engel et al, 2015;Wietrzynski et al, 2020), it has the advantage of providing contextual 3D images of whole cells at a specific time. The FIB-SEM technique does not require a priori knowledge on proteins and genomes to observe cellular structures, making it applicable where other label-dependent methods (e.g.…”
Phytoplankton is a minor fraction of the global biomass playing a major role in primary production and climate. Despite improved understanding of phytoplankton diversity and genomics, we lack nanoscale subcellular imaging approaches to understand their physiology and cell biology. Here, we present a complete Focused Ion Beam -Scanning Electron Microscopy (FIB-SEM) workflow (from sample preparation to image processing) to generate nanometric 3D phytoplankton models. Tomograms of entire cells, representatives of six ecologically-successful phytoplankton unicellular eukaryotes, were used for quantitative morphometric analysis. Besides lineage-specific cellular architectures, we observed common features related to cellular energy management: i) conserved cell-volume fractions occupied by the different organelles; ii) consistent plastid-mitochondria interactions, iii) constant volumetric ratios in these energy-producing organelles. We revealed detailed subcellular features related to chromatin organization and to biomineralization. Overall, this approach opens new perspectives to study phytoplankton acclimation responses to abiotic and biotic factors at a relevant biological scale.
“…The most recent advance of cryo-ET research comes from the combination of ion-milling, cryo-ET and improved image analysis methods (Engel et al 2015 ; Wietrzynski et al 2020 ). These studies have, for the first time, provided a means for obtaining in situ cryo-ET images of thylakoid membranes in vitrified Chlamydomonas cells (Fig.…”
Section: Atomic Force Microscopy Freeze-fracture/etch Electron Micromentioning
confidence: 99%
“…However, because the LHCII complexes do not protrude significantly above the surface of the membranes, their distribution can only be determined by indirect means. Nevertheless, the combination of techniques described in the Wietrzynski et al ( 2020 ) paper has ushered in an exciting new era in thylakoid membrane research. Fig.…”
Section: Atomic Force Microscopy Freeze-fracture/etch Electron Micromentioning
confidence: 99%
“…The margins of several converging thylakoids appear to be attached to a translucent layer associated with the stroma surface of the inner envelope membrane. From Wietrzynski et al ( 2020 ). Bar 0.2 mm Fig.…”
Section: Atomic Force Microscopy Freeze-fracture/etch Electron Micromentioning
Microscopic studies of chloroplasts can be traced back to the year 1678 when Antonie van Leeuwenhoek reported to the Royal Society in London that he saw green globules in grass leaf cells with his single-lens microscope. Since then, microscopic studies have continued to contribute critical insights into the complex architecture of chloroplast membranes and how their structure relates to function. This review is organized into three chronological sections: During the classic light microscope period (1678–1940), the development of improved microscopes led to the identification of green grana, a colorless stroma, and a membrane envelope. More recent (1990–2020) chloroplast dynamic studies have benefited from laser confocal and 3D-structured illumination microscopy. The development of the transmission electron microscope (1940–2000) and thin sectioning techniques demonstrated that grana consist of stacks of closely appressed grana thylakoids interconnected by non-appressed stroma thylakoids. When the stroma thylakoids were shown to spiral around the grana stacks as multiple right-handed helices, it was confirmed that the membranes of a chloroplast are all interconnected. Freeze-fracture and freeze-etch methods verified the helical nature of the stroma thylakoids, while also providing precise information on how the electron transport chain and ATP synthase complexes are non-randomly distributed between grana and stroma membrane regions. The last section (2000–2020) focuses on the most recent discoveries made possible by atomic force microscopy of hydrated membranes, and electron tomography and cryo-electron tomography of cryofixed thylakoids. These investigations have provided novel insights into thylakoid architecture and plastoglobules (summarized in a new thylakoid model), while also producing molecular-scale views of grana and stroma thylakoids in which individual functional complexes can be identified.
“…A potential solution is to provide a priori orientation information prior to alignment. Such information is typically calculated during the particle picking procedure using template matching and/or other automated detection algorithms ( Albert et al, 2020 , Hrabe et al, 2012 , Navarro et al, 2018 , Wietrzynski et al, 2020 ). However, for particularly crowded environments and/or challenging protein targets, these semi-automated procedures can result in high false-positive rates, such that the initial orientation information derived from these methods fail to produce reliable reconstructions.…”
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