Investigation of the role of cell hydrophobicity and EPS production in the aggregation of the marine diatom Cylindrotheca closterium under hypo-saline conditions.

Aggregation of diatoms is of global importance to understand settling of particulate organic carbon in aquatic systems. In this study, we investigate the aggregation of the marine diatom Cylindrotheca closterium during the exponential growth phase under hypo-saline conditions. The results of the flocculation/flotation experiments show that the aggregation of the diatom depends on the salinity. In favorable growth conditions for marine diatoms (salinity of 35), the highest aggregation is achieved. To explain these observations, we used a surface approach combining atomic force microscopy (AFM) and electrochemical methods to characterize both the cell surface properties and the structure of the extracellular polymeric substances (EPS) cell produce, and to quantify the amount of surface-active organic matter released. At a salinity of 35, the results showed that diatoms are soft, hydrophobic and release only small amounts of EPS organized into individual short fibrils. In contrast, diatoms adapt to a salinity of 5 by becoming much stiffer and more hydrophilic, producing larger amounts of EPS that structurally form an EPS network. Both adaptation responses of diatoms, the hydrophobic properties of diatoms and the release of EPS, appear to play an important role in diatom aggregation and explain the behavior observed at different salinities. This biophysical study provides important evidence allowing to get a deep insight into diatom interactions at the nanoscale, which may contribute to a better understanding of large-scale aggregation phenomena in aquatic systems.

Reconstructed membrane vesicles from the microalga Dunaliella as a potential drug delivery system.

The aim of this biophysical study is to characterize reconstructed membrane vesicles obtained from microalgae in terms of their morphology, properties, composition, and ability to transport a model drug. The reconstructed vesicles were either emptied or non-emptied and exhibited a non-uniform distribution of spherical surface structures that could be associated with surface coat proteins, while in between there were pore-like structures of up to 10 nm that could contribute to permeability. The reconstructed vesicles were very soft and hydrophilic, which could be attributed to their composition. The vesicles were rich in proteins and were mostly derived from the cytoplasm and chloroplasts. We demonstrated that all lipid classes of D. tertiolecta are involved in the formation of the reconstructed membrane vesicles, where they play fundamental role to maintain the vesicle structure. The vesicles appeared to be permeable to calcein, impermeable to FITC-ovalbumin, and semipermeable to FITC-concanavalin A, which may be due to a specific surface interaction with glucose/mannose units that could serve as a basis for the development of drug carriers. Finally, the reconstructed membrane vesicles could pave a new way as sustainable and environmentally friendly marine bioinspired carriers and serve for studies on microtransport of materials and membrane-related processes contributing to advances in life sciences and biotechnology.

From algal cells to autofluorescent ghost plasma membrane vesicles.

Plasma membrane vesicles can be effective, non-toxic carriers for microscale material transport, provide a convenient model for probing membrane-related processes, since intracellular biochemical processes are eliminated. We describe here a fine-tuned protocol for isolating ghost plasma membrane vesicles from the unicellular alga Dunaliella tertiolecta, and preliminary characterization of their structural features and permeability properties, with comparisons to giant unilamellar phospholipid vesicles. The complexity of the algal ghost membrane vesicles reconstructed from the native membrane material released after hypoosmotic stress lies between that of phospholipid vesicles and cells. AFM structural characterization of reconstructed vesicles shows a thick envelope and a nearly empty vesicle interior. The surface of the envelope contains a heterogeneous distribution of densely packed, nanometer-scale globules and pore-like structures which may be derived from surface coat proteins. Confocal fluorescence imaging reveals the highly pigmented photosynthetic apparatus located within the thylakoid membrane and retained in the vesicle membrane. Transport of the fluorescent dye calcein into ghost and giant unilamellar vesicles reveals significant differences in permeability. Expanded knowledge of this unique membrane system will contribute to the design of marine bio-inspired carriers for advanced biotechnological applications.

Fluorescence responsiveness of unicellular marine algae Dunaliella to stressors under laboratory conditions.

