Membrane Deformation Research Articles

Multiphysics Analysis of Reversible Electroporation and Electrodeformation of Cervical Cells Using a Nanosecond Pulse Generator

We present an agglomerative in-silico study that explores the feasibility of leveraging nanosecond pulsed electric field on cervical cell samples toward improving cervical cell neoplasia and cancer detection by enhancing the visual fidelity of the screening tests. This comprehensive, multimodal study explores the effect of these pulsed electric fields on membrane electroporation and deformation in cell clusters extracted from real cervical sample images. Apart from performing a quantitative assessment of dye uptake change under the predefined electric stress, we also perform a thermal analysis to check whether the thermal envelope is breached during the application of a pulsed electric field. For the sake of completeness, we also present the hardware details of a Marx generator-based pulsar that provides the necessary stimulus for various multiphysics studies.

Herpesviruses deform membrane during capsid nuclear export by lipid ordering and protein scaffolding.

Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world's population. A critical step in their replication is the export of the viral capsids from the nucleus into the cytoplasm, which occurs by an unusual mechanism termed nuclear egress. Too large to fit through nuclear pores, capsids instead bud at the inner nuclear membrane to form perinuclear enveloped virions, which then fuse with the outer nuclear membrane, releasing the capsids into the cytoplasm. This process is mediated by the virus-encoded nuclear egress complex (NEC) that deforms the membrane around the capsid. To understand how the NEC generates negative membrane curvature, we reconstituted the membrane budding process in vitro using the NEC from herpes simplex virus 1, a prototypical herpesvirus that causes cold sores. To probe its mechanism, we employed a combination of confocal microscopy, electron spin resonance, and structural biology. We found that the NEC uses clusters of positively charged residues to insert into the lipid headgroups, which increases lipid order. We also found that the NEC oligomerizes into a membrane-bound hexagonal coat on the inner surface of the budded vesicles. We propose that the NEC combines lipid ordering and oligomerization to mold the membrane into a spherical shape. Whereas lipid ordering generates negative membrane curvature locally, the NEC oligomerization into a hexagonal scaffold is necessary to achieve negative membrane curvature over a large membrane area. Similar principles may underlie membrane deformation in other systems. Our findings provide a biophysical explanation for the phenomenon of virus-induced nuclear budding.

Microscopic springs: Investigating viscoelastic and elastic tendencies of the cell membrane.

Cell membranes are normally thought of as viscoelastic materials because they exhibit both viscous and elastic properties when deforming, however, some researchers describe membranes as elastic only. Our project seeks to quantify the membrane deformations during a splitting event, and mathematically predict membrane tensions if it were an elastic or viscoelastic material. Knowing the mechanical description that is most appropriate can help future research interested in describing cell mechanics when cells split, change shape, or move. First, we quantified cell shape from experimental fluorescently dyed membranes using ImageJ. We assume that knowing the physical location of the cell membrane over time can be used to quantify how the cell membrane changes due to forces acting on/in the membrane. We built MATLAB functions to import coordinates from ImageJ, center them, segment them based on arctan values into specific angle quadrants, and calculate the force the membrane experiences based on its location relative to its angular quadrant when considered an elastic spring or viscoelastic material. Centering the coordinates allows the functions to apply to multiple different cell experiments, while quadrants allowed the accounting of localized growth and shrinkage of the membrane. To model the elastic properties, we used a Hooke's Law spring equation and a viscoelastic equation with elastic and damper components. Our results suggest that the cell membrane can be treated as a viscoelastic material under small time scales (< 3 minutes) and as an elastic spring for larger time scale deformations. These results can be validated experimentally through laser ablation. By ablating a hole in the cell membrane, we can quantify the recoil of the membrane to determine how much tension was present before the ablation and compare the scale of the experimental tension with our model predicted tension.

Membrane mechanics regulate the cooperativity of proteins in curvature sensing.

