Effect Of Membrane Curvature Research Articles

Understanding the Role of Caveolae in Oxygen Buffering: The Effect of Membrane Curvature.

The "oxygen paradox" can be explained as two opposing biological processes with oxygen (O2) as a reactant. On the one hand, oxygen is essential to aerobic metabolism, powering oxidative phosphorylation in mitochondria. On the other hand, an excess supply of oxygen will generate reactive species which are harmful for the cell. In healthy tissues, the first process must be maximized relative to the second one. We have hypothesized that curved and cholesterol-enriched membrane invaginations called caveolae help maintain the proper oxygen level by taking up oxygen and attenuating its release to the mitochondria. The mechanism by which caveolae may help to buffer the oxygen level in cells is still unclear. Here, we aim to assess how structural aspects of caveolae, the curvature of the membrane, influence the local oxygen abundance and the membrane partitioning. We have modelled a flat bilayer and a liposome composed of dipalmitoylphosphatidylcholine (DPPC), using molecular dynamics simulation. Associated changes in the membrane-level oxygen partition coefficient and free energy profiles will be presented.

Dynamics of force dipoles in curved fluid membranes

How do motor proteins and other biological nanomachines form aggregates in cell membranes and show coordinated activity, essential for so many living processes ? Membrane Fluid mechanics provides a natural explanation. In this work, we show how force dipole motors induce special flows in a spherical fluid membrane and form aggregates on the sphere due to their mutual hydrodynamic interactions. Bulk confinement and membrane curvature effects compete with each other and display novel regimes of coordinated motion.

Membrane curvature-facilitated oncogenic RAS clustering in cells

As a dominant oncogenic protein, Ras GTPases are found segregated into nanoclusters on plasma membrane enabling downstream effector binding and signaling activation. Recent studies shed light on the stimulation effect of membrane curvature as a mechanical trigger for Ras accumulation. However, it remains an unclear picture of why Ras can respond to membrane curvature, especially in live cell environment. It is largely due to the lack of efficient tool to manipulate membrane curvature in cells. Here, we utilized the vertically-aligned nanobar arrays to study the regulation effect of nanoscale membrane curvature on oncogenic Ras clustering in live cells.

Counting permeant crossings to assess the effect of membrane curvature and composition on the permeability rate

Liposomes are spherically shaped phospholipid bilayers that are used to encapsulate hydrophilic or lipophilic drugs. To have efficient drug delivery by a liposome, it should be able to deliver the drug to the specific binding site, where it can affect the binding target. However, sometimes, due to the untimely drug release, this goal cannot be achieved. Therefore, the permeation rate of the drug through the liposomal membrane is important, as it affects the drug’s residence time inside the liposome.

Fluorescent labeling in size-controlled liposomes reveals membrane curvature-induced structural changes in the KcsA potassium channel.

Biological structures with highly curved membranes, such as caveolae and transport vesicles, are essential for signal transduction and membrane trafficking. Although membrane proteins in these structures are subjected to physical stress due to the curvature of the lipid bilayers, the effect of this membrane curvature on protein structure and function remains unclear. In this study, we established an experimental procedure to evaluate membrane curvature-induced structural changes in the prototypical potassium channel KcsA. The effect of a large membrane curvature was estimated using fluorescently labeled KcsA by incorporating it into liposomes with a small diameter (<30nm). We found that a large membrane curvature significantly affects the activation gate conformation of the KcsA channel.

The binding of different model membranes with PKCε C2 domain is not dependent on membrane curvature but affects the sequence of events during unfolding

The C2 domain of novel protein kinases C (nPKC) binds to membranes in a Ca2+-independent way contributing to the activation of these enzymes. We have studied the C2 domain of one of these nPKCs, namely PKCε, and confirmed that it establishes a strong interaction with POPA, which is clearly visible through changes in chemical shifts detected through 31P-MAS-NMR and the protection that it exerts on the domain against thermal denaturation seen through DSC and FT-IR. In this study, using two-dimensional correlation analysis (2D-COS) applied to infrared spectra, we determined the sequence of events that occur during the thermal unfolding of the domain and highlighted some differences when phosphatidic acid or cardiolipin are present. Finally, by means of FRET and DLS experiments, we wanted to determine the effect of membrane curvature on the domain/membrane interaction by using lysophosphatidylcholine to introduce positive curvature as a control and we observed that the effect of these phospholipids on the protein binding is not exerted through the change of membrane curvature.

