Native-state imaging of calcifying and noncalcifying microalgae reveals similarities in their calcium storage organelles

Gal, Assaf; Sorrentino, Andrea; Kahil, Keren; Pereiro, Eva; Faivre, Damien; Scheffel, André

doi:10.1073/pnas.1804139115

Cited by 64 publications

(44 citation statements)

References 38 publications

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“…Finally, we could reconstitute in 3D most of the known steps of the process of biomineralization, known as coccolithogenesis (for reviews see (de Vargas et al, 2015;Taylor et al, 2017)) within a single cell ( Figure 7A). This process starts with the formation of a proccolith ring within Golgi-associated vesicles (during the nucleation phase, Taylor et al, 2017 see also Sviben et al, 2016;Gal et al, 2018), which were clearly visible inside the cell ( Figure 7B). Coccoliths become more structured insofar as the vesicles detached from the Golgi apparatus, in a step called maturation (Taylor et al, 2017).…”

Section: Figure 6 Subcellular Features Of Different Phytoplankton Tamentioning

confidence: 88%

“…) include: i) organelles (nucleusblue, plastid-green and mitochondria-red), ii) the cytosol plus internal vesicular networks (grey): Golgi apparatus, vacuoles and storage compartments. The latter group includes Ca 2+rich storage bodies and forming coccolith in Emiliania(Gal et al, 2018;Sviben et al, 2016), carbon-rich structures in Pelagomonas(Andersen et al, 1993), lipid droplets in Phaeodactylum, starch sheath surrounding the pyrenoid in Micromonas(Lopes Dos Santos et al, 2017) and Symbiodinium and vacuoles of different sizes in Phaeodactylum, Galdieria and Micromonas (the so-called impregnated bodies, (Lopes Dos Santos et al, 2017)).…”

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confidence: 99%

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In-cell quantitative structural imaging of phytoplankton using 3D electron microscopy

Uwizeye

Decelle

Jouneau

et al. 2020

Preprint

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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.

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Section: Figure 6 Subcellular Features Of Different Phytoplankton Tamentioning

confidence: 88%

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confidence: 99%

In-cell quantitative structural imaging of phytoplankton using 3D electron microscopy

Uwizeye

Decelle

Jouneau

et al. 2020

Preprint

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“…Looking at these advances, a complex picture of coccolith formation emerges. Firstly, Ca 2+ ions, which are stored in a calcium and phosphate-rich compartment (Gal et al, 2018;Gal et al, 2017b;Sviben et al, 2016), bind to CAPs and are transported via a yet undiscovered mechanism to a mineralization vesicle pre-equipped with a base plate (Wilbur and Watabe, 1963). Acidic CAPs recognize proteins present on the base plate rim and deliver Ca 2+ ions to this specific area, leading to calcite nucleation.…”

Section: Introductionmentioning

confidence: 99%

Three-dimensional architecture and surface functionality of coccolith base plates

Marzec

Walker

Παναγοπούλου

et al. 2019

Journal of Structural Biology

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Coccolithophores are marine phytoplankton that are among the most prolific calcifiers widespread in Earth's oceans, playing a crucial role in the carbon cycle and in the transport of organic matter to the deep sea. These organisms produce highly complex mineralized scales that are composed of hierarchical assemblies of nano-crystals of calcium carbonate in the form of calcite. Coccolith formation in vivo occurs within compartmentalized mineralisation vesicles derived from the Golgi body, which contain coccolithassociated polysaccharides ('CAPs') providing polymorph selection and mediating crystal growth kinetics, and oval organic mineralisation templates, also known as base plates, which promote heterogenous nucleation and further mechanical interlocking of calcite single crystals. Although the function of coccolith base plates in controlling crystal nucleation have been widely studied, their 3D spatial organization and the chemical functional groups present on the crystal nucleation sites, which are two crucial features impacting biomineralization, remain unsolved. Utilising cryo-electron tomography we show that base plates derived from an exemplary coccolithophore Pleurochrysis carterae (Pcar) in their native hydrated state have a complex 3-layered structure. We further demonstrate, for the first time, the edge and rim of the base plate-where the crystals nucleate-are rich in primary amine functionalities that provide binding targets for negatively charged complexes composed of synthetic macromolecules and Ca 2+ ions. Our results indicate that electrostatic interactions between the negatively charged biogenic CAPs and the positively charged rim of the base plate are sufficient to mediate the transport of Ca 2+ cations to the mineralization sites.