Recruitment of proteins to a membrane surface is critical for many biological processes, such as clathrin-mediated endocytosis and signaling. When interacting with the lipid membrane, many proteins exhibit different affinities to membranes with different curvature, known as curvature sensing. They also generate membrane curvature (curvature induction) by a variety of mechanisms, including the insertion of amphipathic helices. Here we use a continuum membrane model to study how helix insertions can alter the membrane energetics and how the mechanical feedback of the membrane regulates the helix insertions. Our membrane model has two coupled layers of triangular mesh to represent the two monolayers of the bilayer membrane, capturing membrane thickness and tilt. We analyzed the membrane deformation around one helix insertion and compared it to molecular dynamic simulations to optimally define the lipid tilt energies. We then investigate the interactions between insertions and membranes by considering the copy number of insertions and their interval distance, membrane tension or stress before the insertions, membrane curvatures, and lipid composition. Our results showed that multiple insertions exhibit a mechanism of cooperativity; each binding event is not independent of previous events. Our results show that this cooperativity is dependent on membrane tension and curvature. In comparison to experimental data, we validated our simulation results and analyzed the role of lipid flip-flop across the bilayers. Quantifying how protein and membrane interactions feedback on one another is critical for understanding the dynamics and control of proteins that remodel membranes through localization and self-assembly in various cellular processes.

Nuclear membrane deformation via progerin clustering.

The lamina, a structural network of lamin proteins, maintains the shape of the nuclear membrane and is involved in its functions. Lamina is also known to affect gene expression regulation by interacting with chromatin. Therefore, if there is an abnormality in the lamin protein, various diseases are caused. One of the typical diseases caused by lamin defects is premature aging, called Hutchinson-Gilford progeria syndrome (HGPS). HGPS is believed to be caused by progerin generated through alternative splicing of pre-lamin A. In HGPS, progerin is incorporated into the lamina, consequently causing severe nuclear membrane deformation. However, it is still unclear how progerin induces nuclear deformation. Here, we generated a cell line that conditionally induced progerin and observed the process of nuclear membrane deformation in real-time using super-resolution fluorescence imaging. Unlike normal lamin proteins, we found progerin is not evenly distributed in the lamina but formed clusters and confirmed that farnesylation of the progerin c-terminus domain is crucial to the clustering. Moreover, we demonstrated the nuclear membrane deformation by the progerin cluster through invagination rather than the protrusion. Based on the novel layered structure that progerin displaces the peripheral lamin B of the nuclear lamina, we suggest a mechanistic model of nuclear membrane deformation via progerin clustering.

Efficient coarse-grained simulations of multiple Piezo channels in flat membrane and vesicles.

The unique dome shape of mechanosensitive Piezo channels has inspired biophysical studies on whether and how Piezos should cluster under various cell types and physiological conditions. Molecular dynamics (MD) simulation can, in principle, capture Piezo diffusion and clustering stemming from interactions at the near atomistic level. However, due to their very large size, sampling spontaneous inter-channel Piezo interactions is inaccessible by standard MD techniques based on all-atom or Martini coarse-grained (CG) force fields. To overcome this challenge, we developed a minimal CG model for simulating the dynamics of multiple Piezo channels at the submicron scale in membranes and vesicles. In addition, our model enables tuning the shape and mechanical properties of both channel and membrane. Using this model, we investigated the propensity and topology of Piezo clustering under different channel densities, protein conformations, membrane bending rigidity, and membrane curvature. This work provides new insights into the interplay between protein and membrane deformation energy on Piezo clustering, hence bridging the gap between continuum elasticity theory and high-resolution imaging techniques.

Membrane remodeling and vesicle formation by biomolecular condensates: A coarse grained simulation study.

Intrinsically disordered peptides (IDPs) can undergo liquid-liquid phase separation (LLPS) which is essential for membrane-less organelle formation and intracellular compartmentalization. Several experimental studies observed LLPS at or near the lipid membrane surface, leading to the alteration of the membrane morphology. Processes such as tubulation, endocytosis, exocytosis, and multilamellar membrane formation have also been observed in recent experiments. However, the molecular mechanisms and the governing molecular interactions remain unclear. Here we employ molecular dynamics simulations with different levels of coarse-graining to understand these long timescale processes. We study the coacervation of oppositely charged polypeptide sequences, namely, poly-glutamate (E30) and poly-lysine (K30) mixtures in the presence of lipid bilayers containing different ratios of POPC and POPG, with Martini 3.0 explicit solvent forcefield. We find that at least 20% anionic lipids are required to observe successful events of adsorption, followed by wetting, of the EK-coacervate on the bilayer. Upon wetting, the coacervate produces negative curvature to the bilayer and induces local demixing of lipids. Interestingly, the number of contacts between the polyelectrolytes decreases, unlike previous suggestions that predicted an increased overlap among the polyelectrolytes as the driving force of negative curvature formation. Next, we investigate the sensitivity of membrane deformation to the sequence (or charge patterns) of the IDPs. We observe that the IDPs with segregated charge patterns can remodel the membrane more prominently than IDPs with well-mixed charges. In order to understand the biologically relevant length- and time-scales, we developed a solvent-free coarse grained model for such systems. By tuning the IDP-lipid and IDP-IDP interactions, we observe endocytosis, exocytosis, and multilamellarity induced by the LLPS droplet into the bilayer vesicle.

