Supramolecular organization and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

There and back again: bridging meso- and nano-scales to understand lipid vesicle patterning.

We describe a complete methodology to bridge the scales between nanoscale molecular dynamics and (micrometer) mesoscale Monte Carlo simulations in lipid membranes and vesicles undergoing phase separation, in which curving molecular species are furthermore embedded. To go from the molecular to the mesoscale, we notably appeal to physical renormalization arguments enabling us to rigorously infer the mesoscale interaction parameters from its molecular counterpart. We also explain how to deal with the physical timescales at stake at the mesoscale. Simulating the as-obtained mesoscale system enables us to equilibrate the long wavelengths of the vesicles of interest, up to the vesicle size. Conversely, we then backmap from the meso- to the nano-scale, which enables us to equilibrate in turn the short wavelengths down to the molecular length-scales. By applying our approach to the specific situation of patterning a vesicle membrane, we show that macroscopic membranes can thus be equilibrated at all length-scales in achievable computational time offering an original strategy to address the fundamental challenge of timescale in simulations of large bio-membrane systems.

Protein overexpression can induce the elongation of cell membrane nanodomains.

In cell membranes, proteins and lipids are organized into submicrometric nanodomains of varying sizes, shapes, and compositions, performing specific functions. Despite their biological importance, the detailed morphology of these nanodomains remains unknown. Not only can they hardly be observed by conventional microscopy due to their small size, but there is no full consensus on the theoretical models to describe their structuring and their shapes. Here, we use a combination of analytical calculations and Monte Carlo simulations based upon a model coupling membrane composition and shape to show that increasing protein concentration leads to an elongation of membrane nanodomains. The results are corroborated by single-particle tracking measurements on HIV receptors, whose level of expression in the membrane of specifically designed living cells can be tuned. These findings highlight that protein abundance can modulate nanodomain shape and potentially their biological function. Beyond biomembranes, this mesopatterning mechanism is of relevance in several soft-matter systems because it relies on generic physical arguments.

Role of the membrane anchor in the regulation of Lck activity.

Theoretical work suggests that collective spatiotemporal behavior of integral membrane proteins should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction while investigating the mechanism for maintaining a steady amount of the active form of integral membrane protein Lck kinase (LckA) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and pharmacological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that LckA is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady LckA shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behavior fits a phenomenological model of trans-autophosphorylation that becomes more efficient with increasing LckA. We then challenged steady LckA formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in LckA generation, except for the CD45 transmembrane domain that drastically reduced LckA due to its excessive lateral proximity to CD45. Comprehensively, LckA formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with "like/unlike" boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45.

The conical shape of DIM lipids promotes Mycobacterium tuberculosis infection of macrophages.

Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogen Mycobacterium tuberculosis (Mtb). While this lipid promotes the entry of Mtb into macrophages, which occurs via phagocytosis, its molecular mechanism of action is unknown. Here, we combined biophysical, cell biology, and modeling approaches to reveal the molecular mechanism of DIM action on macrophage membranes leading to the first step of Mtb infection. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry showed that DIM molecules are transferred from the Mtb envelope to macrophage membranes during infection. Multiscale molecular modeling and 31P-NMR experiments revealed that DIM adopts a conical shape in membranes and aggregates in the stalks formed between 2 opposing lipid bilayers. Infection of macrophages pretreated with lipids of various shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken together, these results reveal how the molecular shape of a mycobacterial lipid can modulate the biological response of macrophages.

Statistical physics and mesoscopic modeling to interpret tethered particle motion experiments.

Tethered particle motion experiments are versatile single-molecule techniques enabling one to address in vitro the molecular properties of DNA and its interactions with various partners involved in genetic regulations. These techniques provide raw data such as the tracked particle amplitude of movement, from which relevant information about DNA conformations or states must be recovered. Solving this inverse problem appeals to specific theoretical tools that have been designed in the two last decades, together with the data pre-processing procedures that ought to be implemented to avoid biases inherent to these experimental techniques. These statistical tools and models are reviewed in this paper.

