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Abstract
The membrane contact sites (MCSs) enable interorganelle communication by associating organelles at distances of tens of nanometers over extended membrane surfaces and serve to maintain cellular homeostasis through efficient exchange of metabolites, lipid, and calcium between organelles, organelle fission, and movement. Most MCSs and a growing number of tethering proteins especially those involved in mediating the junctions between endoplasmic reticulum (ER) and other organelles have been extensively characterized in mammal and yeast. However, the studies of plant MCSs are still at stages of infancy, at least one reason might be due to the lack of bona fide markers for visualizing these membrane junctions in plant cells. In this study, a series of genetically encoded reporters using split super-folder GFP protein were designed to detect the possible MCSs between ER and three other cellular compartments including chloroplast, mitochondria and plasma membrane (PM) in plant cell. By expressing these genetically encoded reporter in Arabidopsis protoplasts as well as Nicotiana benthamiana leaf, we could intuitively observe the punctate signal surrounding chloroplast upon expression of ER-chloroplast MCS reporter, punctate signal of ER-mitochondria MCS reporter and punctate signal close to the PM upon expression of ER-PM MCS reporter. We also showed that the ER-chloroplast MCSs were dynamic structures that undergo active remodeling with concomitant occurrence of chloroplast dysfunction inside plant cells. This study demonstrates that ER associates with various organelles in close proximity in plant cells and provides tools that might be applicable for visualizing MCSs in plants.
Highlights
Eukaryotic cells are filled with various membranous organelles
We developed a series of reporters to visualize the potential membrane contact sites (MCSs) between endoplasmic reticulum (ER) and three types of membranes including plasma membrane, mitochondria and chloroplast in plant cells
The spGFP11 was fused with fragments from OEP7 that is an outer envelope membrane protein located in chloroplast (Bae et al, 2008), a fragment of OM64 that is a mitochondria-bound outer membrane protein (Chew et al, 2004), and a fragment of plasma membrane (PM) localized membrane protein
Summary
The intracellular membrane confers a specific and relatively stable internal environment to each organelle and consequent compartmentation of the cytoplasm, while it hinders the quick flow of proteins, lipid, ions, or other metabolites between different cellular compartments. To meet the demand of exchange information and materials, ER needs to frequently communicate with almost all classes of membranous organelles inside the cell Such kinds of communications are achieved through vesicular transport within endomembrane system or long-distance transported signaling molecules like calcium and direct physical contacts termed membrane contact sites (MCSs) (Phillips and Voeltz, 2016). A number of proteins called tethers localize to the contact sites and help to maintain the functional integrity of MCS, thereby bridging the ER and respective membranes of other organelles to modulate intracellular calcium dynamics and signaling, lipid exchange, and organelle position (Prinz, 2014; Eisenberg-Bord et al, 2016)
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