Pierre-Alexandre Vidi

Plastoglobules are lipoprotein subcompartments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes.

Plastoglobules are lipoprotein particles inside chloroplasts. Their numbers have been shown to increase during the upregulation of plastid lipid metabolism in response to oxidative stress and during senescence. In this study, we used state-of-the-art high-pressure freezing/freeze-substitution methods combined with electron tomography as well as freeze-etch electron microscopy to characterize the structure and spatial relationship of plastoglobules to thylakoid membranes in developing, mature, and senescing chloroplasts. We demonstrate that plastoglobules are attached to thylakoids through a half-lipid bilayer that surrounds the globule contents and is continuous with the stroma-side leaflet of the thylakoid membrane. During oxidative stress and senescence, plastoglobules form linkage groups that are attached to each other and remain continuous with the thylakoid membrane by extensions of the half-lipid bilayer. Using three-dimensional tomography combined with immunolabeling techniques, we show that the plastoglobules contain the enzyme tocopherol cyclase (VTE1) and that this enzyme extends across the surface monolayer into the interior of the plastoglobules. These findings demonstrate that plastoglobules function as both lipid biosynthesis and storage subcompartments of thylakoid membranes. The permanent structural coupling between plastoglobules and thylakoid membranes suggests that the lipid molecules contained in the plastoglobule cores (carotenoids, plastoquinone, and tocopherol [vitamin E]) are in a dynamic equilibrium with those located in the thylakoid membranes.

Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles.

Chloroplasts contain lipoprotein particles termed plastoglobules. Plastoglobules are generally believed to have little function beyond lipid storage. Here we report on the identification of plastoglobule proteins using mass spectrometry methods in Arabidopsis thaliana. We demonstrate specific plastoglobule association of members of the plastid lipid-associated proteins/fibrillin family as well as known metabolic enzymes, including the tocopherol cyclase (VTE1), a key enzyme of tocopherol (vitamin E) synthesis. Moreover, comparative analysis of chloroplast membrane fractions shows that plastoglobules are a site of vitamin E accumulation in chloroplasts. Thus, in addition to their lipid storage function, we propose that plastoglobules are metabolically active, taking part in tocopherol synthesis and likely other pathways.

Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.

Intersection of the tocopherol and plastoquinol metabolic pathways at the plastoglobule

Plastoglobules, lipid-protein bodies in the stroma of plant chloroplasts, are enriched in non-polar lipids, in particular prenyl quinols. In the present study we show that, in addition to the thylakoids, plastoglobules also contain a considerable proportion of the plastidial PQ-9 (plastoquinol-9), the redox component of photosystem II, and of the cyclized product of PQ-9, PC-8 (plastochromanol-8), a tocochromanol with a structure similar to gamma-tocopherol and gamma-tocotrienol, but with a C-40 prenyl side chain. PC-8 formation was abolished in the Arabidopsis thaliana tocopherol cyclase mutant vte1, but accumulated in VTE1-overexpressing plants, in agreement with a role of tocopherol cyclase (VTE1) in PC-8 synthesis. VTE1 overexpression resulted in the proliferation of the number of plastoglobules which occurred in the form of clusters in the transgenic lines. Simultaneous overexpression of VTE1 and of the methyltransferase VTE4 resulted in the accumulation of a compound tentatively identified as 5-methyl-PC-8, the methylated form of PC-8. The results of the present study suggest that the existence of a plastoglobular pool of PQ-9, along with the partial conversion of PQ-9 into PC-8, might represent a mechanism for the regulation of the antioxidant content in thylakoids and of the PQ-9 pool that is available for photosynthesis.

Ligand-Dependent Oligomerization of Dopamine D2 and Adenosine A2A Receptors in Living Neuronal Cells

Adenosine A2A and dopamine D2 receptors (A2A and D2) associate in homo- and heteromeric complexes in the striatum, providing a structural basis for their mutual antagonism. At the cellular level, the portion of receptors engaging in homo- and heteromers, as well as the effect of persistent receptor activation or antagonism on the cell oligomer repertoire, are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to visualize A2A and D2 oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A2A/A2A, D2/D2, and A2A/D2 BiFC pairs. Robust fluorescence complementation reflecting A2A/D2 heteromers was detected at the cell membrane as well as in endosomes. In contrast, weaker BiFC signals, largely confined to intracellular domains, were detected with A2A/dopamine D1 BiFC pairs. Multicolor BiFC was used to simultaneously visualize A2A and D2 homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D2 stimulation with quinpirole lead to the internalization of D2/D2 and A2A/D2 oligomers and resulted in decreased A2A/D2 relative to A2A/A2A oligomer formation. Opposing effects were observed in cells treated with D2 antagonists or with the A2A agonist 5′-N-methylcarboxamidoadenosine (MECA). Subsequent radioreceptor binding analysis indicated that the drug-induced changes in oligomer formation were not readily explained by alterations in receptor density. These observations support the hypothesis that long-term drug exposure differentially alters A2A/D2 receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.

