Giant Unilamellar Vesicles Membrane Research Articles

Relationship between oligoarginine-induced membrane damage of single Escherichia coli cells and entry of the peptide into the cytoplasm

Cell-penetrating peptides (CPPs) can enter the cytosol of eukaryotic cells without killing them whereas some CPPs exhibit antimicrobial activity against bacterial cells. Here, to elucidate the mode of interaction of the CPP nona-arginine (R9) with bacterial cells, we investigated the interactions of lissamine rhodamine B red-labeled peptide (Rh-R9) with single Escherichia coli cells encapsulating calcein using confocal laser scanning microscopy. After Rh-R9 induced the leakage of a large amount of calcein, the fluorescence intensity of the cytosol due to Rh-R9 greatly increased, indicating that Rh-R9 induces cell membrane damage, thus allowing entry of a significant amount of Rh-R9 into the cytosol. To determine if the lipid bilayer region of the membrane is the main target of Rh-R9, we then investigated the interaction of Rh-R9 with single giant unilamellar vesicles (GUVs) comprising an E. coli polar lipid extract containing small GUVs and AlexaFluor 647 hydrazide (AF647) in the lumen. Rh-R9 entered the GUV lumen without inducing AF647 leakage, but leakage eventually did occur, indicating that GUV membrane damage was induced after the entry of Rh-R9 into the GUV lumen. The Rh-R9 peptide concentration dependence of the fraction of entry of Rh-R9 after a specific interaction time was similar to that of the fraction of leaking GUVs. These results indicate that Rh-R9 can damage the lipid bilayer region of a cell membrane, which may be related to its antimicrobial activity.

Effect of leaflet asymmetry on the stretching elasticity of lipid bilayers with phosphatidic acid

The asymmetry of membranes has a significant impact on their biophysical characteristics and behavior. This study investigates the composition and mechanical properties of symmetric and asymmetric membranes in giant unilamellar vesicles (GUVs) made of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidic acid (POPA). A combination of fluorescence quantification, zeta potential measurements, micropipette aspiration, and bilayer molecular dynamics simulations are used to characterize these membranes. The outer leaflet composition in vesicles is found consistent across the two preparation methods we employed, namely electroformation and inverted emulsion transfer. However, characterizing the inner leaflet poses challenges. Micropipette aspiration of GUVs show that oil residues do not substantially alter membrane elasticity, but simulations reveal increased membrane thickness and decreased interleaflet coupling in the presence of oil. Asymmetric membranes with a POPC:POPA mixture in the outer leaflet and POPC in the inner leaflet display similar stretching elasticity values to symmetric POPC:POPA membranes, suggesting potential POPA insertion into the inner leaflet during vesicle formation and suppressed asymmetry. The inverse compositional asymmetry, with POPC in the outer leaflet and POPC:POPA in the inner one yield less stretchable membranes with higher compressibility modulus compared with their symmetric counterparts. Challenges in achieving and predicting compositional correspondence highlight the limitations of phase-transfer-based methods. In addition, caution is advised when using fluorescently labeled lipids (even at low fractions of 0.5 mol %), as unexpected gel-like domains in symmetric POPC:POPA membranes were observed only with a specific type of labeled DOPE (dioleoylphosphatidylethanolamine) and the same fraction of unlabeled DOPE. The latter suggest that such domain formation may result from interactions between lipids and membrane fluorescent probes. Overall, this study underscores the complexity of factors influencing GUV membrane asymmetry, emphasizing the need for further research and improvement of characterization techniques.

Influence of pH and lipid membrane on the liquid–liquid phase separation of wheat γ-gliadin in aqueous conditions

Protein body (PB) formation in wheat seeds is a critical process influencing seed content and nutritional quality. In this study, we investigate the potential mechanisms governing PB formation through an in vitro approach, focusing on γ-gliadin, a key wheat storage protein. We used a microfluidic technique to encapsulate γ-gliadin within giant unilamellar vesicles (GUVs) and tune the physicochemical conditions in a controlled and rapid way. We examined the influence of pH and protein concentration on LLPS and protein-membrane interactions using various microscopy and spectroscopy techniques. We showed that γ-gliadin encapsulated in GUVs can undergo a pH-triggered liquid–liquid phase separation (LLPS) by two distinct mechanisms depending on the γ-gliadin concentration. At low protein concentrations, γ-gliadins phase separate by a nucleation and growth-like process, while, at higher protein concentration and pH above 6.0, γ-gliadin formed a bi-continuous phase suggesting a spinodal decomposition-like mechanism. Fluorescence and microscopy data suggested that γ-gliadin dense phase exhibited affinity for the GUV membrane, forming a layer at the interface and affecting the reversibility of the phase separation.