We examined the responsiveness of unicellular green alga Dunalliela tertiolecta to selected stressors employing confocal- and time-resolved imaging of endogenous fluorescence. Our aim was to monitor cell endogenous fluorescence changes under exposure to heavy metal Cd, acidification, as well as light by laser-induced photobleaching. The accumulation of Cd in algae cells was confirmed by the secondary ion mass spectroscopy technique. For the first time, custom-made computational techniques were employed to evaluate separately the fluorescence in the flagella vs. the body region. In the presence of Cd, we recorded increase in the green fluorescence in the flagella region in the form of opacities, without change in the fluorescence lifetimes, suggesting higher availability of the fluorescent molecules. Under acidification, we noted significant rise in the green fluorescence in the flagella region, but associated with longer fluorescence lifetimes, pointing to changes in the algae environment. Photobleaching experiments corroborated gathered observations. Obtained data support a differential responsiveness of the flagella vs. the body region to stressors and enable us to better understand the pathophysiological changes of algal cells in culture under stress conditions.

Changes in nanomechanical properties and adhesion dynamics of algal cells during their growth.

Nanomechanical and structural characterisations of algal cells are of key importance for understanding their adhesion behaviour at interfaces in the aquatic environment. We examine here the nanomechanical properties and adhesion dynamics of the algal cells during two phases of their growth using complementary surface methods and the mathematical modelling. Mechanical properties of motile cells are hard to assess while keeping cells viable, and studies to date have been limited. Immobilisation of negatively charged cells to a positively charged substrate enables high-resolution AFM imaging and nanomechanical measurements. Cells were stiffer and more hydrophobic in the exponential than in the stationary phase, suggesting molecular modification of the cell envelope during aging. The corresponding properties of algal cells were in agreement with the increase of critical interfacial tensions of adhesion, determined amperometrically. Cells in exponential phase possessed a larger cell volume, in agreement with the large amount of amperometrically measured displaced charge at the interface. Differences in the kinetics of adhesion and spreading of cells at the interface were attributed to their various volumes and nanomechanical properties that varied during cell aging. Our findings contribute to the present body of knowledge on the biophysics of algal cells on a fundamental level.

Algal cell response to laboratory-induced cadmium stress: a multimethod approach.

We examined the response of algal cells to laboratory-induced cadmium stress in terms of physiological activity, autonomous features (motility and fluorescence), adhesion dynamics, nanomechanical properties, and protein expression by employing a multimethod approach. We develop a methodology based on the generalized mathematical model to predict free cadmium concentrations in culture. We used algal cells of Dunaliella tertiolecta, which are widespread in marine and freshwater systems, as a model organism. Cell adaptation to cadmium stress is manifested through cell shape deterioration, slower motility, and an increase of physiological activity. No significant change in growth dynamics showed how cells adapt to stress by increasing active surface area against toxic cadmium in the culture. It was accompanied by an increase in green fluorescence (most likely associated with cadmium vesicular transport and/or beta-carotene production), while no change was observed in the red endogenous fluorescence (associated with chlorophyll). To maintain the same rate of chlorophyll emission, the cell adaptation response was manifested through increased expression of the identified chlorophyll-binding protein(s) that are important for photosynthesis. Since production of these proteins represents cell defence mechanisms, they may also signal the presence of toxic metal in seawater. Protein expression affects the cell surface properties and, therefore, the dynamics of the adhesion process. Cells behave stiffer under stress with cadmium, and thus, the initial attachment and deformation are slower. Physicochemical and structural characterizations of algal cell surfaces are of key importance to interpret, rationalize, and predict the behaviour and fate of the cell under stress in vivo.

Reaction kinetics and mechanical models of liposome adhesion at charged interface.

Dynamics of adhesion of single liposome at the charged mercury interface is analyzed through its amperometric signal using a reaction kinetics model and a mechanical model. We present analytical solutions of the reaction kinetics model for decoupling and identifying temporal evolution of three distinct states: i) the initial state corresponding to an intact liposome, ii) the intermediate state where the liposome is partly deformed, and iii) the final state of a lipid monolayer. The results obtained with this model indicate that all three states simultaneously evolve from the onset of the adhesion process. The new mechanical model provides a physical interpretation of the three states and emphasizes the role of the forces involved in liposome adhesion process. The main conclusion is that the water content of the liposome is released through the pores formed in the membrane rather than through the channels parallel to the electrode. Both models reproduce the measurements well in the wide potential range and offer a complementary insight into the dynamics of single adhesion event, which can find application in studies of cell adhesion and in drug delivery.