Interorganelle communication and membrane shaping in the early secretory pathway.

Biomolecules in the secretory pathway use membrane trafficking for reaching their final intracellular destination or for secretion outside the cell. This highly dynamic and multipartite process involves different organelles that communicate to one another while maintaining their identity, shape, and function. Recent studies unraveled new mechanisms of interorganelle communication that help organize the early secretory pathway. We highlight how the spatial proximity between endoplasmic reticulum (ER) exit sites and early Golgi elements provides novel means of ER-Golgi communication for ER export. We also review recent findings on how membrane contact sites between the ER and the trans-Golgi membranes can sustain anterograde traffic out of the Golgi complex.

A physical mechanism of TANGO1-mediated bulky cargo export.

The endoplasmic reticulum (ER)-resident protein TANGO1 assembles into a ring around ER exit sites (ERES), and links procollagens in the ER lumen to COPII machinery, tethers, and ER-Golgi intermediate compartment (ERGIC) in the cytoplasm (Raote et al., 2018). Here, we present a theoretical approach to investigate the physical mechanisms of TANGO1 ring assembly and how COPII polymerization, membrane tension, and force facilitate the formation of a transport intermediate for procollagen export. Our results indicate that a TANGO1 ring, by acting as a linactant, stabilizes the open neck of a nascent COPII bud. Elongation of such a bud into a transport intermediate commensurate with bulky procollagens is then facilitated by two complementary mechanisms: (i) by relieving membrane tension, possibly by TANGO1-mediated fusion of retrograde ERGIC membranes and (ii) by force application. Altogether, our theoretical approach identifies key biophysical events in TANGO1-driven procollagen export.

Channeling Effect and Tissue Morphology in a Perfusion Bioreactor Imaged by X-Ray Microtomography.

BACKGROUND: Perfusion bioreactors for tissue engineering hold great promises. Indeed, the perfusion of culture medium enhances species transport and mechanically stimulates the cells, thereby increasing cell proliferation and tissue formation. Nonetheless, their development is still hampered by a lack of understanding of the relationship between mechanical cues and tissue growth. METHODS: Combining tissue engineering, three-dimensional visualization and numerical simulations, we analyze the morphological evolution of neo-tissue in a model bioreactor with respect to the local flow pattern. NIH-3T3 cells were grown under perfusion for one, two and three weeks on a stack of 2 mm polyacetal beads. The model bioreactor was then imaged by X-ray micro-tomography and local tissue morphology was analyzed. To relate experimental observations and mechanical stimulii, a computational fluid dynamics model of flow around spheres in a canal was developed and solved using the finite element method. RESULTS: We observe a preferential tissue formation at the bioreactor periphery, and relate it to a channeling effect leading to regions of higher flow intensity. Additionally, we find that circular crater-like tissue patterns form in narrow channel regions at early culture times. Using computational fluid dynamic simulations, we show that the location and morphology of these patterns match those of shear stress maxima. Finally, the morphology of the tissue is qualitatively described as the tissue grows and reorganizes itself. CONCLUSION: Altogether, our study points out the key role of local flow conditions on the tissue morphology developed on a stack of beads in perfusion bioreactors and provides new insights for effective design of hydrodynamic bioreactors for tissue engineering using bead packings.

Geometric coupling of helicoidal ramps and curvature-inducing proteins in organelle membranes.

Cellular membranes display an incredibly diverse range of shapes, both in the plasma membrane and at membrane bound organelles. These morphologies are intricately related to cellular functions, enabling and regulating fundamental membrane processes. However, the biophysical mechanisms at the origin of these complex geometries are not fully understood from the standpoint of membrane-protein coupling. In this study, we focused on a minimal model of helicoidal ramps representative of specialized endoplasmic reticulum compartments. Given a helicoidal membrane geometry, we asked what is the distribution of spontaneous curvature required to maintain this shape at mechanical equilibrium? Based on the Helfrich energy of elastic membranes with spontaneous curvature, we derived the shape equation for minimal surfaces, and applied it to helicoids. We showed the existence of switches in the sign of the spontaneous curvature associated with geometric variations of the membrane structures. Furthermore, for a prescribed gradient of spontaneous curvature along the exterior boundaries, we identified configurations of the helicoidal ramps that are confined between two infinitely large energy barriers. Overall our results suggest possible mechanisms for geometric control of helicoidal ramps in membrane organelles based on curvature-inducing proteins.

Histological Method to Study the Effect of Shear Stress on Cell Proliferation and Tissue Morphology in a Bioreactor.