Thermoresponsive artificial cells.

We build thermoresponsive phospholipid-based artificial cell model compartments that respond to mild temperature changes in the environment by performing reversible shape deformations, enabling material transfer between entities, and migration. The temperature increase is in the laboratory applied by means of an IR-B-laser, and induces the self-organization of lipid compartments into dynamic clusters. The clusters are motile and can migrate along the temperature gradient. To increase the thermoresponsiveness, we encapsulate poly-N-isopropylacrylamide (PNIPAAm) in the lipid compartments. The polymer solution undergoes reversible gel formation when heated above its lower critical solution temperature (LCST) of 32 °C. We observe the formation of microaggregates inside the lipid compartments which can interact with the membrane and induce membrane deformations. Our experimental system comprises a contactless method to generate compartmentalized multicellular model structures with thermoresponsive properties.

State-specific morphological deformations of the lipid bilayer explain mechanosensitive gating of MSCs ion channels.

The so-called force-from-lipids hypothesis of cellular mechanosensation posits that ion channel gating directly responds to changes in the physical state of the membrane induced for example by lateral tension. Here, we investigate the molecular basis for this transduction mechanism by studying the mechanosensitive ion channel MscS from Escherichia coli and its eukaryotic homolog, MSL1 from Arabidopsis thaliana. First, we use single-particle cryo-EM to determine the structure of a novel open conformation of wild-type MscS, stabilize in thinned lipid nanodisc. Compared with the closed state, the structure shows a reconfiguration of helices TM1, TM2 and TM3a, resulting in widening of the central pore. Based on these structures, we examined how the morphology of the lipid bilayer is altered upon gating, using molecular dynamics simulations. The simulations reveal that, in the absence of extrinsic forces, closed-state MscS causes drastic protrusions in the inner leaflet of the lipid bilayer. These deformations develop to provide adequate solvation to hydrophobic features of the protein surface, and clearly reflect a high energy conformation for the membrane. Strikingly, the protrusions are largely eradicated upon channel opening. An analogous computational study of open and closed states of MSL1 recapitulates these findings. The gating equilibrium of MscS channels thus appears to be dictated by two opposing conformational preferences, namely those of the lipid membrane and of the protein structure. We theorize that membrane tension controls this balance because it increases the energetic cost of the membrane deformations that specifically stabilize the closed channel, shifting the gating equilibrium towards the conductive form.

Regulation of actin-binding proteins by PI(4,5)P2.

The actin cytoskeleton powers membrane deformations during many cellular processes such as cell migration, morphogenesis, and endocytosis. One of the phosphoinositides phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates activities of many actin-binding proteins (ABPs) including profilin, cofilin, Dia2, N-WASP, ezrin, and moesin; however, the underlying molecular mechanisms have remained elusive. Here, we applied biophysical approaches to uncover the molecular principles by which ABPs interact with phosphoinositide-containing membranes. Although ABPs bind to the membranes mainly through electrostatic interactions, we reveal that they show large differences in the affinities and dynamics of membrane interactions, and in the ranges of phosphoinositide densities that they sense. Profilin and cofilin show transient, low-affinity interactions with membranes, whereas F-actin assembly factors Dia2 and N-WASP reside on membranes for longer periods to perform their functions. Ezrin and moesin, which link the actin cytoskeleton to the plasma membrane, bind membranes with high affinities and slow dissociation dynamics. Unlike profilin, cofilin, Dia2, and N-WASP, they do not require high “stimulus-responsive” phosphoinositide density for membrane binding. Together, these findings demonstrate that membrane-interaction mechanisms of ABPs evolved to precisely perform their specific functions in cytoskeletal dynamics.

Using flow to investigate friction and protein transport in lipid membranes.

