Photoswitchable Endocytosis of Biomolecular Condensates in Giant Vesicles

Abstract Interactions between membranes and biomolecular condensates can give rise to complex phenomena such as wetting transitions, mutual remodeling, and endocytosis. In this study, light‐triggered manipulation of condensate engulfment is demonstrated using giant vesicles containing photoswitchable lipids. UV irradiation increases the membrane area, which can be stored in nanotubes. When in contact with a condensate droplet, the UV light triggers rapid condensate endocytosis, which can be reverted by blue light. The affinity of the protein‐rich condensates to the membrane and the reversibility of the engulfment processes is quantified from confocal microscopy images. The degree of photo‐induced engulfment, whether partial or complete, depends on the vesicle excess area and the relative sizes of vesicles and condensates. Theoretical estimates suggest that utilizing the light‐induced excess area to increase the vesicle‐condensate adhesion interface is energetically more favorable than the energy gain from folding the membrane into invaginations and tubes. The overall findings demonstrate that membrane‐condensate interactions can be easily and quickly modulated via light, providing a versatile system for building platforms to control cellular events and design intelligent drug delivery systems for cell repair.

. The scatter in the data are due to fact that the measurements were done using single confocal cross sections rather than 3D scans that would provide projections for the correct determination of the penetration depth and vesicle and condensate radii as explained in the manuscript.The two sets of data (n = 30) are linearly correlated with Pearson correlation coefficient of -0.57(the solid line is a guide to the eye).Two-tailed test of significance was used and the correlation is significant at the 0.05 level as assessed with Origin Pro software.These results demonstrate that the penetration depth values of protein condensates in GUVs under UV irradiation have a statistically significant correlation to     , thus revealing that the adhesion changes are not correlated to size differences of the interacting GUV-protein condensate pairs and that not all UV-induced excess area accumulates at the GUV-condensate interface.

Figure S2 .
Figure S2.Confocal microscopy images showing examples of photoswitchable endocytosis.GUVs composed of equimolar POPC and azo-PC and labelled with 0.1 mol% Atto-647N-DOPE.Glycinin condensates are labelled with 10 µM SRB.(a) The completely engulfed condensate is released after blue light exposure; see Movie S5 for duration of irradiation.(b) Intensity profiles (greenmembrane, magentacondensate) for the dashed line shown in (a) demonstrate that the droplet is fully wrapped by the membrane evidenced by the two peaks in the green profile.(c) The condensates indicated by the yellow arrows become engulfed after 1 s UV light exposure; see Movie S6 for duration of irradiation.(d) Intensity profiles for the dashed lines shown in (c).Scale bars are 5 µm.Further examples for complete engulfment are found in Fig S4.

Figure S6 .
Figure S6.Control experiments with pure POPC GUVs (labelled with 0.1 mol% Atto-647N-DOPE, green) in different irradiation conditions in the absence (a-d) and presence (e-h) of glycinin condensate (labelled with 10 µM SRB, magenta) demonstrating that UV and blue light irradiation do not induce any alterations in the GUV morphology and the protein-lipid interactions.The GUVs were exposed to UV and blue irradiation in (b, f) and (c, g), respectively.Initial illumination conditions of GUVs in the absence of extra UV or blue irradiation are stated as 'dark' in (a, e, d. h).The time stamps show the time in seconds after initiating acquisition.The UV light was turned on at 8 s in (a-d) and 5 s in (e-h), switched to blue at 27 s in (a-d) and 21 s in (e-h).The scale bars in (a) and (e) correspond to 40 µm.Exposing pure POPC GUVs to UV and blue illumination do not induce any changes in the morphology and the membrane area increase of the vesicles.

Figure S7 .
Figure S7.The excess area created upon UV irradiation in larger vesicles leads to higher penetration depth values and smaller condensates penetrate deeper.Alternatively, the larger the condensate is, the more membrane area is required to engulf it and as a result the penetration depth is smaller.To illustrate this, we plot the penetration depth, p, versus the area ratio of the condensate to GUV.The cartoons illustrate various scenarios.The graph displays all penetration depth data under UV light shown in Figure 4 in the main text as a function of the area ratio,   2   2 e. the increase in excess area resulting from UV irradiation in larger vesicles results in greater penetration depth values, allowing smaller condensates to penetrate more deeply.

Figure S8 :
Figure S8: Pictures of electrodeformation chambers before being mounted on a microscope: (a) Eppendorf electrofusion chamber and (b) home-built chamber for electrodeformation assembled from two coverslips sandwiching copper-tape electrodes and parafilm strips.(c) Function generator to apply AC field to induce electrodeformation of GUVs.The two different chambers were used to compare the effects of sample thickness because of concerns regarding the penetration of the UV light.

Figure S9 :
Figure S9: (a) Phase contrast images of a GUV composed of POPC:azo-PC (1:1) in AC field and UV irradiation, as indicated.The arrow indicates the direction of the field.The vesicle was first exposed to an AC field (5 kV m −1 and 1 MHz) to pull out thermal fluctuations and deform them into a prolate ellipsoid with semi-axes a and b.Then, while keeping the AC field on, UV irradiation (365 nm) was initiated.Scale bar is 10 µm.(b) Electrodeformation analysis of the GUV in (a).(c) Quantification of the UV-induced area increase of POPC and POPC:azo-PC (1:1) GUVs via vesicle electrodeformation.10-15 GUVs per condition were analyzed from 3 separate sets of experiments.Each open diamond represents the result of single GUV analysis while the solid circles and line bars are mean values and standard errors.Azo-PC containing GUVs showed ~18±2% of area increase under UV illumination.(d)UV/Vis spectra of large unilamellar vesicles made of azo-PC either in cis or trans state.Multiple irradiation cycles result in overlapping spectra suggesting full reversibility.For clarity, every 5 data point of the spectra in the 2 nd and 3 rd cycle are displayed.(e) Area increase as a result of azo-PC photoswitching is fully reversible as shown for the degree of deformation under UV and blue light (as indicated in the correspondingly shaded regions for the first cycle) for a vesicle containing 10 mol% azo-PC; data reproduced from[2].The GUV is continuously exposed to AC-field (5 kV.m-1 and 1 MHz).