Background: Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold resolves mass transport limitations and provides mechanical stimuli. Despite the recent and important development of bioreactors for tissue engineering, the underlying mechanisms leading to the production of bone substitutes remain poorly understood. Methods: In order to study cell proliferation in a perfusion bioreactor, we propose a simplified experimental set-up using an impermeable scaffold model made of 2 mm diameter glass beads on which mechanosensitive cells, NIH-3T3 fibroblasts are cultured for up to 3 weeks under 10 mL/min culture medium flow. A methodology combining histological procedure, image analysis and analytical calculations allows the description and quantification of cell proliferation and tissue production in relation to the mean wall shear stress within the bioreactor. Results: Results show a massive expansion of the cell phase after 3 weeks in bioreactor compared to static control. A scenario of cell proliferation within the three-dimensional bioreactor porosity over the 3 weeks of culture is proposed pointing out the essential role of the contact points between adjacent beads. Calculations indicate that the mean wall shear stress experienced by the cells changes with culture time, from about 50 mPa at the beginning of the experiment to about 100 mPa after 3 weeks. Conclusion: We anticipate that our results will help the development and calibration of predictive models, which rely on estimates and morphological description of cell proliferation under shear stress.

Lipid Unsaturation Properties Govern the Sensitivity of Membranes to Photoinduced Oxidative Stress.

Unsaturated lipid oxidation is a fundamental process involved in different aspects of cellular bioenergetics; dysregulation of lipid oxidation is often associated with cell aging and death. To study how lipid oxidation affects membrane biophysics, we used a chlorin photosensitizer to oxidize vesicles of various lipid compositions and degrees of unsaturation in a controlled manner. We observed different shape transitions that can be interpreted as an increase in the area of the targeted membrane followed by a decrease. These area modifications induced by the chemical modification of the membrane upon oxidation were followed in situ by Raman tweezers microspectroscopy. We found that the membrane area increase corresponds to the lipids' peroxidation and is initiated by the delocalization of the targeted double bonds in the tails of the lipids. The subsequent decrease of membrane area can be explained by the formation of cleaved secondary products. As a result of these area changes, we observe vesicle permeabilization after a time lag that is characterized in relation with the level of unsaturation. The evolution of photosensitized vesicle radius was measured and yields an estimation of the mechanical changes of the membrane over oxidation time. The membrane is both weakened and permeabilized by the oxidation. Interestingly, the effect of unsaturation level on the dynamics of vesicles undergoing photooxidation is not trivial and thus carefully discussed. Our findings shed light on the fundamental dynamic mechanisms underlying the oxidation of lipid membranes and highlight the role of unsaturations on their physical and chemical properties.

Gaussian curvature directs the distribution of spontaneous curvature on bilayer membrane necks.

Formation of membrane necks is crucial for fission and fusion in lipid bilayers. In this work, we seek to answer the following fundamental question: what is the relationship between protein-induced spontaneous mean curvature and the Gaussian curvature at a membrane neck? Using an augmented Helfrich model for lipid bilayers to include membrane-protein interaction, we solve the shape equation on catenoids to find the field of spontaneous curvature that satisfies mechanical equilibrium of membrane necks. In this case, the shape equation reduces to a variable coefficient Helmholtz equation for spontaneous curvature, where the source term is proportional to the Gaussian curvature. We show how this latter quantity is responsible for non-uniform distribution of spontaneous curvature in minimal surfaces. We then explore the energetics of catenoids with different spontaneous curvature boundary conditions and geometric asymmetries to show how heterogeneities in spontaneous curvature distribution can couple with Gaussian curvature to result in membrane necks of different geometries.

Solubilization kinetics determines the pulsatory dynamics of lipid vesicles exposed to surfactant.

We establish a biophysical model for the dynamics of lipid vesicles exposed to surfactants. The solubilization of the lipid membrane due to the insertion of surfactant molecules induces a reduction of membrane surface area at almost constant vesicle volume. This results in a rate-dependent increase of membrane tension and leads to the opening of a micron-sized pore. We show that solubilization kinetics due to surfactants can determine the regime of pore dynamics: either the pores open and reseal within a second (short-lived pore), or the pore stays open up to a few minutes (long-lived pore). First, we validate our model with previously published experimental measurements of pore dynamics. Then, we investigate how the solubilization kinetics and membrane properties affect the dynamics of the pore and construct a phase diagram for short and long-lived pores. Finally, we examine the dynamics of sequential pore openings and show that cyclic short-lived pores occur with a period inversely proportional to the solubilization rate. By deriving a theoretical expression for the cycle period, we provide an analytical tool to estimate the solubilization rate of lipid vesicles by surfactants. Our findings shed light on some fundamental biophysical mechanisms that allow simple cell-like structures to sustain their integrity against environmental stresses, and have the potential to aid the design of vesicle-based drug delivery systems. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

Pulsatile Gating of Giant Vesicles Containing Macromolecular Crowding Agents Induced by Colligative Nonideality.