Many cells display lipid-anchored proteins on their plasma membranes. Fluid flow parallel to the cells may displace these proteins. Examples of this have been observed, but lateral flow transport of membrane proteins has not been widely studied. Measurements of the shear force, membrane friction, and resulting protein velocity will make it possible to determine whether this phenomenon is common and contributes to flow sensing. Our lab has developed a method for measuring flow transport of lipid-anchored proteins in a simplified model system: supported bilayer patches. We show that this method is robust and that the flow mobility observed for lipid-anchored proteins under shear flow depends on the ratio of the hydrodynamic force applied to the protein to the frictional drag provided by the lipid anchor. We show that the expected results are observed when these factors are independently varied. One factor that contributes to membrane drag on lipid-anchored proteins is the interleaflet friction. Interleaflet friction is a fundamental parameter governing membrane mechanics: along with membrane viscosity, it determines the time and energy scales for membrane deformations. However, measurements of this parameter are sparse and have previously been accomplished by a large variety of different experimental techniques, making it difficult to compare them or to identify trends. We adapted a recently developed method to determine how interleaflet friction varies with membrane lipid composition. We show that this method gives reproducible results and can detect changes to interleaflet friction resulting from even small differences in acyl chain structure. In particular, we observe dramatic changes to friction when cholesterol is included in the membrane. We look forward to adapting these techniques to investigate how membrane mechanics impact the mobility of proteins and membranes in living cells.

State-specific morphological deformations of the lipid bilayer explain mechanosensitive gating of MscS ion channels.

The force-from-lipids hypothesis of cellular mechanosensation posits that membrane channels open and close in response to changes in the physical state of the lipid bilayer, induced for example by lateral tension. Here, we investigate the molecular basis for this transduction mechanism by studying the mechanosensitive ion channel MscS from Escherichia coli and its eukaryotic homolog MSL1 from Arabidopsis thaliana. First, we use single-particle cryo-electron microscopy to determine the structure of a novel open conformation of wild-type MscS, stabilized in a thinned lipid nanodisc. Compared with the closed state, the structure shows a reconfiguration of helices TM1, TM2, and TM3a, and widening of the central pore. Based on these structures, we examined how the morphology of the membrane is altered upon gating, using molecular dynamics simulations. The simulations reveal that closed-state MscS causes drastic protrusions in the inner leaflet of the lipid bilayer, both in the absence and presence of lateral tension, and for different lipid compositions. These deformations arise to provide adequate solvation to hydrophobic crevices under the TM1-TM2 hairpin, and clearly reflect a high-energy conformation for the membrane, particularly under tension. Strikingly, these protrusions are largely eradicated upon channel opening. An analogous computational study of open and closed MSL1 recapitulates these findings. The gating equilibrium of MscS channels thus appears to be dictated by opposing conformational preferences, namely those of the lipid membrane and of the protein structure. We propose a membrane deformation model of mechanosensation, which posits that tension shifts the gating equilibrium towards the conductive state not because it alters the mode in which channel and lipids interact, but because it increases the energetic cost of the morphological perturbations in the membrane required by the closed state.

An analytical model describing the mechanics of erythrocyte membrane wrapping during active invasion of a plasmodium falciparum merozoite

The invasion of a merozoite into an erythrocyte by membrane wrapping is a hallmark of malaria pathogenesis. The invasion involves biomechanical interactions whereby the merozoite exerts actomyosin-based forces to push itself into and through the erythrocyte membrane while concurrently inducing biochemical damage to the erythrocyte membrane. Whereas the biochemical damage process has been investigated, the detailed mechanistic understanding of the invasion mechanics remains limited. Thus, the current study aimed to develop a mathematical model describing the mechanical factors involved in the merozoite invasion into an erythrocyte and explore the invasion mechanics.A shell theory model was developed comprising constitutive, equilibrium and governing equations of the deformable erythrocyte membrane to predict membrane mechanics during the wrapping of an entire non-deformable ellipsoidal merozoite. Predicted parameters include principal erythrocyte membrane deformations and stresses, wrapping and indentation forces, and indentation work. The numerical investigations considered two limits for the erythrocyte membrane deformation during wrapping (4% and 51% areal strain) and erythrocyte membrane phosphorylation (decrease of membrane elastic modulus from 1 to 0.5 kPa).For an intact erythrocyte, the maximum indentation force was 1 and 8.5 pN, and the indentation work was 1.92 × 10−18 and 1.40 × 10−17 J for 4% and 51% areal membrane strain. Phosphorylation damage in the erythrocyte membrane reduced the required indentation work by 50% to 0.97 × 10−18 and 0.70 × 10−17 J for 4% and 51% areal strain.The current study demonstrated the developed model's feasibility to provide new knowledge on the physical mechanisms of the merozoite invasion process that contribute to the invasion efficiency towards the discovery of new invasion-blocking anti-malaria drugs.

