Signature of a non-harmonic potential as revealed from a consistent shape and fluctuation analysis of an adherent membrane

The interaction of fluid membranes with a scaffold, which can be a planar surface or a more complex structure, is intrinsic to a number of systems - from artificial supported bilayers and vesicles to cellular membranes. In principle, these interactions can be either discrete and protein mediated, or continuous. In the latter case, they emerge from ubiquitous intrinsic surface interaction potentials as well as nature-designed steric contributions of the fluctuating membrane or from the polymers of the glycocalyx. Despite the fact that these nonspecific potentials are omnipresent, their description has been a major challenge from experimental and theoretical points of view. Here we show that a full understanding of the implications of the continuous interactions can be achieved only by expanding the standard superposition models commonly used to treat these types of systems, beyond the usual harmonic level of description. Supported by this expanded theoretical framework, we present three independent, yet mutually consistent, experimental approaches to measure the interaction potential strength and the membrane tension. Upon explicitly taking into account the nature of shot noise as well as of finite experimental resolution, excellent agreement with the augmented theory is obtained, which finally provides a coherent view of the behavior of the membrane in a vicinity of a scaffold.Comment: 15 pages, 12 figures, accepted by Physical Review

Single-molecule analysis of dynamics and interactions of the SecYEG translocon

Protein translocation and insertion into the bacterial cytoplasmic membrane are the essential processes mediated by the Sec machinery. The core machinery is composed of the membrane-embedded translocon SecYEG that interacts with the secretion-dedicated ATPase SecA and translating ribosomes. Despite the simplicity and the available structural insights on the system, diverse molecular mechanisms and functional dynamics have been proposed. Here, we employ total internal reflection fluorescence microscopy to study the oligomeric state and diffusion of SecYEG translocons in supported lipid bilayers at the single-molecule level. Silane-based coating ensured the mobility of lipids and reconstituted translocons within the bilayer. Brightness analysis suggested that approx. 70% of the translocons were monomeric. The translocons remained in a monomeric form upon ribosome binding, but partial oligomerization occurred in the presence of nucleotide-free SecA. Individual trajectories of SecYEG in the lipid bilayer revealed dynamic heterogeneity of diffusion, as translocons commonly switched between slow and fast mobility modes with corresponding diffusion coefficients of 0.03 and 0.7 µm2·s−1. Interactions with SecA ATPase had a minor effect on the lateral mobility, while bound ribosome:nascent chain complexes substantially hindered the diffusion of single translocons. Notably, the mobility of the translocon:ribosome complexes was not affected by the solvent viscosity or macromolecular crowding modulated by Ficoll PM 70, so it was largely determined by interactions within the lipid bilayer and at the interface. We suggest that the complex mobility of SecYEG arises from the conformational dynamics of the translocon and protein:lipid interactions

Zwitterionic polymer ligands: An ideal surface coating to totally suppress protein-nanoparticle corona formation?

International audienceIn the last few years, zwitterionic polymers have been developed as antifouling surface coatings. However, their ability to completely suppress protein adsorption at the surface of nanoparticles in complex biological media remains undemonstrated. Here we investigate the formation of hard (irreversible) and soft (reversible) protein corona around model nanoparticles (NPs) coated with sulfobetaine (SB), phosphorylcholine (PC) and carboxybetaine (CB) polymer ligands in model albumin solutions and in whole serum. We show for the first time a complete absence of protein corona around SB-coated NPs, while PC-and CB-coated NPs undergo reversible adsorption or partial aggregation. These dramatic differences cannot be described by naïve hard/soft acid/base electrostatic interactions. Single NP tracking in the cytoplasm of live cells corroborate these in vitro observations. Finally, while modification of SB polymers with additional charged groups lead to consequent protein adsorption, addition of small neutral targeting moieties preserves antifouling and enable efficient intracellular targeting

Analyses of Adhesion Topography and Fluctuations in Bio-Membranes by Advanced Optical Microscopy