Molecular Interactions Between Egg White Peptides and Giant Unilamellar Vesicle Membranes: Effect of Peptide Localization on Membrane Fluidity.

Bioactive peptides have been shown to affect cell membrane fluidity, which is an important indicator of the cell membrane structure and function. However, the underlying mechanism of egg white-derived bioactive peptide regulation of cell membrane fluidity has not been elucidated yet. The cell membrane fluidity was investigated by giant unilamellar vesicles in the present study. The results showed that peptides TCNW, ADWAK, ESIINF, VPIEGII, LVEEY, and WKLC connect to membranes through intermolecular interactions, such as hydrogen bonding and regulated membrane fluidity, in a concentration-dependent way. In addition, peptides prefer to localize in the hydrophobic core of the bilayers. This study provides a theoretical basis for analyzing the localization of egg white bioactive peptides in specific cell membrane regions and their influence on the cell membrane fluidity.

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Effect of Phosphatidylethanolamine on Pore Formation Induced by the Antimicrobial Peptide PGLa.

Most antimicrobial peptides (AMPs) induce pore formation and a burst of lipid bilayers and plasma membranes. This causes severe leakage of the internal contents and cell death. The AMP PGLa forms nanopores in giant unilamellar vesicles (GUVs) comprising dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG). We here elucidated the effect of the line tension of a prepore rim on PGLa-induced nanopore formation by investigating the interaction of PGLa with single GUVs comprising dioleoylphosphatidylethanolamine (DOPE)/DOPG (6:4) in buffer using the single GUV method. We found that PGLa forms nanopores in the GUV membrane, which evolved into a local burst and burst of GUVs. The rate of pore formation in DOPE/DOPG-GUVs was smaller than that in DOPC/DOPG-GUVs. PGLa is located only in the outer leaflet of a GUV bilayer just before a fluorescent probe AF647 leakage from the inside, indicating that this asymmetric distribution induces nanopore formation. PGLa-induced local burst and burst of GUVs were observed at 10 ms-time resolution. After nanopore formation started, dense particles and small vesicles appeared in the GUVs, followed by a decrease in the GUV diameter. The GUV was finally converted into smaller GUV or lipid membrane aggregates. We discuss the mechanisms of PGLa-induced nanopore formation and its direct evolution to a GUV burst.

Dynamic Light-Induced Protein Patterns at Model Membranes.

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

Rearrangement of GUV-confined actin networks in response to micropipette aspiration.

Although diverse actin network architectures found inside the cell have been individually reconstituted outside of the cell, how different types of actin architectures reorganize under applied forces is not entirely understood. Recently, bottom-up reconstitution has enabled studies where dynamic and phenotypic characteristics of various actin networks can be recreated in an isolated cell-like environment. Here, by creating a giant unilamellar vesicle (GUV)-based cell model encapsulating actin networks, we investigate how actin networks rearrange in response to localized stresses applied by micropipette aspiration. We reconstitute actin bundles and branched bundles in GUVs separately and mechanically perturb them. Interestingly, we find that, when aspirated, protrusive actin bundles that are otherwise randomly oriented in the GUV lumen collapse and align along the axis of the micropipette. However, when branched bundles are aspirated, the network remains intact and outside of the pipette while the GUV membrane is aspirated into the micropipette. These results reveal distinct responses in the rearrangement of actin networks in a network architecture-dependent manner when subjected to physical forces.

Bottom-up approach to explore alpha-amylase assisted membrane remodelling

Soluble alpha-amylases play an important role in the catabolism of polysaccharides. In this work, we show that the malt α -amylase can interact with the lipid membrane and further alter its mechanical properties. Vesicle fluctuation spectroscopy is used for quantitative measurement of the membrane bending rigidity of phosphatidylcholines lipid vesicles from the shape fluctuation based on the whole contour of Giant Unilamellar Vesicles (GUVs). The bending rigidity of the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid vesicles in water increases significantly with the presence of 0.14 micromolar alpha-amylase (AA) in the exterior solution. It appears that the enzyme present in the external solution interacts with the outer layer of the bilayer membrane, leading to an asymmetry of the solution on either side of the bilayer membrane and altering its elasticity. At AA concentration of 1.5 micromolars and above, changes in the morphology of the GUV membrane are observed. The interaction between AA in the external solution and the external leaflet causes the bilayer membrane to curve spontaneously, leading to the formation of outbuds, giving a positive spontaneous curvature of C0 ≤ 0.05 μm−1 at ≈ 1 mg / ml of the AA concentration. We validate and characterize its concentration-dependent role in stabilizing the membrane curvature. Our findings indicate that the involvement of the enzyme, depending on the concentration, can have a considerable effect on the mechanical characteristics of the membrane.

