Screening the Dynamics of Membrane Constituents in Intact Microalgal Cells by Solid-State NMR

Abstract

Microalgae are unicellular organisms protected by a lipid-rich plasma membrane. In most species, this bilayer is covered by a cellulose, carbonate or silica wall. Microalgae are at the basis of the aquatic food chain; therefore the action of contaminants from human activities can impact all the trophic food web. To exert their biological effect, these molecules can target or traverse the microalgal membranes to access internal activity sites, with important consequences on cell survival. Solid-state nuclear magnetic resonance (SS-NMR) is a non- intrusive method that can provide information at the molecular level on the membrane constituents. Because of the complex composition of natural membranes, SS-NMR generally relies on the use of mimetics made of phospholipids. The objective of our work was thus to provide a tool to probe all membrane components by 13C SS-NMR using isotopically-labeled intact microalgae. To do so, we have established a strategy to sort specific constituents according to their dynamics. Using experiments based on through-space (cross polarization) or through-bond (RINEPT) magnetization transfer, rigid and dynamic molecules, respectively, can be identified and studied. For this purpose, we have developed protocols to 13C-enrich microalgae up to an average labeling of 90%. Our 13C SS-NMR study of saltwater (I. galbana, C. gracilis) and freshwater (C. vulgaris, C. reinhardtii) microalgae revealed important differences in the dynamics of their cell wall constituents. Notably, C. vulgaris used in biofuel production presents the most dynamic membrane due to its high lipid content. Also, the integrity of species without cell walls, such as I. galbana, seems to be ensured by a more rigid organization. Our dynamic filter strategy was also tested to verify the effect of nanocrystalline cellulose and could reveal membrane stiffening at high nanoparticle concentrations.