The ability of large macromolecules to exhibit nontrivial deviations in colligative properties of their aqueous solutions is well-appreciated in polymer physics. Here, we show that this colligative nonideality subjects giant lipid vesicles containing inert macromolecular crowding agents to osmotic pressure differentials when bathed in small-molecule osmolytes at comparable concentrations. The ensuing influx of water across the semipermeable membrane induces characteristic swell-burst cycles: here, cyclical and damped oscillations in size, tension, and membrane phase separation occur en route to equilibration. Mediated by synchronized formation of transient pores, these cycles orchestrate pulsewise ejection of macromolecules from the vesicular interior reducing the osmotic differential in a stepwise manner. These experimental findings are fully corroborated by a theoretical model derived by explicitly incorporating the contributions of the solution viscosity, solute diffusivity, and the colligative nonideality of the osmotic pressure in a previously reported continuum description. Simulations based on this model account for the differences in the details of the noncolligatively induced swell-burst cycles, including numbers and periods of the repeating cycles, as well as pore lifetimes. Taken together, our observations recapitulate behaviors of vesicles and red blood cells experiencing sudden osmotic shocks due to large (hundreds of osmolars) differences in the concentrations of small molecule osmolytes and link intravesicular macromolecular crowding with membrane remodeling. They further suggest that any tendency for spontaneous overcrowding in single giant vesicles is opposed by osmotic stresses and requires independent specific interactions, such as associative chemical interactions or those between the crowders and the membrane boundary.

Systems biology of cellular membranes: a convergence with biophysics.

Systems biology and systems medicine have played an important role in the last two decades in shaping our understanding of biological processes. While systems biology is synonymous with network maps and '-omics' approaches, it is not often associated with mechanical processes. Here, we make the case for considering the mechanical and geometrical aspects of biological membranes as a key step in pushing the frontiers of systems biology of cellular membranes forward. We begin by introducing the basic components of cellular membranes, and highlight their dynamical aspects. We then survey the functions of the plasma membrane and the endomembrane system in signaling, and discuss the role and origin of membrane curvature in these diverse cellular processes. We further give an overview of the experimental and modeling approaches to study membrane phenomena. We close with a perspective on the converging futures of systems biology and membrane biophysics, invoking the need to include physical variables such as location and geometry in the study of cellular membranes. WIREs Syst Biol Med 2017, 9:e1386. doi: 10.1002/wsbm.1386 For further resources related to this article, please visit the WIREs website.

Pulsatile Lipid Vesicles under Osmotic Stress.

The response of lipid bilayers to osmotic stress is an important part of cellular function. Recent experimental studies showed that when cell-sized giant unilamellar vesicles (GUVs) are exposed to hypotonic media, they respond to the osmotic assault by undergoing a cyclical sequence of swelling and bursting events, coupled to the membrane's compositional degrees of freedom. Here, we establish a fundamental and quantitative understanding of the essential pulsatile behavior of GUVs under hypotonic conditions by advancing a comprehensive theoretical model of vesicle dynamics. The model quantitatively captures the experimentally measured swell-burst parameters for single-component GUVs, and reveals that thermal fluctuations enable rate-dependent pore nucleation, driving the dynamics of the swell-burst cycles. We further extract constitutional scaling relationships between the pulsatile dynamics and GUV properties over multiple timescales. Our findings provide a fundamental framework that has the potential to guide future investigations on the nonequilibrium dynamics of vesicles under osmotic stress.

Averaged model for momentum and dispersion in hierarchical porous media.

Hierarchical porous media are multiscale systems, where different characteristic pore sizes and structures are encountered at each scale. Focusing the analysis to three pore scales, an upscaling procedure based on the volume-averaging method is applied twice, in order to obtain a macroscopic model for momentum and diffusion-dispersion. The effective transport properties at the macroscopic scale (permeability and dispersion tensors) are found to be explicitly dependent on the mesoscopic ones. Closure problems associated to these averaged properties are numerically solved at the different scales for two types of bidisperse porous media. Results show a strong influence of the lower-scale porous structures and flow intensity on the macroscopic effective transport properties.