The cell membrane not only mechanically separates the interior of the cell from the exterior environment but is also an active organelle involved in a host of functions like maintaining osmotic balance, aiding in endo/exocytosis, regulating cell shape and mediating cell adhesion. The cell membrane is soft and easily deformable and hence may be subject to active deformations or even thermal fluctuations. The present work aims at a quantitative understanding of fluctuations of model cell membranes which are either adhered or free. The model system consisted of giant unilamellar vesicles (GUVs). Adhesion was effected via specific ligand-receptor interaction of biotin-neutravidin. Special structured adhesive substrates were developed where the receptors were presented in the form of grids or lines. Two light microscopic techniques were employed to probe the membrane (i) Dual-Wavelength Reflection Interference Contrast Microscopy (DW-RICM), which measures membrane-substrate distances with an accuracy of few nanometer and up to a range of one micrometer, with temporal resolution of about 50 ms. Here, significant progress in automating and refining the analysis of DWRICM data was made. (ii) Fluctuation Correlation Spectroscopy (FluCS), which is an entirely novel method developed during this thesis and measures membrane fluctuations with spatial and temporal resolution of 20 nm and 10 µs, respectively. It is based on the set up of Fluorescence Correlation Spectroscopy, and like FCS measures the decay of the correlation in fluorescence signal, but in FluCS the decay is due to membrane fluctuations rather than diffusion. DW-RICM measurements revealed that GUVs on the structured adhesive substrates exhibit regions of bound and fluctuating membrane, in accordance with the underlying pattern. In the fluctuating zone, the membrane presented itself as a flat-topped hill with the membrane-substrate distance saturated in a plateau of 79 +/- 9 nm. In this plateau, the fluctuation amplitude was found to be 12 +/- 2 nm. Variation of the shape (grid verses lines) or size (grids of 3.5 or 7 µm lattice constant) influenced neither the height nor the fluctuation amplitude in the plateau. Theoretical analysis (collaboration with Daniel Schmidt, Universität Stuttgart/Prof. Udo Seifert, Universität Stuttgart/Prof. Ana Smith, Universität Erlangen-Nürnberg) of the membrane shape and fluctuations permitted us to infer the membrane tension (3.7 +/- 0.7 µJ/m²) and the stiffness of the unspecific interaction potential (equivalent to the double derivative at minimum ; 2.3+/- 0.2 x 10^8 J/m^4). Fourier analysis revealed that modes of preferentially 2 µm wavelength developed. The plateau height could be tuned from 0 to 538 nm by changing the effective membrane tension via a change in the osmotic gradient between the inside and outside of the GUV. Corresponding fluctuation amplitude ranges from non-detectable to a maximum of 16 nm. FluCS can probe fluctuation far away as well as near a substrate. Using FluCS, the tension far from the substrate was measured to be 0.4 +/- 0.2 µJ/m² in non-adhered vesicles, and 0.5 +/- 0.4 µJ/m² in vesicles very weakly adhering to structured substrates. Compared with RICM measurements, where the vesicles studied adhered relatively more strongly and exhibited higher tension and smaller amplitudes, FluCS allowed for quantification of adhesion in the limit of soft, weakly adhering vesicles. The stiffness of the unspecific interaction potential was measured to be 7 +/- 3 x 10^5 J/m^4 for the case of free vesicles. Analysis of the decay of correlation with FluCS can also give information about damping which we interpreted as an effective viscosity amounting to 1.3+/-0.1 x 10^{-3} kg/(m s) in both free and adhered vesicles. As expected, close to the substrate, damping was even higher. Fluctuations in membranes of living cells (HEK and macrophages) was probed with FluCS. A novel liposome-fusion method was developed to over-saturate the cell membrane with fluorescent dye molecules (developed in our institute by Dr. Agnes Csiszár) ; a prerequisite for FluCS. Fluctuation amplitudes above the resolution limit were detected. The decay in correlation was found to be different from those measured for vesicles and varied from cell to cell. We believe that this variation arises due to active and directed cell motion. We present some possible future advancement of FluCS including better acquisition and treatment of data in particular for application to out of equilibrium systems like cells

