Reversible Social Self-Sorting of Colloidal Cell-Mimics with Blue Light

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Reversible Social Self-Sorting of Colloidal CellMimics with Blue Light Switchable Proteins Elizaveta Chervyachkova, and Seraphine Valeska Wegner ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00250 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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ACS Synthetic Biology

Reversible Social Self-Sorting of Colloidal Cell-Mimics with Blue Light Switchable Proteins Elizaveta Chervyachkovaa and Seraphine V. Wegnera* a

Max Planck Institute for Polymer Research, Mainz, 55128, Germany

* [email protected]

KEYWORDS Self-assembly, social self-sorting, photoswitchable proteins, reversibility, heterodimerization

ABSTRACT Towards the bottom-up assembly of synthetic cells from molecular building blocks it is an ongoing challenge to assemble micrometer sized compartments that host different processes into precise multicompartmental assemblies, also called prototissues. The difficulty lies in controlling interactions between different compartments dynamically both in space and time, as these interactions determine how they organize with respect to each other and how they work together. In this study, we have been able to control the self-assembly and social selfsorting of four different types of colloids, which we use as a model for synthetic cells, into two separate families with visible light. For this purpose we used two photoswitchable protein pairs (iLID/Nano and nHagHigh/pMagHigh) that both reversibly heterodimerize upon blue light exposure and dissociate from each other in the dark. These photoswitchable proteins provide non-invasive, dynamic and reversible remote control under biocompatible conditions over the self-assembly process with unprecedented spatial and temporal precision. In addition, each protein pair brings together specifically two different types of colloids. The orthogonality of the two protein pairs enables social self-sorting of a four component mixture into two distinct families of colloidal aggregates with controlled arrangements. These results

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will ultimately pave the way for the bottom-up assembly of multicompartment synthetic prototissues of a higher complexity, enabling us to control precisely and dynamically the organization of different compartments in space and time.

In the search to produce a minimal synthetic cell from non-living components, many compartments that mimic real life processes are being developed.1-3 It has become evident that one compartment can only house a few reactions without interference, causing a major bottleneck.4-5 Nature has solved this problem during evolution by placing incompatible processes into organelles within a cell and by forming multicellular organisms from cells with different capabilities.6-7 These evolutionary developments have greatly increased the complexity of life and the tasks that cells can perform. The reason behind these breakthroughs is the controlled interaction between compartments (organelles, vesicles or cells), which in turn led to the self-assembly of multicompartmental architectures such as tissues. Fundamental features of these interactions include the precise control in space and time needed to define the exchange between compartments, high specificity needed to operate in complex mixtures, and reversibility needed to enable dynamic adjustment to the environment. Similarly, it is a worthwhile challenge to find a way of precisely modulating interactions between different minimal synthetic cells to guide their self-assembly into prototissues and protocell communities with complex behavior. 8-9 Concepts established in colloidal self-assembly into larger structures are an important guide for the self-assembly of prototissues from minimal synthetic cells based on GUVs, polymersomes,

proteinosomes and coacervates,

which are all micrometer sized

compartments.9-13 Colloids are highly suitable models that can be used not only to study the self-assembly of nano- and micrometer sized objects into higher order structures such as but not limited to minimal synthetic cells.14-15 Various external triggers, including temperature,


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pH and light, have been used to remote control the self-assembly of colloids. Colloids decorated with thermo-responsive peptides16-17 and polymers18 as well as complementary DNA strands19-25 can be reversibly self-assembled in a temperature dependent fashion. DNAbased strategies, in particular, allow for specific assemblies with great diversity on the nanoand micrometer scale. Nonetheless, temperature responsive assemblies lack the high spatial and temporal control found in nature and are limited by high transition temperatures. Light is an attractive stimulus to remote control systems since the delivery of light is noninvasive, different wavelengths can be used to trigger orthogonal reactions and it provides the greatest

spatiotemporal resolution.26 Light-induced self-assembly of colloids and

nanoparticles is possible based on the light-induced changes in polarity of azobenzenes27-28 or spiropyrans,26, 29-30 dimerization of coumarins,31 cleavage of nitrobenzenes32 and host-guest interactions between azobenzenes/cyclodextrines33-35 or methyl viologen/curcubit[8]urils.36-38 Some of these light-induced self-assemblies are also reversible and rely on specific interactions that are orthogonal to others, acquiring other key features of self-assembly found in nature. However, all of these self-assembly processes require the use of UV light and are mostly formed in organic solvents, making them incompatible with biological molecules.14, 2631, 33-38