A hierarchical 3D finite element model of osteocyte: The spectrin membrane skeleton in mechanical transmission

Osteocytes in vivo are subjected to fluid shear stress and respond by initiating downstream mechano-transduction. Our previous studies demonstrated that the reticulate spectrin-based membrane skeleton (Spectrin-MS) beneath the cell membrane plays an important role in the mechanical property maintenance, as well as in the mechano-transduction of osteocytes. But how does the gridding extent of the spectrin-MS affect the mechanical transmission inside the cell remains unclear. To address this issue, a 3D finite element model (FEM) of osteocytes containing Spectrin-MS of varied gridding extent (representing polymerized/normal/depolymerized state) was established. The stress-strain responses of different components of the osteocyte to 15 dyn/cm2 fluid shear stress were obtained. Results showed that the depolymerized Spectrin-MS provided less support for the cell membrane and thus led more membrane deformation under shear stress. The depolymerization of Spectrin-MS also altered the mechanical transmission pathway inside the cell, and ultimately increased the stress transmitted to the nucleus. A compensative effect between the Spectrin-MS and cytoskeleton could be found in the mechanical transmission. These results indicate that the spectrin-MS is not only acting as a structural support, but also as a connecting linkage between the cell membrane and cytoskeleton. Future research of the mechanotransduction of osteocytes may benefit from considerations of the integration of the Spectrin-MS.

Intermediate conductance Ca2+-activated potassium channels are activated by functional coupling with stretch-activated nonselective cation channels in cricket myocytes.

Cooperative gating of localized ion channels ranges from fine-tuning excitation-contraction coupling in muscle cells to controlling pace-making activity in the heart. Membrane deformation resulting from muscle contraction activates stretch-activated (SA) cation channels. The subsequent Ca2+ influx activates spatially localized Ca2+-sensitive K+ channels to fine-tune spontaneous muscle contraction. To characterize endogenously expressed intermediate conductance Ca2+-activated potassium (IK) channels and assess the functional relevance of the extracellular Ca2+ source leading to IK channel activity, we performed patch-clamp techniques on cricket oviduct myocytes and recorded single-channel data. In this study, we first investigated the identification of IK channels that could be distinguished from endogenously expressed large-conductance Ca2+-activated potassium (BK) channels by adding extracellular Ba2+. The single-channel conductance of the IK channel was 62 pS, and its activity increased with increasing intracellular Ca2+ concentration but was not voltage-dependent. These results indicated that IK channels are endogenously expressed in cricket oviduct myocytes. Second, the Ca2+ influx pathway that activates the IK channel was investigated. The absence of extracellular Ca2+ or the presence of Gd3+ abolished the activity of IK channels. Finally, we investigated the proximity between SA and IK channels. The removal of extracellular Ca2+, administration of Ca2+ to the microscopic region in a pipette, and application of membrane stretching stimulation increased SA channel activity, followed by IK channel activity. Membrane stretch-induced SA and IK channel activity were positively correlated. However, the emergence of IK channel activity and its increase in response to membrane mechanical stretch was not observed without Ca2+ in the pipette. These results strongly suggest that IK channels are endogenously expressed in cricket oviduct myocytes and that IK channel activity is regulated by neighboring SA channel activity. In conclusion, functional coupling between SA and IK channels may underlie the molecular basis of spontaneous rhythmic contractions.

Nonlinear FE aeroelastic analysis of membrane airfoils with fixed and elastic supports

This paper presents a nonlinear aeroelastic analysis of membrane airfoils using the finite element method. The model uses the aerodynamic potential flow models based on the Prandtl–Glauert and Theodorsen’s thin-airfoil theories. The structural model accounts for the nonlinear strain due to the membrane deformation. The final aeroelastic system is presented in the time-domain which enables both static and transient response analysis. A comparison between the linear and the nonlinear aeroelastic results is presented. The effect of the elastic supports and their stiffness ratio on the aeroelastic characteristics is also investigated for static and dynamic responses due to step or harmonic wind loadings with a comparison to the fixed-ends membranes.

Modulation of self-organizing circuits at deforming membranes by intracellular and extracellular factors.

Mechanical forces exerted to the plasma membrane induce cell shape changes. These transient shape changes trigger, among others, enrichment of curvature-sensitive molecules at deforming membrane sites. Strikingly, some curvature-sensing molecules not only detect membrane deformation but can also alter the amplitude of forces that caused to shape changes in the first place. This dual ability of sensing and inducing membrane deformation leads to the formation of curvature-dependent self-organizing signaling circuits. How these cell-autonomous circuits are affected by auxiliary parameters from inside and outside of the cell has remained largely elusive. Here, we explore how such factors modulate self-organization atthe micro-scale and its emerging properties at the macroscale.