Simple Physical Model Unravels Influences of Chemokine on Shape Deformation and Migration of Human Hematopoietic Stem Cells

We studied the dynamic behavior of human hematopoietic stem cells (HSC) on the in vitro model of bone marrow surfaces in the absence and presence of chemokine (SDF1α). The deformation and migration of cells were investigated by varying the chemokine concentration and surface density of ligand molecules. Since HSC used in this study were primary cells extracted from the human umbilical cord blood, it is not possible to introduce molecular reporter systems before or during the live cell imaging. To account for the experimental observations, we propose a simple and general theoretical model for cell crawling. In contrast to other theoretical models reported previously, our model focuses on the nonlinear coupling between shape deformation and translational motion and is free from any molecular-level process. Therefore, it is ideally suited for the comparison with our experimental results. We have demonstrated that the results in the absence of SDF1α were well recapitulated by the linear model, while the nonlinear model is necessary to reproduce the elongated migration observed in the presence of SDF1α. The combination of the simple theoretical model and the label-free, live cell observations of human primary cells opens a large potential to numerically identify the differential effects of extrinsic factors such as chemokines, growth factors, and clinical drugs on dynamic phenotypes of primary cells

Nanometric thermal fluctuations of weakly confined biomembranes measured with microsecond time-resolution

International audienceWe probe the bending fluctuations of bio-membranes using highly deflated giant unilamellar vesicles (GUVs) bound to a substrate by a weak potential arising from generic interactions. The substrate is either homogeneous, with GUVs bound only by the weak potential, or is chemically functionalized with a micro-pattern of very strong specific binders. In both cases, the weakly adhered membrane is seen to be confined at a well-defined distance above the surface while it continues to fluctuate strongly. We quantify the fluctuations of the weakly confined membrane at the substrate proximal surface as well as of the free membrane at the distal surface of the same GUV. This strategy enables us to probe in detail the damping of fluctuations in the presence of the substrate, and to independently measure the membrane tension and the strength of the generic interaction potential. Measurements were done using two complementary techniques - dynamic optical displacement spectroscopy (DODS, resolution: 20 nm, 10 ms), and dual wavelength reflection interference contrast microscopy (DW-RICM, resolution: 4 nm, 50 ms). After accounting for the spatio-temporal resolution of the techniques, an excellent agreement between the two measurements was obtained. For both weakly confined systems we explore in detail the link between fluctuations on the one hand and membrane tension and the interaction potential on the other hand

Fluorescence Correlation Spectroscopy Reveals Interaction of Some Microdomain-Associated Lipids with Cellular Focal Adhesion Sites

Cells adhere to the extracellular matrix at distinct anchoring points, mostly focal adhesions. These are rich in immobile transmembrane- and cytoskeletal-associated proteins, some of which are known to interact with lipids of the plasma membrane. To investigate their effect on lipid mobility and molecular interactions, fluorescently labeled lipids were incorporated into the plasma membranes of primary myofibroblasts using fusogenic liposomes. With fluorescence correlation spectroscopy, we tested mobilities of labeled microdomain-associated lipids such as sphingomyelin (SM), ganglioside (GM1), and cholesterol as well as of a microdomain-excluded phospholipid (PC) and a lipid-like molecule (DiIC18(7)) in focal adhesions (FAs) and in neighboring non-adherent membrane areas. We found significantly slower diffusion of SM and GM1 inside FAs but no effect on cholesterol, PC, and DiIC18(7). These data were compared to the molecular behavior in Lo/Ld-phase separated giant unilamellar vesicles, which served as a model system for microdomain containing lipid membranes. In contrast to the model system, lipid mobility changes in FAs were molecularly selective, and no particle enrichment occurred. Our findings suggest that lipid behavior in FAs cannot be described by Lo/Ld-phase separation. The observed slow-down of some molecules in FAs is potentially due to transient binding between lipids and some molecular constituent(s)