Photoactivation of LOV domains with chemiluminescence.

Optogenetics has opened new possibilities in the remote control of diverse cellular functions with high spatiotemporal precision using light. However, delivering light to optically non-transparent systems remains a challenge. Here, we describe the photoactivation of light-oxygen-voltage-sensing domains (LOV domains) with in situ generated light from a chemiluminescence reaction between luminol and H2O2. This activation is possible due to the spectral overlap between the blue chemiluminescence emission and the absorption bands of the flavin chromophore in LOV domains. All four LOV domain proteins with diverse backgrounds and structures (iLID, BcLOV4, nMagHigh/pMagHigh, and VVDHigh) were photoactivated by chemiluminescence as demonstrated using a bead aggregation assay. The photoactivation with chemiluminescence required a critical light-output below which the LOV domains reversed back to their dark state with protein characteristic kinetics. Furthermore, spatially confined chemiluminescence produced inside giant unilamellar vesicles (GUVs) was able to photoactivate proteins both on the membrane and in solution, leading to the recruitment of the corresponding proteins to the GUV membrane. Finally, we showed that reactive oxygen species produced by neutrophil like cells can be converted into sufficient chemiluminescence to recruit the photoswitchable protein BcLOV4-mCherry from solution to the cell membrane. The findings highlight the utility of chemiluminescence as an endogenous light source for optogenetic applications, offering new possibilities for studying cellular processes in optically non-transparent systems.

Effect of membrane tension on antimicrobial peptide PGLa-induced pore formation in lipid bilayers

The osmotic pressure (Π) method has recently been developed to quantitatively examine the effect of membrane tension (σ) on pore formation in giant unilamellar vesicles (GUVs) induced by antimicrobial peptides (AMPs). Here, we used the Π method to reveal the effect of σ on the interaction of an AMP, PGLa, with lipid bilayers comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6). PGLa induced leakage of fluorescent probes from single GUVs under Π, indicating nanopore formation. Membrane tension did not transform a PGLa-induced nanopore into a micropore nor cause GUV burst up to 3.4 mN/m, which is in contrast with the effect of σ on another AMP, magainin 2-induced pore formation, where lower σ resulted in GUV burst. The fraction of leaking GUVs at a specific time increased with increasing σ, indicating that the rate of PGLa-induced pore formation increases with increasing σ. The rate of transfer of fluorescent probe-labeled PGLa across the lipid bilayer without pore formation also increased with increasing σ. PGLa-induced pore formation requires a symmetric distribution of peptides in both leaflets of the GUV bilayer, and thus we infer that the increase in the rate of PGLa transfer from the outer leaflet to the inner leaflet underlies the increase in the rate of pore formation with increasing σ. On the basis of these results, we discuss the difference between the effect of σ on nanopore formation in GUV membranes induced by PGLa and that by magainin 2.

Membrane Tubulation with a Biomembrane Force Probe.

Tubulation is a common cellular process involving the formation of membrane tubes ranging from 50 nm to 1 µm in diameter. These tubes facilitate intercompartmental connections, material transport within cells and content exchange between cells. The high curvature of these tubes makes them specific targets for proteins that sense local geometry. In vitro, similar tubes have been created by pulling on the membranes of giant unilamellar vesicles. Optical tweezers and micromanipulation are typically used in these experiments, involving the manipulation of a GUV with a micropipette and a streptavidin-coated bead trapped in optical tweezers. The interaction forms streptavidin/biotin bonds, leading to tube formation. Here, we propose a cost-effective alternative using only micromanipulation techniques, replacing optical tweezers with a Biomembrane Force Probe (BFP). The BFP, employing a biotinylated erythrocyte as a nanospring, allows for the controlled measurement of forces ranging from 1 pN to 1 nN. The BFP has been widely used to study molecular interactions in cellular processes, extending beyond its original purpose. We outline the experimental setup, tube formation and characterization of tube dimensions and energetics, and discuss the advantages and limitations of this approach in studying membrane tubulation.

CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs.

For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.

Effects of membrane potentials on the electroporation of giant unilamellar vesicles.