Effects of Membrane Curvature on Amyloid-Beta Aggregation

Effects of Membrane Curvature on Amyloid-Beta Aggregation

Reaction-diffusion waves coupled with membrane curvature.

The reaction-diffusion waves of proteins are known to be involved in fundamental cellular functions, such as cell migration, cell division, and vesicular transportation. In some of these phenomena, pattern formation on the membranes is induced by the coupling between membrane deformation and the reaction-diffusion system through curvature-inducing proteins that bend the biological membranes. Although the membrane shape and the dynamics of the curvature-inducing proteins affect each other in these systems, the effect of such mechanochemical feedback loops on the waves has not been studied in detail. In this study, reaction-diffusion waves coupled with membrane deformation are investigated using simulations combining a dynamically triangulated membrane model with the Brusselator model extended to include the effect of membrane curvature. It is found that the propagating wave patterns change into nonpropageting patterns and spiral wave patterns due to the mechanochemical effects. Moreover, the wave speed is positively or negatively correlated with the local membrane curvature depending on the spontaneous curvature and bending rigidity. In addition, self-oscillation of the vesicle shape occurs, associated with the reaction-diffusion waves of curvature-inducing proteins. This agrees with the experimental observation of GUVs with a reconstituted Min system, which plays a key role in the cell division of Escherichia coli. The findings of this study demonstrate the importance of mechanochemical coupling in biological phenomena.

Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature.

Lipases are important hydrolytic enzymes used in a spectrum of technological applications, such as the pharmaceutical and detergent industries. Because of their versatile nature and ability to accept a broad range of substrates, they have been extensively used for biotechnological and industrial applications. Current assays to measure lipase activity primarily rely on low-sensitivity measurements of pH variations or visible changes of material properties, like hydration, and often require high amounts of proteins. Fluorescent readouts, on the other hand, offer high contrast and even single-molecule sensitivity, albeit they are reliant on fluorogenic substrates that structurally resemble the native ones. Here we present a method that combines the highly sensitive readout of fluorescent techniques while reporting enzymatic lipase function on native substrates. The method relies on embedding the environmentally sensitive fluorescent dye pHrodo and native substrates into the bilayer of liposomes. The charged products of the enzymatic hydrolysis alter the local membrane environment and thus the fluorescence intensity of pHrodo. The fluorescence can be accurately quantified and directly assigned to product formation and thus enzymatic activity. We illustrated the capacity of the assay to report the function of diverse lipases and phospholipases both in a microplate setup and at the single-particle level on individual nanoscale liposomes using total internal reflection fluorescence (TIRF). The parallelized sensitive readout of microscopy combined with the inherent polydispersity in sizes of liposomes allowed us to screen the effect of membrane curvature on lipase function and identify how mutations in the lid region control the membrane curvature-dependent activity. We anticipate this methodology to be applicable for sensitive activity readouts for a spectrum of enzymes where the product of the enzymatic reaction is charged.

