Biology subjects

Lesniewska, E.

Publications and source records attributed to Lesniewska, E..

3 recordsLinked to original sources

bioRxiv2025

Large extracellular vesicles subsets and contents discrimination: the potential of morpho mechanical approaches at single EV level

Extracellular vesicles (EVs) are heterogenous lipid bound membranous structures released by different cells, showing a great potential to be used as biomarkers. They have also been explored for their role in the context of environmental toxicity. When endothelial cells are exposed to pollutants like Polycyclic Aromatic Hydrocarbons (PAH) - the most common being benzo[a]pyrene (B[a]P) - EVs released from those cells undergo surface and cargo modifications. Subpopulations of large EVs (lEVs) have shown to contain either damaged or intact mitochondria which is inexorably linked to oxidative stress conditions. In this paper, we studied B[a]P induced modifications in lEVs derived from endothelial cells, through morpho mechanical characterization with atomic force microscopy (AFM). Colocalizing AFM with fluorescence microscopy allowed us to differentiate between EVs containing mitochondria and those that did not. EVs containing mitochondria had a larger size (maximum diameter) when coming from treated cells (1.8 {+/-} 0.89 {micro}m) as compared to control cells (1.63 {+/-} 0.76 {micro}m). Moreover, their Youngs moduli were higher in the treated condition (3.09 {+/-} 2.54 MPa in average) as compared to the control condition (1.25 {+/-} 0.92 MPa in average). We also observed a heterogeneity within single vesicles, with most Youngs modulus values ranging from 0.1 up to 30 MPa for the treated condition and from 0.1 to 5 MPa for the control condition. Finally, applying linear discriminant analysis (LDA) and Random Forest (RF) algorithms on maximum diameter, height, and distribution of Youngs modulus values, we demonstrated the possibility to discriminate between EV subpopulations. Indeed, we successfully managed to a) distinguish EVs containing mitochondria from the "empty" ones, with an accuracy of 84% and b) discriminate whether these mitochondria-containing EVs originated from control or treated conditions, with an accuracy of 76%. These findings highlight the power of combining morpho-mechanical analysis and machine learning for identifying and discriminating EV subpopulations, no longer requiring any EVs fluorescence labelling.

biophysics↗

bioRxiv2022

Inferring assembly-curving trends of bacterial micro-compartment shell hexamers from crystal structure arrangements

Bacterial microcompartments (BMC) are complex macromolecular assemblies that participate to varied chemical processes in about one fourth of bacterial species. BMC-encapsulated enzymatic activities are segregated from other cell contents by means of semipermeable shells, justifying why BMC are viewed as prototype nano-reactors for biotechnological applications. Herein, we undertook a comparative study of trends of self-assembly of BMC hexamers (BMC-H), the most abundant shell constituents. Published and new microscopy data show that some BMC-H, like {beta}-carboxysomal CcmK, tend to assemble flat whereas other BMC-H often build curved-implying objects. Inspection of available crystal structures presenting BMC-H in tiled arrangements permitted to identify two major assembly modes with a striking connection with experimental trends. All-atom molecular dynamics (MD) supported that BMC-H bending is triggered robustly only from the disposition adopted by BMC-H that form curved objects experimentally, conducting to almost identical arrangements to those found in structures of recomposed BMC shells. Simulations on ensembles of planar-behaving hexamers, which were previously reconfigured to comply with such disposition, confirmed that bending is defined by assembly details, rather than by BMC-H identity. Finally, although no common atomic determinants could be identified as responsible of BMC-H spontaneous curvature, an inter-hexamer ionic pair was pinpointed as contributor to hold a subset of BMC-H in low bending dispositions. These results are expected to improve our understanding of the variable mechanisms of biogenesis characterized for BMC, and of possible strategies to regulate BMC size and shape.

biochemistry↗

bioRxiv2019

Occurrence and stability of hetero-hexamer associations formed by β-carboxysome CcmK shell components

The carboxysome is a bacterial micro-compartment (BMC) subtype that encapsulates enzymatic activities necessary for carbon fixation. Carboxysome shells are composed of a relatively complex cocktail of proteins, their precise number and identity being species dependent. Shell components can be classified in two structural families, the most abundant class associating as hexamers (BMC-H) that are supposed to be major players for regulating shell permeability. Up to recently, these proteins were proposed to associate as homo-oligomers. Genomic data, however, demonstrated the existence of paralogs coding for multiple shell subunits. Here, we studied cross-association compatibilities among BMC-H CcmK proteins of Synechocystis sp. PCC6803. Co-expression in Escherichia coli proved a consistent formation of hetero-hexamers combining CcmK1 and CcmK2 or, remarkably, CcmK3 and CcmK4 subunits. Unlike CcmK1/K2 hetero-hexamers, the stoichiometry of incorporation of CcmK3 in associations with CcmK4 was low. Cross-interactions implicating other combinations were weak, highlighting a structural segregation of the two groups that could relate to gene organization. Sequence analysis and structural models permitted to localize interactions that would favor formation of CcmK3/K4 hetero-hexamers. Attempts to crystallize these CcmK3/K4 associations conducted to the unambiguous elucidation of a CcmK4 homo-hexamer structure. Yet, subunit exchange could not be demonstrated in vitro. Biophysical measurements showed that hetero-hexamers are thermally less stable than homo-hexamers, and impeded in forming larger assemblies. These novel findings are discussed in frame with reported data to propose a functional scenario in which minor CcmK3/K4 incorporation in shells would introduce sufficient local disorder as to allow shell remodeling necessary to adapt rapidly to environmental changes.

biochemistry↗