Figure S10 .
Figure S10.(a) Confocal and (b) phase contrast images of 50 mol% azo-PC containing GUVs during electrodeformation analysis.Initial state of GUVs before the exposure to AC field and UV light is demonstrated on the left panels.The middle panels show the morphology of GUVs in AC field only.In the right panel, GUVs are exposed to UV illumination as well as AC field.In order to monitor vesicles in confocal microscopy, GUVs are labelled with 0.1 mol% Atto-647N-DOPE.The scale bars are 10 µm.Quantification of light-induced membrane area under confocal and phase contrast microscopy based on vesicle electrodeformation.(c)The deformation of the vesicles in AC-field and UV light are measured through the changes in the vesicle aspect ratio.Membrane area of the GUVs are calculated through the area of the ellipsoid.By subtracting the initial membrane area in AC field from the membrane area under the influence of both UV light and AC field, we could obtain the UV induced area increase in the membrane.In addition to different microscopes, the effects of usage of Eppendorf and home-made chambers are also compared.Based on the ANOVA and T-tests (p-values are 0.76 and 0.082, respectively), the differences between different methods are not significant.Each filled square and diamond symbol represents the analysis of a single GUV while filled circles and line bars are the mean values and standard deviation.The average area increase of vesicles under UV illumination shows no dependence on the microscope mode or the used experimental chamber.

Figure S11 . 2 ,
Figure S11.UV-induced GUV area increase versus adhered area increase of the glycinin condensates.GUVs composed of 1:1 POPC:Azo-PC and labelled with 0.1 mol% Atto-647N-DOPE.The adhered area changes detected through confocal screenshots in the absence and presence of UV irradiation were measured through Fiji software.The calculations were based on the spherical cap geometries.Each filled square is an individual data point generated from the pair of interacting GUV-condensate system in which a single vesicle interacted with a single condensate.The quadratic proportionality of the radius of the interacting condensate to the radius of the GUV,   2   2 , were indicated on top of the each datapoint.Red line corresponds to the y=x line representing the data points when all the excess area accumulates on the membrane-condensate interface.The data points are distributed above the y=x line and their distribution does not show any dependence on   2   2

Figure S12 .
Figure S12.Blue (a)  and UV (b) light induced shape transformations of 50 mol% azo-PC containing GUVs in low concentrations of sugar solution and in the absence of any salt asymmetry between internal and external GUV media.GUVs labeled with 0.1 mol% Atto-647N-DOPE were grown in 100 mM sucrose solution and 1:1 diluted to 105 mM glucose solution in absence of any salts.Upon UV irradiation (365 nm), the GUVs underwent complex shape transformations over time which were distinct from the internal tubulation events observed in the presence of high salt asymmetry like in Figure1cin the main text.The time stamps of changing UV-induced morphological transitions of GUVs are shown in the upper part of the images.The scale bar is same for all these confocal images and corresponds to 10 µm.
Release of a completely engulfed condensate in blue light.The GUV composed of POPC:Azo-PC 1:1 and labelled with 0.1 mol% Atto-647N-DOPE were sequentially exposed to UV and blue light.Glycinin condensate was labelled with 10 µM SRB.The time stamps show the time in seconds after initiating acquisition.The periods of irradiation of the sample are indicated on the upper part of the video.The scale bar is 5 µm.Movie S6: Engulfment of multiple condensates in UV light.The GUV composed of POPC:Azo-PC 1:1 and labelled with 0.1 mol% Atto-647N-DOPE was exposed to UV light.Glycinin condensates were labelled with 10 µM SRB.The time stamps show the time in seconds after initiating acquisition.The periods of irradiation of the sample are indicated on the upper part of the video.The scale bar is 5 µm.The video was processed with LAS X and Fiji software.Movie S7: Condensate partial engulfment when exposed to UV light, displaying a large change in the contact angle between the condensate and the membrane.The GUV composed of POPC:Azo-PC 1:1 and labelled with 0.1 mol% Atto-647N-DOPE was exposed to UV light.Glycinin condensate was labelled with 10 µM SRB.The time stamps show the time in seconds after initiating acquisition.The periods of irradiation of the sample are indicated on the upper part of the video.The scale bar is 5 µm.Movie S8: Condensate partial engulfment in UV light.The GUV composed of POPC:Azo-PC 1:1 and labelled with 0.1 mol% Atto-647N-DOPE was exposed to UV light.Glycinin condensate was labelled with 10 µM SRB.The time stamps show the time in seconds after initiating acquisition.The periods of irradiation of the sample are indicated on the upper part of the video.The scale bar is 5 µm.

Table S1 .
Full width at half maximum (FWHM) input values in cm −1 and their physically plausible ranges expected for each type of secondary structure as measured with ATR-FTIR.[1]