Domain formation in bicomponent vesicles induced by composition-curvature coupling.

Lipid vesicles composed of a mixture of two types of lipids are studied by intensive Monte Carlo numerical simulations. The coupling between the local composition and the membrane shape is induced by two different spontaneous curvatures of the components. We explore the various morphologies of these biphasic vesicles coupled to the observed patterns such as nano-domains or labyrinthine mesophases. The effect of the difference in curvatures, the surface tension, and the interaction parameter between components is thoroughly explored. Our numerical results quantitatively agree with the previous analytical results obtained by Gueguen et al. [Eur. Phys. J. E 37, 76 (2014)] in the disordered (high temperature) phase. Numerical simulations allow us to explore the full parameter space, especially close to and below the critical temperature, where analytical results are not accessible. Phase diagrams are constructed and domain morphologies are quantitatively studied by computing the structure factor and the domain size distribution. This mechanism likely explains the existence of nano-domains in cell membranes as observed by super-resolution fluorescence microscopy.

How does temperature impact the conformation of single DNA molecules below melting temperature?

The double stranded DNA molecule undergoes drastic structural changes during biological processes such as transcription during which it opens locally under the action of RNA polymerases. Local spontaneous denaturation could contribute to this mechanism by promoting it. Supporting this idea, different biophysical studies have found an unexpected increase in the flexibility of DNA molecules with various sequences as a function of the temperature, which would be consistent with the formation of a growing number of locally denatured sequences. Here, we take advantage of our capacity to detect subtle changes occurring on DNA by using high throughput tethered particle motion to question the existence of bubbles in double stranded DNA under physiological salt conditions through their conformational impact on DNA molecules ranging from several hundreds to thousands of base pairs. Our results strikingly differ from previously published ones, as we do not detect any unexpected change in DNA flexibility below melting temperature. Instead, we measure a bending modulus that remains stable with temperature as expected for intact double stranded DNA.

Dependence of DNA Persistence Length on Ionic Strength and Ion Type.

Even though the persistence length L_{P} of double-stranded DNA plays a pivotal role in cell biology and nanotechnologies, its dependence on ionic strength I lacks a consensual description. Using a high-throughput single-molecule technique and statistical physics modeling, we measure L_{P} in the presence of monovalent (Li^{+}, Na^{+}, K^{+}) and divalent (Mg^{2+}, Ca^{2+}) metallic and alkyl ammonium ions, over a large range 0.5 mM≤I≤5 M. We show that linear Debye-Hückel-type theories do not describe even part of these data. By contrast, the Netz-Orland and Trizac-Shen formulas, two approximate theories including nonlinear electrostatic effects and the finite DNA radius, fit our data with divalent and monovalent ions, respectively, over the whole I range. Furthermore, the metallic ion type does not influence L_{P}(I), in contrast to alkyl ammonium monovalent ions at high I.

Fluctuation tension and shape transition of vesicles: renormalisation calculations and Monte Carlo simulations.