Living organisms maintain a resting membrane potential, which plays an important role in various biophysical and biological processes. In the context of medical applications, irreversible electroporation (IRE) is a non-thermal and minimally invasive technique that utilizes precisely controlled electric field pulses of micro- to millisecond durations to effectively ablate cancer and tumor cells. Previous studies on IRE-induced rupture of cell-mimetic giant unilamellar vesicles (GUVs) have primarily been conducted in the absence of membrane potentials. In this study, we investigated the electroporation of GUVs, including parameters such as the rate constant of rupture and the probability of rupture, in the presence of various negative membrane potentials. The membranes of GUVs were prepared using lipids and channel forming proteins. As the membrane potential increased from 0 to -90 mV, the rate constant of rupture showed a significant increase from (7.5 ± 1.6)×10-3 to (35.6 ± 5.5)×10-3 s-1. The corresponding probability of rupture also exhibited a notable increase from 0.40 ± 0.05 to 0.68 ± 0.05. To estimate the pore edge tension, the electric tension-dependent logarithm of the rate constant was fitted with the Arrhenius equation for different membrane potentials. The presence of membrane potential did not lead to any significant changes in the pore edge tension. The increase in electroporation is reasonably explained by the decrease in the prepore free energy barrier. The choice of buffer used in GUVs can significantly influence the kinetics of electroporation. This study provides valuable insights that can contribute to the application of electroporation techniques in the biomedical field.

Membrane inward/outward budding and transition pathway induced by the asymmetric solutions

The study of membrane morphology transitions is crucial for understanding various biological processes, such as cell division, and intracellular vesicle transport. In this work, we aim to clarify the molecular mechanism underlying the asymmetric solution-induced membrane shape transition in lipid bilayers. Using giant unilamellar vesicles (GUVs) as model membranes, we investigated the interaction of fully hydrated lipid membranes with two distinct sugar solutions—glucose and sucrose. We applied advanced imaging techniques and theoretical analyses to study membrane deformation and its transition pathways. Our experimental observations revealed that asymmetric sugar solutions induce spontaneous curvature in the phospholipid bilayer, resulting in the formation of outward(/inward) budding vesicles over a wide range of parameters. The direction of the curvature can be reversed by exchanging the internal and external solutions, indicating that the sugar-lipid interactions generate a significant positive(/negative) spontaneous curvature. Theoretically, we attempted to determine the parameter range of stable membrane shapes (i.e., phase diagram) using two dimensionless physical parameters—volume-to-area ratio and normalized spontaneous curvature. Obtaining a phase diagram helps identify the transition pathways of GUVs membrane shapes (experimentally, we captured two possible outward budding vesicles and one inward budding vesicle transition pathway). Our findings can be extended to other types of lipid membranes and provide new insights into the deformation of cell-like micron-sized vesicles, such as exosomes and endosomes, which are increasingly used as biomarkers and drug delivery systems. Moreover, lipid vesicles containing anticancer drugs exhibit fewer side effects than non-liposomal anticancer agents and can effectively target solid tumors.

DNA Origami Signaling Units Transduce Chemical and Mechanical Signals in Synthetic Cells

AbstractTransmembrane proteins transmit chemical signals as well as mechanical cues. The latter is often achieved by coupling to the cytoskeleton. The incorporation of fully engineerable membrane‐spanning structures for the transduction of chemical and, in particular, mechanical signals is therefore a critical aim for bottom‐up synthetic biology. Here, a membrane‐spanning DNA origami signaling units (DOSUs) is designed and mechanically coupled to DNA cytoskeletons encapsulated within giant unilamellar vesicles (GUVs). The incorporation of the DOSUs into the GUV membranes is verified and clustering upon external stimulation is achieved. Dye‐influx assays reveal that clustering increases the insertion efficiency. The transmembrane‐spanning DOSUs act as pores to allow for the transport of single‐stranded DNA into the GUVs. This is employed to trigger the reconfiguration of DNA cytoskeletons within GUVs. In addition to chemical signaling, mechanical coupling of the DOSUs to the internal DNA cytoskeletons is induced. With chemical cues from the environment, clustering of the DOSUs is induced, which triggers a symmetry break in the organization of the DNA cytoskeleton inside of the GUV. DNA‐based transmembrane structures are engineered that transduce signals without transporting the signaling molecule itself—providing a route toward signal processing and adaptive synthetic cells.

Bacterial Outer-Membrane-Mimicking Giant Unilamellar Vesicle Model for Detecting Antimicrobial Permeability.