Membrane Curvature Effects on Rhodopsin Activation Investigated by Time-Resolved Electronic Spectroscopy

Membrane Curvature Effects on Rhodopsin Activation Investigated by Time-Resolved Electronic Spectroscopy

Light-Inducible Generation of Membrane Curvature in Live Cells with Engineered Bar Domain Proteins

Nanoscale membrane curvature is now understood to play an active role in many subcellular processes. Studies of nanoscale membrane curvature effects on protein function thus far have primarily consisted of in vitro studies, as few methods are able to induce membrane curvature in live cells. A new method of generating nanoscale membrane curvature that is dynamic, reversible, and capable of stimulating a large population of cells is desired. Here, we report the development of two optogenetic systems: opto-FBAR and opto-IBAR. Derived from the human FBP17 and IRSp53 proteins, respectively, these systems allow us to induce the formation of positive and negative membrane curvature with fine spatiotemporal precision. In opto-FBAR, blue light activation results in the formation of membrane tubules, controllable down to the subcellular level. Using a modified image analysis program, we are able to quantify levels of plasma membrane tubulation in cells. opto-IBAR activation results in the formation or elongation of cellular membrane protrusions and filopodia. These systems present a novel approach for controlling nanoscale membrane curvature in live cells.

Membrane curvature induces cardiolipin sorting

Cardiolipin is a cone-shaped lipid predominantly localized in curved membrane sites of bacteria and in the mitochondrial cristae. This specific localization has been argued to be geometry-driven, since the CL’s conical shape relaxes curvature frustration. Although previous evidence suggests a coupling between CL concentration and membrane shape in vivo, no precise experimental data are available for curvature-based CL sorting in vitro. Here, we test this hypothesis in experiments that isolate the effects of membrane curvature in lipid-bilayer nanotubes. CL sorting is observed with increasing tube curvature, reaching a maximum at optimal CL concentrations, a fact compatible with self-associative clustering. Observations are compatible with a model of membrane elasticity including van der Waals entropy, from which a negative intrinsic curvature of −1.1 nm−1 is predicted for CL. The results contribute to understanding the physicochemical interplay between membrane curvature and composition, providing key insights into mitochondrial and bacterial membrane organization and dynamics.

Comparative Study of Pore Formation Energy by High Intensity, Nanosecond Electrical Pulse.

Nanosecond, high intensity electric pulses create nanopores in the cell membrane. Pore formation energy is probed by taking account of the strain energy based on the continuum model. Maxwell stress acting on the cell membrane is included in the 3D model calculation as well as the effect of membrane curvature. In addition, comparison between cylindrical and toroidal pores were made to explore the difference of strain energy and force over the pores at a range of radii. Through the analyses the transmembrane potential were kept constant in order to obtain a transient response in that the electric pulse has a ultrashort duration and pore-evolving process is rapid as well. Our results demonstrate that under the same circumstances toroidal pores have higher strain energy than cylindrical pores due to the surface area and volume of the pore shape.

Effects of membrane curvature and pH on proton pumping activity of single cytochrome bo3 enzymes

The molecular mechanism of proton pumping by heme-copper oxidases (HCO) has intrigued the scientific community since it was first proposed. We have recently reported a novel technology that enables the continuous characterisation of proton transport activity of a HCO and ubiquinol oxidase from Escherichia coli, cytochrome bo3, for hundreds of seconds on the single enzyme level (Li et al. J Am Chem Soc 137 (2015) 16055–16063). Here, we have extended these studies by additional experiments and analyses of the proton transfer rate as a function of proteoliposome size and pH at the N- and P-side of single HCOs. Proton transport activity of cytochrome bo3 was found to decrease with increased curvature of the membrane. Furthermore, proton uptake at the N-side (proton entrance) was insensitive to pH between pH6.4–8.4, while proton release at the P-side had an optimum pH of ~7.4, suggesting that the pH optimum is related to proton release from the proton exit site. Our previous single-enzyme experiments identified rare, long-lived conformation states of cytochrome bo3 where protons leak back under turn-over conditions. Here, we analyzed and found that ~23% of cytochrome bo3 proteoliposomes show ΔpH half-lives below 50s after stopping turnover, while only ~5% of the proteoliposomes containing a non-pumping mutant, E286C cytochrome bo3 exhibit such fast decays. These single-enzyme results confirm our model in which HCO exhibit heterogeneous pumping rates and can adopt rare leak states in which protons are able to rapidly flow back.