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“…To evaluate the extent of alteration in calciumcontaining organelles in T. brucei cells deficient for TbRrp44, we have used cryo spectromicroscopy at MISTRAL to obtain X-ray absorption near-edge structure spectra (XANES) on fully hydrated cells. Sequential images were acquired from the same field of view starting below the Ca L2,3 absorption edge, up to 356 eV, which is above the Ca absorption edge [61,62] (Fig 10A). For localization of calcium-containing organelles, images were acquired at pre-edge and calcium L3-edge energies for several control and knockdown cells.…”

Section: Acidocalcisomes Present a Larger Size In Tbrrp44 Knockdown Cmentioning

confidence: 99%

Trypanosoma bruceiultra-structure cell modifications caused by knockdown of the ribonuclease Rrp44/Dis3

Cesaro

Hiraiwa

Carneiro

et al. 2020

Preprint

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Rrp44 is a conserved eukaryotic protein showing endonuclease and exoribonuclease activity that plays essential functions in RNA maturation and degradation. In Trypanosoma brucei, depletion of Rrp44 (TbRrp44) blocks processing of large subunit ribosomal RNA precursors, leading to disruption of ribosome synthesis and inhibition of cell proliferation. We used a combination of molecular and chemical markers to investigate the fate of T. brucei cells upon knockdown of TbRrp44. In addition, synchrotron deep UV microscopy and cryo soft X-ray tomography were used to investigate cell morphology and ultra-structure modifications. Downregulation of TbRrp44 results in induction of autophagy, inactivation of mitochondria and expansion of acidic and lysosome-derived vacuoles in parallel with enlargement of cell size. Nuclei also increase in size without changes in DNA content. 3D tomographic reconstructions revealed extreme vacuolation of the cytoplasm of TbRrp44 knockdown cells and general alteration of organelles. Calcium-containing vesicles (acidocalcisomes) were identified by Xray absorption near-edge structure spectra (XANES) of calcium L2,3 edges on fully hydrated cells. The volumes of acidocalcisomes and lipid droplet were quantified from 3D reconstructions. Both were found in higher number and with larger volumes in TbRrp44 knockdown cells. These multiple defects indicate that a combination of signals, starting from nucleolar stress and activation of autophagy, converge to induce lysosome expansion. With time, the cytoplasm is taken up by lysosome-derived vacuoles, which may be one of the final stages leading to cell death triggered by TbRrp44 depletion. These studies provide the first evidence on the ultra-structure cell modifications caused by Rrp44 ribonuclease deficiency. Author summaryTrypanosoma brucei is a parasitic protozoan belonging to the Kinetoplastidae Class, which displays distinct cellular, genomic and molecular features. These features include extra intervening sequences in the large subunit ribosomal RNA (rRNA) precursor that are not found in the homologous rRNA precursor of host cells. Genetic downregulation of the T. brucei Rrp44 (TbRrp44) ribonuclease shows that it is required for accurate excision of these intervening sequences to produce mature rRNA molecules. Here, we have used a multidisciplinary approach based on molecular and chemical markers, synchrotron deep UV microscopy and cryo soft X-ray tomography to show that downregulation of TbRrp44 leads to a series of cellular alterations that eventually result in cell death. Mitochondrial activity is particularly affected in parallel with induction of autophagy response, increase in size of the cell and of organelles such as acidocalcisomes and lipid droplets, and with a massive expansion of lysosome-derived vacuoles. The ultrastructural modifications that take place in T. brucei cells are nicely highlighted in the 3D reconstructions generated using cryo soft X-ray tomography images. This study provides new insights into the multiple cellular cons...

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Native-state imaging of calcifying and noncalcifying microalgae reveals similarities in their calcium storage organelles

Cited by 64 publications

References 38 publications

In-cell quantitative structural imaging of phytoplankton using 3D electron microscopy

In-cell quantitative structural imaging of phytoplankton using 3D electron microscopy

Three-dimensional architecture and surface functionality of coccolith base plates

Trypanosoma bruceiultra-structure cell modifications caused by knockdown of the ribonuclease Rrp44/Dis3

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