It has been known for long that the fluctuation surface tension of membranes r, computed from the height fluctuation spectrum, is not equal to the bare surface tension σ, which is introduced in the theory either as a Lagrange multiplier to conserve the total membrane area or as an external constraint. In this work we relate these two surface tensions both analytically and numerically. They are also compared to the Laplace tension γ, and the mechanical frame tension τ. Using the Helfrich model and one-loop renormalisation calculations, we obtain, in addition to the effective bending modulus κeff, a new expression for the effective surface tension σeff = σ - εkBT/(2ap) where kBT is the thermal energy, ap the projected cut-off area, and ε = 3 or 1 according to the allowed configurations that keep either the projected area or the total area constant. Moreover we show that the crumpling transition for an infinite planar membrane occurs for σeff = 0, and also that it coincides with vanishing Laplace and frame tensions. Using extensive Monte Carlo (MC) simulations, triangulated membranes of vesicles made of N = 100-2500 vertices are simulated within the Helfrich theory. As compared to alternative numerical models, no local constraint is applied and the shape is only controlled by the constant volume, the spontaneous curvature and σ. It is shown that the numerical fluctuation surface tension r is equal to σeff both with radial MC moves (ε = 3) and with corrected MC moves locally normal to the fluctuating membrane (ε = 1). For finite vesicles of typical size R, two different regimes are defined: a tension regime for [small sigma, Greek, circumflex]eff = σeffR2/κeff > 0 and a bending one for -1 < [small sigma, Greek, circumflex]eff < 0. A shape transition from a quasi-spherical shape imposed by the large surface energy, to more deformed shapes only controlled by the bending energy, is observed numerically at [small sigma, Greek, circumflex]eff ≃ 0. We propose that the buckling transition, observed for planar supported membranes in the literature, occurs for [small sigma, Greek, circumflex]eff ≃ -1, the associated negative frame tension playing the role of a compressive force. Hence, a precise control of the value of σeff in simulations cannot but enhance our understanding of shape transitions of vesicles and cells.

Probing a label-free local bend in DNA by single molecule tethered particle motion.

Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high-throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of one to seven 6A-tracts, we find that the sequence CA6CGG induces a bend angle of 19° ± 4°. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well.

Where Biology Meets Physics--A Converging View on Membrane Microdomain Dynamics.

For several decades, the phenomenon of membrane component segregation into microdomains has been a well-known and highly debated subject, and varying concepts including the raft hypothesis, the fence-and-picket model, hydrophobic-mismatch, and specific protein-protein interactions have been offered as explanations. Here, we review the level of insight into the molecular architecture of membrane domains one is capable of obtaining through biological experimentation. Using SNARE proteins as a paradigm, comprehensive data suggest that several dozens of molecules crowd together into almost circular spots smaller than 100 nm. Such clusters are highly dynamical as they constantly capture and lose molecules. The organization has a strong influence on the functional availability of proteins and likely provides a molecular scaffold for more complex protein networks. Despite this high level of insight, fundamental open questions remain, applying not only to SNARE protein domains but more generally to all types of membrane domains. In this context, we explain the view of physical models and how they are beneficial in advancing our concept of micropatterning. While biological models generally remain qualitative and descriptive, physics aims towards making them quantitative and providing reproducible numbers, in order to discriminate between different mechanisms which have been proposed to account for experimental observations. Despite the fundamental differences in biological and physical approaches as far as cell membrane microdomains are concerned, we are able to show that convergence on common points of views is in reach.

DNA denaturation bubbles: free-energy landscape and nucleation/closure rates.

The issue of the nucleation and slow closure mechanisms of non-superhelical stress-induced denaturation bubbles in DNA is tackled using coarse-grained MetaDynamics and Brownian simulations. A minimal mesoscopic model is used where the double helix is made of two interacting bead-spring rotating strands with a prescribed torsional modulus in the duplex state. We demonstrate that timescales for the nucleation (respectively, closure) of an approximately 10 base-pair bubble, in agreement with experiments, are associated with the crossing of a free-energy barrier of 22 kBT (respectively, 13 kBT) at room temperature T. MetaDynamics allows us to reconstruct accurately the free-energy landscape, to show that the free-energy barriers come from the difference in torsional energy between the bubble and duplex states, and thus to highlight the limiting step, a collective twisting, that controls the nucleation/closure mechanism, and to access opening time scales on the millisecond range. Contrary to small breathing bubbles, those more than 4 base-pair bubbles are of biological relevance, for example, when a pre-existing state of denaturation is required by specific DNA-binding proteins.

The extracellular δ-domain is essential for the formation of CD81 tetraspanin webs.