Membrane-active Antimicrobial Peptides as Template Structures for Novel Antibiotic Agents

The increase of pathogens being resistant to antibiotics represents a global health problem and therefore it is a pressing need to develop antibiotics with novel mechanisms of action. Host defense peptides, which have direct antimicrobial activity (also termed antimicrobial peptides) or immune modulating activity, are valuable template structures for the development of such compounds. Antimicrobial peptides exhibit remarkably different structures as well as biological activity profiles with multiple targets. A large fraction of these peptides interfere physically with the cell membrane of bacteria (focus of this review), but can also translocate into the cytosol, where they interact with nucleic acids, ribosomes and proteins. Several potential interaction sites have to be considered on the route of the peptides from the environment to the cytoplasmic membrane. Translocation of peptides through the cell wall may not be impaired by the thick but relatively porous peptidoglycan layer. However, interaction with lipopolysaccharides of the outer membrane of Gram-negative bacteria and (lipo)teichoic acids of Gram-positive bacteria may reduce the effective concentration at the cytoplasmic membrane, where supposedly the killing event takes place. On a molecular level several mechanisms are discussed, which are important for the rational design of improved antimicrobial compounds: toroidal pore formation, carpet model (coverage of membrane surface by peptides), interfacial activity, void formation, clustering of lipids and effects of membrane curvature. In summary, many of these models just represent special cases that can be interrelated to each other and depend on both the nature of lipids and peptides.

Polarized Localization Microscopy Detects Membrane Curvature and Reveals the Interplay between Membrane Orientation and Protein Dynamics

Nanoscale membrane curvature is a necessary component of countless cellular processes. However, the understanding of these processes is limited by the experimental techniques and its inability to detect and resolve nanoscale membrane curvature. Here we present Polarized Localization Microscopy (PLM), a novel optical microscopy technique that detects and resolves membrane curvature with 15 nm resolution. PLM combines the advantages of polarized total internal reflection fluorescence microscopy (TIRF) and fluorescence localization microscopy to reveal single-fluorophore locations and orientations without sacrificing the localization precision of the point spread function. We demonstrate PLM capability of resolving membrane orientations by draping a supported lipid bilayer over polystyrene nanoparticles of varying sizes on a glass coverslip, thus creating a model membrane with coexisting flat and curved regions and membrane radii of curvature as small as 20 nm. Further, we introduce theoretical analysis that enables mapping out the membrane topology from the density of detected localizations in fluorescence excitation polarization. Using this approach, the interplay between membrane topology and cholera toxin subunit B (CTxB) dynamics was addressed. CTxB partitioned to curved membrane over the nanoparticle and further drove formation of lipid tubules. PLM detected the binding of an exocytosis protein complex to nanoscale membrane curvature. PLM was utilized to perform high throughput single particle tracking to reveal the correlated effects of membrane curvature, dynamics, and protein distribution. By providing super-resolution of membrane topology, PLM is able to reveal the critical components in various cellular processes such as endocytosis/exocytosis, viral infections, and neurotransmission.

Coarse-grained molecular dynamics study of membrane fusion: Curvature effects on free energy barriers along the stalk mechanism.

The effects of membrane curvature on the free energy barrier for membrane fusion have been investigated using coarse-grained molecular dynamics (CG-MD) simulations, assuming that fusion takes place through a stalk intermediate. Free energy barriers were estimated for stalk formation as well as for fusion pore formation using the guiding potential method. Specifically, the three different geometries of two apposed membranes were considered: vesicle-vesicle, vesicle-planar, and planar-planar membranes. The free energy barriers for the resulting fusion were found to depend importantly on the fusing membrane geometries; the lowest barrier was obtained for vesicular membranes. Further, lipid sorting was observed in fusion of the mixed membranes of dimyristoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine (DOPE). Specifically, DOPE molecules were found to assemble around the stalk to support the highly negative curved membrane surface. A consistent result for lipid sorting was observed when a simple continuum model (CM) was used, where the Helfrich energy and mixing entropy of the lipids were taken into account. However, the CM predicts a much higher free energy barrier than found using CG-MD. This discrepancy originates from the conformational changes of lipids, which were not considered in the CM. The results of the CG-MD simulations reveal that a large conformational change in the lipid takes place around the stalk region, which results in a reduction of free energy barriers along the stalk mechanism of membrane fusion.

