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Light-Controlled Membrane Mechanics and Shape Transitions of Photoswitchable Lipid Vesicles Carla Pernpeintner, James Allen Frank, Patrick Urban, Christian Roman Roeske, Stefanie Pritzl, Dirk Trauner, and Theobald Lohmueller Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01020 • Publication Date (Web): 31 Mar 2017 Downloaded from http://pubs.acs.org on April 2, 2017
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Light-Controlled Membrane Mechanics and Shape Transitions of Photoswitchable Lipid Vesicles Carla Pernpeintner†,§,#, James A. Frank‡,#, Patrick Urban†, Christian R. Roeske†, Stefanie Pritzl†, Dirk Trauner‡,§,*, and Theobald Lohmüller†,§,* †
Photonics and Optoelectronics Group, Department of Physics and CeNS, Ludwig Maximilians University Munich, München, Amalienstraße 54, 80799 Munich, Germany
‡
Department of Chemistry and Center for Integrated Protein Science, Ludwig Maximilians University Munich, Butenandtstraße 5-13, 81377 Munich, Germany
§
Nanosystems Initiative Munich, Schellingstraße 4, 80799 Munich, Germany
ABSTRACT: Giant unilamellar vesicles (GUVs) represent a versatile model system to emulate the fundamental properties and functions associated with the plasma membrane of living cells. Deformability and shape transitions of lipid vesicles are closely linked to the mechanical properties of the bilayer membrane itself, and are typically difficult to control under physiological conditions. Here, we developed a protocol to form cell-sized vesicles from an azobenzene-containing phosphatidylcholine (azoPC), that undergoes photoisomerization on irradiation with UV-A and visible light. Photoswitching within the photolipid vesicles enabled rapid and precise control of the mechanical properties of the membrane. By varying the intensity and dynamics of the optical stimulus, controlled vesicle shape changes such as budding transitions, invagination, pearling, or the formation of membrane tubes was achieved. With this system, we could mimic the morphology changes normally seen in cells, in the absence of any molecular machines associated with the cytoskeleton. Furthermore, we devised a mechanism to utilize photoswitchable lipid membranes for storing mechanical energy, and then releasing it on command as locally usable work.
INTRODUCTION. Cellular membranes can undergo complex morphological changes, such as budding1-2, invagination2, tube formation3, pearling4, or division5, that are fundamental for the orchestration of organic life. The plasma membrane consists of a fluid bilayer of lipid molecules and membrane components that display a high level of lateral mobility6. From a biochemical perspective, altering the cell shape or triggering membrane deformations involves the reorganization of the lipid molecules and membrane proteins7. Many cellular processes, however, are also closely tied to mechanical factors and force regulation8. Here, the mechanical properties of the plasma membrane itself may play a pivotal role in controlling cellular shape transitions and cell morphologies that determine biological functions. It has been shown that certain bacterial cells are able to divide without the existence of a protein cell division machinery9. Instead, these bacteria utilize a mechanism that involves the formation of membrane protrusions and a subsequent resolution of the cell body into smaller daughter cells. These events, that involve membrane budding and self-reproduction of cellular vesicles seemingly without the requirement of active force generation, might represent an early milestone in the evolution of cellular life. In light of these findings, the question arises how cellular shape transformations such as budding, invagination, division
or fusion, could be initiated and regulated in response to a mechanical stimulus of the plasma membrane alone10. Giant unilamellar vesicles (GUVs)11 made of phospholipid molecules are a widely used model system to mimic the shape and outer plasma membrane of living cells12-13. When exposed to physical stimuli such as force, changes in temperature, and pressure14-16, they display complex shape transitions that resemble the shape changes observed in living cells17. Building on a comprehensive theoretical framework, the shape dynamics of GUVs can be controlled by varying the area-tovolume ratio of the vesicles, or by chemically inducing membrane bending moments18-19. Experimentally, the regulation of temperature20 or osmotic conditions21 provides efficient control of vesicle shape. Nanoparticle adhesion22 or the application of electromagnetic force fields23-26 have also been used. However, these methods lack reversibility, physiological compatibility or the possibility to control vesicle shape in a spatiotemporally defined manner. Yuan et al.21 have suggested in a theoretical model that not only the intensity, but also the time dependent rate change of a stimulus could be a possible regulation factor for vesicle shape. To date, this theory could not be confirmed experimentally, as it would require a quick and homogeneous method for manipulating membranes under tightly controlled experimental conditions.