CD81 is a ubiquitously expressed member of the tetraspanin family. It forms large molecular platforms, so-called tetraspanin webs that play physiological roles in a variety of cellular functions and are involved in viral and parasite infections. We have investigated which part of the CD81 molecule is required for the formation of domains in the cell membranes of T-cells and hepatocytes. Surprisingly, we find that large CD81 platforms assemble via the short extracellular δ-domain, independent from a strong primary partner binding and from weak interactions mediated by palmitoylation. The δ-domain is also essential for the platforms to function during viral entry. We propose that, instead of stable binary interactions, CD81 interactions via the small δ-domain, possibly involving a dimerization step, play the key role in organizing CD81 into large tetraspanin webs and controlling its function.

Mixed lipid bilayers with locally varying spontaneous curvature and bending.

A model of lipid bilayers made of a mixture of two lipids with different average compositions on both leaflets, is developed. A Landau Hamiltonian describing the lipid-lipid interactions on each leaflet, with two lipidic fields ψ 1 and ψ 2, is coupled to a Helfrich one, accounting for the membrane elasticity, via both a local spontaneous curvature, which varies as C 0 + C 1(ψ 1 - ψ 2/2), and a bending modulus equal to κ 0 + κ 1(ψ 1 + ψ 2)/2. This model allows us to define curved patches as membrane domains where the asymmetry in composition, ψ 1 - ψ 2, is large, and thick and stiff patches where ψ 1 + ψ 2 is large. These thick patches are good candidates for being lipidic rafts, as observed in cell membranes, which are composed primarily of saturated lipids forming a liquid-ordered domain and are known to be thick and flat nano-domains. The lipid-lipid structure factors and correlation functions are computed for globally spherical membranes and planar ones and for a whole set of parameters including the surface tension and the coupling in the two leaflet compositions. Phase diagrams are established, within a Gaussian approximation, showing the occurrence of two types of Structure Disordered phases, with correlations between either curved or thick patches, and an Ordered phase, corresponding to the divergence of the structure factor at a finite wave vector. The varying bending modulus plays a central role for curved membranes, where the driving force κ 1 C 0 (2) is balanced by the line tension, to form raft domains of size ranging from 10 to 100 nm. For planar membranes, raft domains emerge via the cross-correlation with curved domains. A global picture emerges from curvature-induced mechanisms, described in the literature for planar membranes, to coupled curvature- and bending-induced mechanisms in curved membranes forming a closed vesicle.

LFA-1 nanoclusters integrate TCR stimulation strength to tune T-cell cytotoxic activity.

T-cell cytotoxic function relies on the cooperation between the highly specific but poorly adhesive T-cell receptor (TCR) and the integrin LFA-1. How LFA-1-mediated adhesion may scale with TCR stimulation strength is ill-defined. Here, we show that LFA-1 conformation activation scales with TCR stimulation to calibrate human T-cell cytotoxicity. Super-resolution microscopy analysis reveals that >1000 LFA-1 nanoclusters provide a discretized platform at the immunological synapse to translate TCR engagement and density of the LFA-1 ligand ICAM-1 into graded adhesion. Indeed, the number of high-affinity conformation LFA-1 nanoclusters increases as a function of TCR triggering strength. Blockade of LFA-1 conformational activation impairs adhesion to target cells and killing. However, it occurs at a lower TCR stimulation threshold than lytic granule exocytosis implying that it licenses, rather than directly controls, the killing decision. We conclude that the organization of LFA-1 into nanoclusters provides a calibrated system to adjust T-cell killing to the antigen stimulation strength.

Mesoscopic models for DNA stretching under force: New results and comparison with experiments.