What is currently missing is a way to reversibly self-assemble colloids with spatial and

temporal control into higher order structures with non-invasive visible light and under biocompatible conditions. In biology it is not enough for cells to self-assemble, they also have to self-sort into precise arrangements in order to work together. In model colloidal mixtures the self-sorting can be divided up according to the level of sorting complexity. 15, 26, 33, 39-41 In indiscriminate sorting all types of colloids self-assemble into one aggregate, which just requires non-selective interactions (e.g. electrostatic interactions). In asocial (narcissistic) sorting each type of colloid assembles into isolated aggregates, which require self-complementary and orthogonal


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interactions. In social self-sorting pairs of colloids, so called families, form separate coaggregates, which require complementary and orthogonal interactions. For example, Walther and coworkers demonstrated the social-sorting into two families of colloids based on the orthogonal interactions of α-cyclodextrin with azobenzenes and β-cyclodextrin with ferrocene.33 Social self-sorting enables the formation of order in complex mixtures of colloids and hence will also enable the assembly of minimal synthetic cells into prototissues with high complexity. In this work, we aim to achieve the social self-sorting between two families of colloids as models to be used for minimal synthetic cells with non-invasive visible light under biocompatible conditions. We aim to capture the fundamental features of interactions between cells that are observed in nature. More specifically, these interactions should be precisely controlled in space and time, orthogonal to other interactions and reversible. For this purpose we will use two photoswitchable proteins that specifically bind to their interaction partners under blue light illumination and dissociate from these partners in the dark. A number of such photoswitchable proteins have recently been developed in the field of optogenetics to control various processes (e.g. gene activation, protein localization, enzyme activity, protein clustering) inside the cell using visible light.42-46 In this study, the first protein we use is called iLID (an engineered version of the LOV2 (light-oxygen-voltage-sensing) domain from Avena sativa, with an incorporated SsrA peptide), which reversibly binds to Nano (wild-type SspB peptide) upon blue light illumination.44, 47 The second blue light sensitive protein pair used in this study, nMagHigh and pMagHigh, has been derived from the fungal photoreceptor Vivid (VVD) from Neurospora crassa, and also specifically heterodimerizes with each other under blue light.42, 48

These protein interactions respond to low intensity blue light, which is non-invasive, they

are reversible in the dark and operate under biocompatible conditions. Our hypothesis is that the orthogonality and high specificity of these protein pairs will enable the social self-sorting


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of micrometer-sized cell-mimics on demand in a four component mixture under blue light control.

RESULTS AND DISCUSSION Blue light-dependent self-assembly based on photoswitchable proteins. Two different blue light-dependent interaction pairs, iLID/Nano and nMagHigh/pMagHigh, have been previously used to control various processes with light in cells.44, 48-49 However, they have not yet been used to mediate interactions in synthetic systems in general, or to induce lightdependent self-assembly of particles in particular. Therefore, we began by investigating whether each of these two different blue light sensitive protein pairs could independently induce aggregation of colloids (Figure 1a). To do this, we immobilized separately on 2 µm Ni2+-NTA functionalized polystyrene beads (Figure S1) each of the purified proteins through their His-tags (Figure S3). There were approximately 200 proteins per bead, which were estimated by using a His-tagged green fluorescent protein (His-GFP) as a model protein (Figure S2a). In fluorescence-activated cell sorting (FACS) experiments, there was a single population of His-GFP functionalized beads with a significantly higher fluorescence signal compared to unfunctionalized beads, demonstrating a homogenous functionalization of the colloids (Figure S2b). Subsequently, we mixed bead populations with complementary interaction partners in equal proportions and incubated them either in the dark or under blue light (460 nm). Both bead mixtures with immobilized iLID and Nano (Figure 1b, Figure S4a) as well as nMagHigh and pMagHigh (Figure 1c, Figure S4b) aggregate significantly more under blue light than in the dark. Yet, the two blue light sensitive protein pairs form differently shaped clusters under blue light illumination. The clusters based on the iLID/Nano protein dimerization are very compact indicating a reaction limited aggregation (Figure 1b, Figure S4a). This is further supported by the broad distribution in cluster sizes (Figure S5c).


