Vacuole is a multifunctional compartment central to a large number of functions (storage, catabolism, maintenance of the cell homeostasis) in oxygenic phototrophs including microalgae. Still, microalgal cell vacuole is much less studied than that of higher plants although knowledge of the vacuolar structure and function is essential for understanding physiology of nutrition and stress tolerance of microalgae. Here, we combined the advanced analytical and conventional transmission electron microscopy methods to obtain semi-quantitative, spatially resolved at the subcellular level information on elemental composition of the cell vacuoles in several free-living and symbiotic chlorophytes. We obtained a detailed record of the changes in cell and vacuolar ultrastructure in response to environmental stimuli under diverse conditions. We suggested that the vacuolar inclusions could be divided into responsible for storage of phosphorus (mainly in form of polyphosphate) and those accommodating non-protein nitrogen (presumably polyamine) reserves, respectively.The ultrastructural findings, together with the data on elemental composition of different cell compartments, allowed us to speculate on the role of the vacuolar membrane in the biosynthesis and sequestration of polyphosphate. We also describe the ultrastructural evidence of possible involvement of the tonoplast in the membrane lipid turnover and exchange of energy and metabolites between chloroplasts and mitochondria. These processes might play a significant role in acclimation in different stresses including nitrogen starvation and extremely high level of CO and might also be of importance for microalgal biotechnology. Advantages and limitations of application of analytical electron microscopy to biosamples such as microalgal cells are discussed.

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Coacervates are dense microdroplets formed by liquid-liquid phase separation (LLPS) of macromolecules that have gained increasing attention as drug delivery vehicles. Recently, we have reported a new intracellular delivery system based on self-coacervating histidine (His)-rich beak peptides (HBpep and HBpep-SP) inspired by beak proteins of the Humboldt squid. These peptide microdroplets combine excellent encapsulation efficiency of therapeutics with high transfection rate and low cytotoxicity. However, the mechanism by which they cross the cell membrane remains elusive. Previous inhibitor studies provided incomplete clues into the detail uptake pathway, although they suggested a cholesterol-dependent and, possibly, an energy-independent non-classical mechanism of internalization. In this study, we improved our understanding of coacervates/cell membrane interactions using model membranes, namely Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs). We also employ a combination of electron microscopy techniques to gain detailed structural insights into the cell uptake of HBpep and HBpep-SP coacervates. We demonstrate that modulating lipid charge and cholesterol level influence coacervate attachment to GUVs. However, they are not able to cross the GUV's lumen in an energy-independent manner. We then show that the coacervates enter HeLa and HepG2 cells via a mechanism sharing morphological features of macropinocytosis and phagocytosis, in particular involving cytoskeleton rearrangement and capture by filipodia. Our study provides key insights into the interaction of HPpep and HBpep-SP coacervates with model membranes as well as their cellular uptake pathway.

We established a new simple approach to study phosphorus (P) and nitrogen (N) reserves at subcellular level potentially applicable to various types of cells capable of accumulating P- and/or N-rich inclusions. Here, we report on using this approach for locating and assessing the abundance of the P and N reserves in microalgal and cyanobacterial cells. The approach includes separation of the signal from P- or N-rich structures from noise on the energy-filtered transmission electron microscopy (EFTEM) P- or N-maps. The separation includes (i) relative entropy estimation for each pixel of the map, (ii) binary thresholding of the map, and (iii) segmenting the image to assess the inclusion relative area and localization in the cell section. The separation is based on comparing the a posteriori probability that a pixel of the map contains information about the sample vs. Gaussian a priori probability that the pixel contains noise. The difference is expressed as relative entropy value for the pixel; positive values are characteristic of the pixels containing the payload information about the sample. This is the first known method for quantification and locating at a subcellular level P-rich and N-rich inclusions including tiny (< 180 nm) structures. We demonstrated the applicability of the proposed method both to the cells of eukaryotic green microalgae and cyanobacteria. Using the new method, we elucidated the heterogeneity of the studied cells in accumulation of P and N reserves across different species. The proposed approach will be handy for any cytological and microbiological study requiring a comparative assessment of subcellular distribution of cyanophycin, polyphosphates or other type of P- or N-rich inclusions. An added value is the potential of this approach for automation of the data processing and evaluation enabling an unprecedented increase of the EFTEM analysis throughput.

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