The construction of bacterial outer membrane models with native lipids like lipopolysaccharide (LPS) is a barrier to understanding antimicrobial permeability at the membrane interface. Here, we engineer bacterial outer membrane (OM)-mimicking giant unilamellar vesicles (GUVs) by constituting LPS under different pH conditions and assembled GUVs with controlled dimensions. We quantify the LPS reconstituted in GUV membranes and reveal their arrangement in the leaflets of the vesicles. Importantly, we demonstrate the applications of OM vesicles by exploring antimicrobial permeability activity across membranes. Model peptides, melittin and magainin-2, are examined where both peptides exhibit lower membrane activity in OM vesicles than vesicles devoid of LPS. Our findings reveal the mode of action of antimicrobial peptides in bacterial-membrane-mimicking models. Notably, the critical peptide concentration required to elicit activity on model membranes correlates with the cell inhibitory concentrations that revalidate our models closely mimic bacterial membranes. In conclusion, we provide an OM-mimicking model capable of quantifying antimicrobial permeability across membranes.

Effect of monolayer spontaneous curvature on constant tension-induced pore formation in lipid bilayers.

The methodology of constant tension-induced rupture of giant unilamellar vesicles (GUVs) has provided information on tension-induced pore formation. This method was used to investigate the effect of spontaneous curvature (H0) for a lipid monolayer on the rate constant (kr) for constant tension (σ)-induced rupture, which originates from pore formation in lipid bilayers. Lipids were incorporated with different H0 values into GUV membranes to change the overall H0 value for the GUV monolayer. The dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylethanolamine (DOPE) (4/6, molar ratio, here and elsewhere) monolayer has a negative H0, whereas the DOPG/dioleoylphosphatidylcholine (DOPC) (4/6) monolayer has an essentially zero H0. A higher tension was required to induce the rupture of DOPG/DOPE (4/6)-GUVs compared with DOPG/DOPC (4/6)-GUVs. The line tension (Γ) for a pre-pore in DOPG/DOPE (4/6)-GUVs, determined by the analysis of the tension dependence of kr, was 1.5 times larger than that in DOPG/DOPC (4/6)-GUVs. The kr values for GUVs comprising DOPG/DOPC/18:1 lysophosphatidylcholine (LPC) (40/55/10), which has a positive H0, were larger than those for DOPG/DOPC (4/6)-GUVs under the same tension. The Γ value for DOPG/DOPC/LPC (40/55/10)-GUVs was almost half that for DOPG/DOPC (4/6)-GUVs. These results indicate that Γ decreases with increasing H0, which results in an increase in kr. Based on these results, the effect of H0 on kr and Γ is discussed.

Branched actin cortices reconstituted in vesicles sense membrane curvature

The actin cortex is a complex cytoskeletal machinery that drives and responds to changes in cell shape. It must generate or adapt to plasma membrane curvature to facilitate diverse functions such as cell division, migration, and phagocytosis. Due to the complex molecular makeup of the actin cortex, it remains unclear whether actin networks are inherently able to sense and generate membrane curvature, or whether they rely on their diverse binding partners to accomplish this. Here, we show that curvature sensing is an inherent capability of branched actin networks nucleated by Arp2/3 and VCA. We develop a robust method to encapsulate actin inside giant unilamellar vesicles (GUVs) and assemble an actin cortex at the inner surface of the GUV membrane. We show that actin forms a uniform and thin cortical layer when present at high concentration and distinct patches associated with negative membrane curvature at low concentration. Serendipitously, we find that the GUV production method also produces dumbbell-shaped GUVs, which we explain using mathematical modeling in terms of membrane hemifusion of nested GUVs. We find that branched actin networks preferentially assemble at the neck of the dumbbells, which possess a micrometer-range convex curvature comparable with the curvature of the actin patches found in spherical GUVs. Minimal branched actin networks can thus sense membrane curvature, which may help mammalian cells to robustly recruit actin to curved membranes to facilitate diverse cellular functions such as cytokinesis and migration.

Locally induced shockwaves for selective perforation of cargo loaded lipid vesicles with temporal and spatial control.

Controlled poration of lipid membranes is crucial for numerous biomimetic applications such as targeted drug delivery. Although several chemical and physical mechanisms have been proposed for the poration of synthetic membranes, achieving good temporal and spatial control remains a challenge. In this study, we introduce a novel method for membrane poration that utilizes the mechanical shockwave generated by the photo-acoustic effect, which occurs when an optically opaque microparticle is illuminated by a near-infrared laser of optical tweezers. We show that the shockwave effectively porates membranes of giant unilamellar vesicles in close proximity to the microparticle without damaging nearby cells, which is a desirable outcome for potential targeted drug delivery. The poration effect is nonspecific and operates on both liquid and gel phase membranes. Since the photo-acoustic effect can be triggered by standard optical tweezers, this method holds broad applicability in various experimental settings within the field of soft matter research.