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On the other hand, the nMagHigh/pMagHigh clusters are loosely packed aggregates (Figure 1c, Figure S4b) and the cluster size distribution is narrow (Figure S5d), showing a diffusion limited aggregation. An explanation for this difference in cluster shape is the difference in the reaction kinetics of these two protein pairs. The iLID/Nano protein interaction has fast reversion kinetics (half-life < 30 seconds), which allows the beads to rearrange in the cluster and pack tighter. On the contrary, the nMagHigh/pMagHigh interaction has very slow reversion kinetics (half-life ≈ 4.8 hours) and thus clusters form in a diffusion limited manner. 44, 48

All these observations demonstrated that the blue light-dependent interaction between

iLID and Nano as well as nMagHigh and pMagHigh were sufficiently strong to mediate light-dependent bead self-assembly but can lead to different cluster structures. Figure 1. Blue light-dependent self-assembly of beads. (a) iLID (or nMagHigh)

a Blue

Dark

b Blue

Dark

c Blue

Dark

functionalized beads (blue) and Nano (or pMagHigh) functionalized beads (green) selfassemble under blue light due to the blue light-dependent heterodimerization of these proteins. (b) Bright field images of a mixture of iLID and Nano functionalized 2 µm polystyrene beads in the dark and under blue light. (c) Bright field images of a mixture of


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nMagHigh and pMagHigh functionalized 2 µm polystyrene beads in the dark and under blue light. The scale bar is 25 µm. Reversibility of blue light-dependent self-assembly. Self-assembly in nature is highly dynamic and the relative arrangement of compartments changes over time. Given this, it is important to understand how the dynamics and reversibility work in synthetic systems. The protein pairs that we were using here reversibly dissociate from each other in the dark, which should, in theory, also lead to the disassembly of the light-induced bead aggregates in the dark. To investigate the light-dependent self-assembly and its reversibility, we quantified the aggregation behavior of bead mixtures with both the iLID/Nano pair and the nMagHigh/pMagHigh pair under low agitation (50 rpm) to prevent beads from settling. We compared the aggregation of beads kept in the dark or under blue light for 2 hours with beads that were first kept under blue light for 2 hours and then placed in the dark for 1 hour (reversed). Given these conditions, we calculated the aggregation ratio as the area of clusters (aggregates with a projected area bigger than 10 beads), divided by the total area of all beads in the same bright field image (Figure 2a-b).50 First of all, in the case of both photoswitchable protein pairs the aggregation ratios were higher under blue light than in the dark. For example, for the iLID/Nano pair the aggregation ratio increased from 27 % in the dark to 37 % under blue light, and for the nMagHigh/pMagHigh pair the aggregation ratio increased from 30 % in the dark to 42 % under blue light. Secondly, light-dependent aggregation for both systems proved reversible in the dark as in the reversed samples the aggregation ratio decreased upon withdrawal of the blue light. For instance, for the iLID/Nano pair the aggregation ratio decreased from 37 % under blue light to 29 % in the reversed sample, which is comparable to the dark value of 27 %. For the nMagHigh/pMagHigh pair, the aggregation ratio went down from 42 % under blue light to 32% in the reversed sample. In parallel, the number of clusters as well as their average size are also higher under blue light


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illumination than in the dark and in the reversed samples (Figure S5a-d). Attempts to analyze the bead aggregation with FACS failed. We suspect that this is due to high shear forces during the measurements, which lead to the disruption of the aggregates. Moreover, the blue light-dependent reversible self-assembly mediated by these two blue light switchable protein pairs exhibit comparable aggregation behavior.

a

b

Figure 2. Light-induced reversible aggregation of the beads functionalized with: (a) iLID/Nano and (b) nMagHigh/pMagHigh. Samples kept in the dark are depicted in grey; samples kept under blue light are depicted in blue; samples that were reversed are depicted in green. One-Way ANOVA test (significance level 0.05) was performed to analyze the statistical difference followed by Dunn-Sidak post hoc test (significance level 0.05). Error bars are the standard error from > 60 images.