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Here, we report an approach to stimulate vesicles with light using photoswitchable lipid molecules that can undergo a fast and reversible change in their molecular configuration upon spectrally defined illumination. Photoswitchable amphiphilic azobenzene derivatives have been used to control the permeability of synthetic bilayer membranes27-29. Yet, so far only a few proof-of-principle studies have investigated the possibility to observe shape transformations of vesicles that were doped with custom-designed photoswitchable amphiphiles30-33. Importantly, for these studies, the photosensitive part of the molecules was often located at the hydrophilic end of the bilayer membrane, and was not embedded within the hydrophobic center. In addition, phospholipid molecules, in which one or both of the acyl chains were functionalized with an azobenzene group, have been synthesized, and their optical properties and photoreactivity has been investigated34. However, the formation of lipid bilayer membranes and vesicles composed of entirely photoswitchable lipids has not yet been reported. In this work, we took these investigations a step further and prepared GUVs from 100% photoswitchable lipids. We could reversibly control the photostationary states (cis and trans) of the lipids and hence, the mechanics of the entire vesicle. The immediate effect of photoswitching on membrane deformability and bending rigidity was characterized using optical tweezers and flow measurements. We observed that the bending rigidity of GUV membranes could be quickly and reversibly tuned by almost two orders of magnitude, depending on the illumination intensity. Based on these findings, we devised a mechanism to utilize photoswitchable lipid membranes for storing energy and then releasing it as locally usable work, an effect that is controlled solely by light.
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Merck. Synthetic details and spectroscopic characterization of the compounds can be found in the Supporting Information. Preparation of small unilamellar azo-PC vesicles (azo-PC SUVs). We dissolved azo-PC in CHCl3 (Sigma-Aldrich Chemie GmbH, Munich/Germany) at a final concentration of 6.36 mM. We use a protocol based on tip sonification35 for the formation of small unilamellar vesicles. 100 µL of the azo-PC lipid solution was diluted with CHCl3 to a final volume of 1 mL. The solvent was then removed under reduced pressure, depositing layers of lipids on the wall of the round bottom flask. The flask was filled with 2 mL ddH2O. The flask was then briefly sonicated (Elma Schmidbauer GmbH, Singen/Germany) until the solution appeared milky, and was then transferred to a centrifuge tube. Next, tip sonication (BANDELIN electronic GmbH & Co. KG, Berlin/Germany) was performed for two times 30 s on ice. Finally, the sample was centrifuged for 10 min at 10,000 rpm. The supernatant was stored at 4 °C until further use. Preparation of giant unilamellar azo-PC vesicles (azo-PC GUVs). For the formation of GUVs, we adapted the electroformation method36-37 using a homebuilt electroformation chamber. Two Pt wires, 3 mm apart, were run through nine reaction wells with a volume of 1.5 mL each. A few µL of the 6.36 mM lipid solution was pipetted onto the wires. After evaporation of the CHCl3, the wells were filled with 1.5 mL of a 300 mM sucrose solution or 1 M sorbitol solution. Before starting the electroformation protocol, the chamber was sealed and heated to 70 °C. Then, an actuated electric field (5 Hz, 180 min) was applied to the wires with a function generator. The solution containing GUVs was stored at 4 °C until further use.