Three-Dimensional Culture of Human Breast Epithelial Cells: The How and the Why

Organs are made of the organized assembly of different cell types that contribute to the architecture necessary for functional differentiation. In those with exocrine function, such as the breast, cell-cell and cell-extracellular matrix (ECM) interactions establish mechanistic constraints and a complex biochemical signaling network essential for differentiation and homeostasis of the glandular epithelium. Such knowledge has been elegantly acquired for the mammary gland by placing epithelial cells under three-dimensional (3D) culture conditions.Three-dimensional cell culture aims at recapitulating normal and pathological tissue architectures, hence providing physiologically relevant models to study normal development and disease. The specific architecture of the breast epithelium consists of glandular structures (acini) connected to a branched ductal system. A single layer of basoapically polarized luminal cells delineates ductal or acinar lumena at the apical pole. Luminal cells make contact with myoepithelial cells and, in certain areas at the basal pole, also with basement membrane (BM) components. In this chapter, we describe how this exquisite organization as well as stages of disorganization pertaining to cancer progression can be reproduced in 3D cultures. Advantages and limitations of different culture settings are discussed. Technical designs for induction of phenotypic modulations, biochemical analyses, and state-of-the-art imaging are presented. We also explain how signaling is regulated differently in 3D cultures compared to traditional two-dimensional (2D) cultures. We believe that using 3D cultures is an indispensable method to unravel the intricacies of human mammary functions and would best serve the fight against breast cancer.

Adenosine A2A receptors assemble into higher‐order oligomers at the plasma membrane

MINT‐6797156, MINT‐6797142: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by bimolecular fluorescence complementation (MI:0809) MINT‐6797129: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by fluorescent resonance energy transfer (MI:0055)

Plastid Proteome Assembly without Toc159: Photosynthetic Protein Import and Accumulation of N -Acetylated Plastid Precursor Proteins

Proteome analysis and genome-wide transcript profiling in mutant lines deficient in Toc159 define putative Toc159-independent and Toc159-dependent precursor proteins and provide insight into Toc159 receptor function and regulation of plastid protein import. Import of nuclear-encoded precursor proteins from the cytosol is an essential step in chloroplast biogenesis that is mediated by protein translocon complexes at the inner and outer envelope membrane (TOC). Toc159 is thought to be the main receptor for photosynthetic proteins, but lacking a large-scale systems approach, this hypothesis has only been tested for a handful of photosynthetic and nonphotosynthetic proteins. To assess Toc159 precursor specificity, we quantitatively analyzed the accumulation of plastid proteins in two mutant lines deficient in this receptor. Parallel genome-wide transcript profiling allowed us to discern the consequences of impaired protein import from systemic transcriptional responses that contribute to the loss of photosynthetic capacity. On this basis, we defined putative Toc159-independent and Toc159-dependent precursor proteins. Many photosynthetic proteins accumulate in Toc159-deficient plastids, and, surprisingly, several distinct metabolic pathways are negatively affected by Toc159 depletion. Lack of Toc159 furthermore affects several proteins that accumulate as unprocessed N-acetylated precursor proteins outside of plastids. Together, our data show an unexpected client protein promiscuity of Toc159 that requires a far more differentiated view of Toc159 receptor function and regulation of plastid protein import, in which cytosolic Met removal followed by N-terminal acetylation of precursors emerges as an additional regulatory step.

Fluorescent and Bioluminescent Protein-Fragment Complementation Assays in the Study of G Protein-Coupled Receptor Oligomerization and Signaling

Most cellular functions, including signaling by G protein-coupled receptors (GPCRs), are mediated by protein-protein interactions, making the identification and localization of protein complexes key to the understanding of cellular processes. In complement to traditional biochemical techniques, noninvasive resonance energy transfer (RET) and protein-fragment complementation assays (PCAs) now allow protein interactions to be detected in the context of living cells. In this review, fluorescent and bioluminescent PCAs are discussed and their application illustrated with studies on GPCR signaling. Newly developed techniques combining PCA and RET assays for the detection of ternary and quaternary protein complexes are also presented.

Plastoglobules: a new address for targeting recombinant proteins in the chloroplast

BackgroundThe potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures) to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary.ResultsHere, we evaluated plastoglobule lipoprotein particles as a new subchloroplastic destination for recombinant proteins. The yellow fluorescent protein as a trackable cargo was targeted to plastoglobules when fused to plastoglobulin 34 (PGL34) as the carrier. Similar to adipocyte differentiation related protein (ADRP) in animal cells, most of the protein sequence of PGL34 was necessary for targeting to lipid bodies. The recombinant protein was efficiently enriched in plastoglobules isolated by simple flotation centrifugation. The viability of plants overproducing the recombinant protein was not affected, indicating that plastoglobule targeting did not significantly impair photosynthesis or sugar metabolism.ConclusionOur data identify plastoglobules as a new targeting destination for recombinant protein in leaf crops. The wide-spread presence of plastoglobules and plastoglobulins in crop species promises applications comparable to those of transgenic oilbody-oleosin technology in molecular farming.