Single-molecule experiments on double-stranded B-DNA stretching have revealed one or two structural transitions, when increasing the external force. They are characterized by a sudden increase of DNA contour length and a decrease of the bending rigidity. The nature and the critical forces of these transitions depend on DNA base sequence, loading rate, salt conditions and temperature. It has been proposed that the first transition, at forces of 60-80 pN, is a transition from B to S-DNA, viewed as a stretched duplex DNA, while the second one, at stronger forces, is a strand peeling resulting in single-stranded DNAs (ssDNA), similar to thermal denaturation. But due to experimental conditions these two transitions can overlap, for instance for poly(dA-dT). In an attempt to propose a coherent picture compatible with this variety of experimental observations, we derive an analytical formula using a coupled discrete worm-like chain-Ising model. Our model takes into account bending rigidity, discreteness of the chain, linear and non-linear (for ssDNA) bond stretching. In the limit of zero force, this model simplifies into a coupled model already developed by us for studying thermal DNA melting, establishing a connection with previous fitting parameter values for denaturation profiles. Our results are summarized as follows: i) ssDNA is fitted, using an analytical formula, over a nano-Newton range with only three free parameters, the contour length, the bending modulus and the monomer size; ii) a surprisingly good fit on this force range is possible only by choosing a monomer size of 0.2 nm, almost 4 times smaller than the ssDNA nucleobase length; iii) mesoscopic models are not able to fit B to ssDNA (or S to ss) transitions; iv) an analytical formula for fitting B to S transitions is derived in the strong force approximation and for long DNAs, which is in excellent agreement with exact transfer matrix calculations; v) this formula fits perfectly well poly(dG-dC) and λ-DNA force-extension curves with consistent parameter values; vi) a coherent picture, where S to ssDNA transitions are much more sensitive to base-pair sequence than the B to S one, emerges. This relatively simple model might allow one to further study quantitatively the influence of salt concentration and base-pairing interactions on DNA force-induced transitions.

Boundary fluctuation dynamics of a phase-separated domain in planar geometry.

Using phase-ordering kinetics and of renormalization group theories, we derive analytically the relaxation times of the long wavelength fluctuations of a phase-separated domain boundary in the vicinity of (and below) the critical temperature in the planar Ising universality class. For a conserved order parameter, the relaxation time grows like Λ^{3} at wavelength Λ and can be expressed in terms of parameters relevant at the microscopic scale: lattice spacing, bulk diffusion coefficient of the minority phase, and temperature. These results are supported by numerical simulations of 2D Ising models, enabling in addition calculating the nonuniversal numerical prefactor. We discuss the applications of these findings to the determination of the real timescale associated with elementary Monte Carlo moves from the measurement of long wavelength relaxation times on experimental systems or molecular dynamics simulations.

Physical constraints in polymer modeling of chromatin associations with the nuclear periphery at kilobase scale.

Interactions of chromatin with the nuclear lamina imposes a radial genome distribution important for nuclear functions. How physical properties of chromatin affect these interactions is unclear. We used polymer simulations to model how physical parameters of chromatin affect its interaction with the lamina. Impact of polymer stiffness is greater than stretching on its configurations at the lamina; these are manifested as trains describing extended interactions, and loops describing desorbed regions . Conferring an attraction potential leads to persistent interaction and adsorption-desorption regimes manifested by fluctuations between trains and loops. These are modulated by polymer stiffness and stretching, with a dominant impact of stiffness on resulting structural configurations. We infer that flexible euchromatin is more prone to stochastic interactions with lamins than rigid heterochromatin characterizing constitutive LADs. Our models provide insights on the physical properties of chromatin as a polymer which affect the dynamics and patterns of interactions with the nuclear lamina.

Probing DNA conformational changes with high temporal resolution by tethered particle motion.

The tethered particle motion (TPM) technique informs about conformational changes of DNA molecules, e.g. upon looping or interaction with proteins, by tracking the Brownian motion of a particle probe tethered to a surface by a single DNA molecule and detecting changes of its amplitude of movement. We discuss in this context the time resolution of TPM, which strongly depends on the particle-DNA complex relaxation time, i.e. the characteristic time it takes to explore its configuration space by diffusion. By comparing theory, simulations and experiments, we propose a calibration of TPM at the dynamical level: we analyze how the relaxation time grows with both DNA contour length (from 401 to 2080 base pairs) and particle radius (from 20 to 150 nm). Notably we demonstrate that, for a particle of radius 20 nm or less, the hydrodynamic friction induced by the particle and the surface does not significantly slow down the DNA. This enables us to determine the optimal time resolution of TPM in distinct experimental contexts which can be as short as 20 ms.