EXPERIMENTAL. Compound synthesis. All reagents and solvents were purchased from commercial sources (Sigma-Aldrich, TCI Europe N.V., Strem Chemicals, etc.) and were used without further purification unless otherwise noted. Tetrahydrofuran (THF) was distilled under a N2 atmosphere from Na/benzophenone prior to use. Triethylamine (NEt3), was distilled under a N2 atmosphere from CaH2 prior to use. Further dry solvents such as ethyl acetate (EtOAc), benzene (PhH), dichloromethane (CH2Cl2), toluene (PhMe), ethanol (EtOH), acetonitrile (MeCN) and methanol (MeOH) were purchased from Acros Organics as "extra dry" reagents and used as received. Solvents were degassed by sparging the freshly distilled solvent with argon gas in a Schlenk flask under ultra-sonication using a Bandelin Sonorex RK510H ultra-sonic bath for 20 min prior to use. Reactions were monitored by TLC on pre-coated, Merck Silica gel 60 F254 glass-backed plates and the chromatograms were first visualized by UV irradiation at 254 nm, followed by staining with aqueous ninhydrin, anisaldehyde or ceric ammonium molybdate solution (CAM), and finally gentle heating with a heat gun. Flash silica gel chromatography was performed using silica gel (SiO2, particle size 40-63 µm) purchased from
UV-A illumination. For UV-A illumination throughout all experiments, we used a fiber coupled LED (λ = 365 nm, Prismatix, Israel) with a maximum total output power of 70 mW measured at the end of the fiber. Typical power densities used varied between 0.1-5 kW/cm². In Figure 1B and 3B, a λ = 465 nm LED was used to isomerize the molecules back to the trans-state. Optical spectroscopy. For optical spectroscopy experiments, the liquid samples were kept in Quartz cuvettes. UV/Vis spectra of azo-PC in CHCl3 were obtained with a Cary 60 UVVis spectrometer (Agilent Technologies). Illumination periods with λ = 365 nm or 465 nm light were > 30 s, to guarantee that either the UV-A- or blue-adapted photostationary states were reached. The latter was controlled by recording spectra after different illumination periods. The dynamic light scattering data shown in Figure 3B was obtained using a Zetasizer Nano Series ‘L’ instrument (Malvern, Worcestershire/UK). Laser microscopy setup. Microscopy and optical tweezer experiments were performed with a custom-built laser
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microscopy setup based on an Axiotech 100 microscope (ZEISS, Oberkochen/Germany). For dark-field microscopy, a dark-field condenser (NA 1,2/1,4) was used in combination with a water immersion objective (100x or 63x). For image acquisition, we used a Canon 6D digital camera. For optical tweezer experiments, a near-infrared laser (λ = 1064 nm; Cobolt RumbaTM, Cobolt AB, Stockholm/Sweden) was guided through the 100x water immersion objective. The net laser power directly below the objective was 120–240 mW. A piezo motor (Controller: CU30, LINOS Photonics) controlled by a LabVIEW procedure was used to move the stage. Bending rigidity measurements. In order to determine the bending rigidity for the closed surface of a vesicle, which is closely related to shape, stability, strength, and structural phases38, we used the expression for curvature energy developed by Helfrich (1973)39
̅ ⋅
(1)
yet neglecting spontaneous and Gaussian curvatures and vesicle stretching. Without the application of an external force, any vesicle appears in the shape of a sphere with constant, size-independent curvature energy 8 4̅ . Considering the application of the work W on a spherical membrane that causes a membrane bending, results in a difference in curvature energy following equation (1) and thus leads to
(2)
For a controlled vesicle deformation, we use a piezo stage to induce a microfluidic flow (0 – 60 µm/s), while at the same time holding the vesicle in a defined position with optical tweezers. The procedure was recorded at a video framerate of 30-60 fps. The resulting change in the vesicle’s shape projection was measured and fitted to an ellipse, while stepwise varying the stage speed. The length of these steps was chosen so that the vesicle evolved into its new equilibrium shape (static case), which was also considered for the determination of the resulting shape. In order to verify that vesicle stretching did not take place, we measured the circumference of each vesicle. For high illumination intensities and flow speeds, an increase of the circumference was observed, in the other cases no significant increase could be found. Hence, in these cases equation (2) was applicable. To determine W, we simulated the total force acting on the vesicles (simplified as prolate spheroids) for a given flow speed and vesicle shape in the respective equilibrium case. The simulations were done by numerically solving the NavierStokes equations (finite element method) using the software COMSOL Multiphysics. Due to small lengths and velocities (Re