Macrophages modulate stiffness-related foreign body responses through plasma membrane deformation

Implants are widely used in medical applications and yet macrophage-mediated foreign body reactions caused by implants severely impact their therapeutic effects. Although the extensive use of multiple surface modifications has been introduced to provide some mitigation of fibrosis, little is known about how macrophages recognize the stiffness of the implant and thus influence cell behaviors. Here, we demonstrated that macrophage stiffness sensing leads to differential inflammatory activation, resulting in different degrees of fibrosis. The potential mechanism for macrophage stiffness sensing in the early adhesion stages tends to involve cell membrane deformations on substrates with different stiffnesses. Combining theory and experiments, we show that macrophages exert traction stress on the substrate through adhesion and altered membrane curvature, leading to the uneven distribution of the curvature-sensing protein Baiap2, resulting in cytoskeleton remodeling and inflammation inhibition. This study introduces a physical model feedback mechanism for early cellular stiffness sensing based on cell membrane deformation, offering perspectives for future material design and targeted therapies.

Fluid-structure interaction characteristics of inflatable reentry aeroshell at subsonic speed

Inflatable aeroshells, made of flexible membranes and pressurized inflatable torus, experience significant aerodynamic loads during atmospheric entry. The aerodynamic forces deform the lightweight, flexible membrane, and such deformation changes the flow field, changing the aerodynamic characteristics and increasing the deformation even further. Thus, fluid-structure coupled modeling of these problems is crucial for the reliable performance prediction of such reentry vehicles. This study provides wind tunnel experimental results on subsonic fluid-structure interaction (FSI), focusing on the aeroshell deformation and oscillatory behavior designed to validate coupled simulations. Wind tunnel experiments were conducted using a scaled membrane aeroshell model for subsonic speed. Aerodynamic coefficients, pressures at the rear of the model, and structural vibrations were measured for freestream Mach number of 0.3. A two-way coupled FSI model was set up in a partitioned manner for analyzing detailed distributions of the flow field properties, which was validated by the experimental data. The model was based on the open-source fluid solver OpenFOAM, computational structural solver CalculiX and coupling library preCICE. The present FSI analysis model well reproduced fundamental features such as swing motion, membrane deformation, and the wake in the flow field simulation, which were observed in the experiment. The results indicated that the membrane surface deforms elastically by aerodynamic force caused by the large pressure difference between the front and rear sides of the vehicle. The continuous generation and shedding of the wake vortex caused unsteady behavior in the flow field, followed by the small amplitude oscillation of the aeroshell. An external aerodynamic force caused the oscillation because the frequency of this oscillation did not correspond to that of natural frequencies.

Aerodynamics and fluid–structure interaction of an airfoil with actively controlled flexible leeward surface

Piezoelectric macro-fibre composite (MFC) actuators are employed onto the flexible leeward surface of an airfoil for active control. Time-resolved aerodynamic forces, membrane deformations and flow fields are synchronously measured at low Reynolds number (Re = 6 × 104). Mean aerodynamics show that the actively controlled airfoil can achieve lift-enhancement and drag-reduction simultaneously in the angle of attack range of 10° ≤ α ≤ 14°, where the rigid airfoil encounters stall. The maximum increments of lift and lift-to-drag ratio are 27.1 % and 126 % at the reduced actuation frequency of ${f^ + } = 3.52$ . The unsteady coupling features are further analysed at α = 12°, where the maximum lift-enhancement occurs. It is newly discovered that the membrane vibrations and flow fields are locked into half of the actuation frequency when ${f^ + } &gt; 3$ . The shift of the dominant vibration mode from bending to inclining is the reason for the novel ‘half-frequency lock-in’ phenomenon. To the fluid–structure interaction, there are three characteristic frequencies for the actively controlled airfoil: $S{t_1} = 0.5{f^ + }$ , $S{t_2} = {f^ + }$ , and $S{t_3} = 1.5{f^ + }$ . Here, St1 and its harmonics (St2, St3) are coupled with the natural frequencies of the leading-edge shear layer, resulting in the generation of multi-scale flow structures and suppression of flow separation. The lift presents comparable dominant frequencies between St1 and St3, which means the instantaneous lift is determined by the flow structures of St1 and St3. The local membrane bulge and dent affect the instantaneous swirl strength of flow structures near the maximum vibration amplitude location, which is the main reason for the variation of instantaneous lift.