SNAP-Tag-Reactive Lipid Anchors Enable Targeted and Spatiotemporally Controlled Localization of Proteins to Phospholipid Membranes.

The natural mechanisms that direct proteins to membranes are typically complex, requiring multiple steps and accessory components. It would be advantageous to develop simplified methods to direct proteins of interest to phospholipid membranes in a single step. Here we report a modular method for membrane localization of proteins by using chemically modified phospholipid anchors capable of covalent attachment to O(6)-methylguanine DNA methyltransferase (SNAP-tag) fusion proteins. To our knowledge, this is the first use of SNAP-tag reactions to modify benzylguanine-functionalized lipid membranes. We demonstrate that photocaged lipid precursors enable light-triggered spatial and temporal control over protein localization. The anchoring system is compatible with cell-free expression, allowing for genetic targeting of proteins to lipid membranes of giant unilamellar vesicles. This technique can be used to control membrane curvature effects, similar to what has been previously observed with certain membrane-bound proteins. This work addresses a current need in synthetic biology for simplified and robust methods to control membrane localization of expressed proteins and shows promise as a general tool for protein targeting to lipid vesicles and cellular membranes.

Nanoscale phase behavior on flat and curved membranes

The diverse physical properties of membranes play a critical role in many membrane associated biological processes. Proteins responsible for membrane transport can be affected by the lateral membrane order and lateral segregation of proteins is often controlled by the preference of certain membrane anchors for membrane phases having a physically ordered state. The dynamic properties of coexisting membrane phases are often studied by investigating their thermal behavior. Optical trapping of gold nanoparticles is a useful tool to generate local phase transitions in membranes. The high local temperatures surrounding an irradiated gold nanoparticle can be used to melt a part of a giant unilamellar lipid vesicle (GUV) which is then imaged using phase sensitive fluorophores embedded within the bilayer. By local melting of GUVs we reveal how a protein-free, one component lipid bilayer can mediate passive transport of fluorescent molecules by localized and transient pore formation. Also, we show how tubular membrane curvatures can be generated by optical pulling from the melted region on the GUV. This will allow us to measure the effect of membrane curvature on the phase transition temperature.

An investigation of the effect of membrane curvature on transmembrane-domain dependent protein sorting in lipid bilayers.

Sorting of membrane proteins within the secretory pathway of eukaryotic cells is a complex process involving discrete sorting signals as well as physico-chemical properties of the transmembrane domain (TMD). Previous work demonstrated that tail-anchored (TA) protein sorting at the interface between the Endoplasmic Reticulum (ER) and the Golgi complex is exquisitely dependent on the length and hydrophobicity of the transmembrane domain, and suggested that an imbalance between TMD length and bilayer thickness (hydrophobic mismatch) could drive long TMD-containing proteins into curved membrane domains, including ER exit sites, with consequent export of the mismatched protein out of the ER. Here, we tested a possible role of curvature in TMD-dependent sorting in a model system consisting of Giant Unilamellar Vesicles (GUVs) from which narrow membrane tubes were pulled by micromanipulation. Fluorescent TA proteins differing in TMD length were incorporated into GUVs of uniform lipid composition or made of total ER lipids, and TMD-dependent sorting and diffusion, as well as the bending rigidity of bilayers made of microsomal lipids, were investigated. Long and short TMD-containing constructs were inserted with similar orientation, diffused equally rapidly in GUVs and in tubes pulled from GUVs, and no difference in their final distribution between planar and curved regions was detected. These results indicate that curvature alone is not sufficient to drive TMD-dependent sorting at the ER-Golgi interface, and set the basis for the investigation of the additional factors that must be required.