Area Increase and Budding in Giant Vesicles Triggered by Light: Behind the Scene

Abstract Biomembranes are constantly remodeled and in cells, these processes are controlled and modulated by an assortment of membrane proteins. Here, it is shown that such remodeling can also be induced by photoresponsive molecules. The morphological control of giant vesicles in the presence of a water‐soluble ortho‐tetrafluoroazobenzene photoswitch (F‐azo) is demonstrated and it is shown that the shape transformations are based on an increase in membrane area and generation of spontaneous curvature. The vesicles exhibit budding and the buds can be retracted by using light of a different wavelength. In the presence of F‐azo, the membrane area can increase by more than 5% as assessed from vesicle electrodeformation. To elucidate the underlying molecular mechanism and the partitioning of F‐azo in the membrane, molecular dynamics simulations are employed. Comparison with theoretically calculated shapes reveals that the budded shapes are governed by curvature elasticity, that the spontaneous curvature can be decomposed into a local and a nonlocal contribution, and that the local spontaneous curvature is about 1/(2.5 µm). The results show that exo‐ and endocytotic events can be controlled by light and that these photoinduced processes provide an attractive method to change membrane area and morphology.


S1.2. Photoisomerization of F-azo
UV/Vis absorption spectra were recorded using quartz cuvettes on a Cary 50 spectrophotometer equipped with a Peltier-thermostated cell holder (temperature accuracy ±0.1 K). The solvents used were of spectrophotometric grade. Irradiation experiments were performed using a LOT-Oriel 1000 W mediumpressure Xe/Hg lamp equipped with band-pass filters.

2:
A solution of 1 [1] (  was then dissolved in dry THF and 60% w/w NaH/mineral oil (0.17 mM, 6 mg) was added at 0°C. The resulting mixture was allowed to come back to room temperature and stirred for 1 h. Hexane was then added, and the precipitate was filtered and dried to give F-azo (72 mg, 81%) as an orange solid. 1 Figure S3. Spectrum of a 100 W high pressure mercury lamp. The intensity at 365 nm (the used UV range at which F-azo isomerizes to the cis form) is much stronger than the intensity at 488 nm (the visible range where trans-to-cis F-azo isomerization takes place). The power intensity at 546 nm measured above the objective was 0.85 mW/mm 2 . It was used to estimate the power intensity at 365 nm according to the spectrum intensity. Figure S4. Time-lapse of F-azo-free GUV. A quasi-spherical vesicle was exposed to UV light in the absence of F-azo. The UV irradiation started at the 7th second. The vesicle does not undergo visible morphological changes for ~50 s of UV irradiation. The scale bar corresponds to 10 µm. Figure S5. Size distribution of F-azo aggregates in filtered, 0.25 mM F-azo solution measured by DLS (see section 11.3 for details on measurement).

S5. Elution profile of LUVs and F-azo molecules.
The   Figure. S7. Deformation of a vesicle in the presence of electric field and in the absence of F-azo. Snapshots show the dynamic of the electrodeformation. The AC field was switched on at 0 s (snapshot B) and continued for ~35 s (C and D). The graph shows the apparent aspect ratio as a function of time. The GUV reaches its maximum deformation within the first ~7 s (C). The scale bar corresponds to 10 µm.

S7. Reversible budding in the presence of an AC field.
The vesicle in Fig. S10 expels buds during UV light irradiation (snapshot B). The buds are reabsorbed by the vesicle upon blue light irradiation (snapshots C and D). The budding process repeats, when the vesicle S7 is irradiated with UV light for a second time (snapshot E). An AC field (1 MHz, 5 Vpp) was constantly applied. The graph shows the apparent aspect ratio of the vesicle during the process. After the budding event the vesicle deformation decreases (B and E). During the blue light irradiation, the deformation reaches its previous level due to the reabsorption of the buds. Note that the gaps in the curve, after the first UV light irradiation and before the second one, are due to the change of the filters for blue and UV light.

S10. Spontaneous curvature of outward budding GUVs
The membrane area A of a vesicle defines the vesicle size ≡ √ /(4 ) .
(1) In general, for a smooth membrane surface the mean curvature is where C1 and C2 are the principle curvatures.

Curvature elasticity according to the spontaneous curvature model:
In this model, the bending energy of any smooth shape is given by (3) which depends only on two parameters, the bending rigidity and the spontaneous curvature . The notation { } is used to indicate that the bending energy represents a functional of the shape.

Curvature elasticity according to the area-difference-elasticity (ADE) model:
The ADE model is defined by the energy functional (4) with the local energy term ℰ defined in Eq. 3 corresponding to the spontaneous curvature model and the nonlocal area-difference-elasticity term where Δ { } represents the area difference between the two leaflets of the vesicle with shape S, ℐ { } ≡ ∫ (6) is the integrated mean curvature, κ′ the nonlocal bending rigidity and ,0 = ∫ 1 = 4 represents the integrated mean curvature which characterizes the relaxed vesicle shape with an optimal packing of the molecules in both leaflets.

Effective spontaneous curvature:
Since the shapes that minimize the energy functional (4)

Neck closure condition for a spherical out-bud:
Consider a vesicle with a spherical out-bud where the mother and the bud radii are and , respectively. The closing neck (between the mother vesicle and the out-bud) is characterized by the Therefore, we can obtain the local spontaneous curvature , if we observe the geometry of the vesicle during neck closure and use the relation Sphere to out-but transition curves

S11.2. Vesicle preparation
Giant unilamellar vesicles were grown using the electroformation method. [2] In Before the experiments, the LUVs were diluted 5 × in a 0.1 M sucrose solution.

S11.3. Dynamic light scattering (DLS) of F-azo samples and LUVs
The size distribution of F-azo aggregates and the extruded LUVs were determined at 25°C with a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK), operating with a 4 mW HeNe laser (632.8 nm), a detector positioned at the scattering angle of 173°, and a temperature-control jacket for the cuvette.
Aliquot of 1 ml F-azo with concentration of 0.25 mM was degassed for 10 min with ThermoVac (MicroCal, MA). Three DLS measurements consisting of 20 runs with duration of 5 s were performed.
DLS measurements were performed also on degassed LUV suspensions to determine the vesicle size.

S12. F-azo aggregates
In aqueous solutions, F-azo aggregates due to its limited water solubility. Crystal-like structures were observed when using higher concentrated (> 0.5 mM) F-azo solutions (Fig. S3). In order to exclude these S12 crystal-like structures we filtered the F-azo stock solution (through a filter with pore size: 0.22 µm). The latter was stable and the crystal-like structures were not observed after filtration. We were concerned that the total concentration of F-azo molecules might have been reduced during the filtration step. We measured the absorbance at 320 nm of both unfiltered F-azo (in the concentration range of 0-0.1 mM) and filtered F-azo (for two concentrations: 0.025 mM and 0.05 mM). Then the extinction coefficients of the filtered and unfiltered F-azo (for 0.025 mM and 0.05 mM) were compared. The concentration of the filtered F-azo was ~ 3% lower than the one of the unfiltered F-azo solutions. This small concentration difference was considered in the calculation of the concentration of the stock F-azo solution.

S13. Aggregation in filtered F-azo solutions under UV light irradiation
During prolonged (~ 2 min) UV light irradiation, the F-azo molecules start form aggregates of a few μm in size (Fig. S4). Note that during our experiments, the samples were irradiated with UV light for less than a minute, which is below the threshold of the appearing of the aggregates.