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Self-assembly and disassembly kinetics. When the protein pairs iLID and Nano as well as nMagHigh and pMagHigh are used to control protein interactions in the cell, they interact with each other within a few minutes when exposed to blue light.44,

48

However, the

immobilization of the proteins on a bead and the self-assembly itself can lead to different dynamics.51 When we observe the aggregation dynamics, in the case of the iLID/Nano pair, the aggregation ratio differed only by 2 % in dark and light conditions after 30 minutes (Figure 3a). With longer blue light illumination of up to 2 hours the aggregation ratio increased to 37 % and the difference between dark and light samples became more prominent (10

%).

Likewise,

similar

self-assembly

dynamics

were

observed

for

the

nMagHigh/pMagHigh pair (Figure 3b). While there was no significant difference between the dark and light samples after 30 minutes of blue light exposure, aggregation ratio increased to 42 % after 2 hours of blue light illumination and was 12 % higher compared to the dark sample. Moreover, we could observe that for both protein pairs that aggregation reaches a plateau after 2 hours of illumination with blue light. The extent of bead aggregation also depends on the blue light intensity (Figure S6). The blue light intensity of 0.4 mW/cm2 used in all the experiments is sufficient for both protein pairs to achieve maximal aggregation but already at 0.3 mW/cm2 the blue light dependent aggregation decreases. These light intensities are very low and not toxic to living systems. Overall, these results demonstrate that we can modulate the degree of self-assembly by adjusting the illumination time or light intensity and that the bead aggregation and not the photoswitching of the proteins is the rate limiting step. Comparing the aggregation kinetics of protein functionalized beads in the dark and under blue light to unfunctionalized beads gives insight into the driving forces behind the selfassembly (Figure 3a-b). First of all, the beads aggregate to a certain level (aggregation ratio

≈ 17 %) as soon as they are functionalized with proteins. This is probably due to unspecific


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interactions between the proteins since it is independent of the type of protein on the bead (Figure 3a-b). Secondly, the unfunctionalized beads do not significantly aggregate and remain dispersed in buffer over time, demonstrating their colloidal stability and that the observed aggregation is due to the protein functionalization. Thirdly, the iLID/Nano and nMagHigh/pMagHigh functionalized beads also aggregate to some extend in the dark over 2 hours, although much less and slower than under blue light. This is expected as both iLID and Nano as well as nMagHigh and pMagHigh have also some affinity for each other in the dark, which increases under blue light illumination 36- and 17- fold, respectively.44, 48 The addition of 0.2 % (w/v) BSA or 0.2 % (w/v) Pluronic® F-127 to the bead mixtures in order to block unspecific interactions completely suppress any aggregation (Figure S7). Figure 3. Aggregation dynamics of: a) iLID/Nano and b) nMagHigh/pMagHigh a

b


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functionalized beads. Samples kept in the dark are depicted in black; samples kept under blue light are depicted in blue; unfunctionalized beads are depicted in orange. Error bars are the standard error from > 30 images.

Beside the aggregation dynamics under blue light, we also investigated the kinetics of the disassembly in the dark afterwards and the repeated switchability of these interactions. For the reversibility studies, a mixture of iLID/Nano or nMagHigh/pMagHigh beads was first aggregated under blue light for 2 hours before placing them into the dark. For both protein pairs it takes 30 minutes in the dark to reverse the aggregation by ≈ 80 % (Figure S8a-b). The blue-light dependent interactions between these proteins and hence the aggregation of the beads can be switched on and off multiple times in blue light/dark cycles. For example, for the iLID/Nano pair the aggregation ratio changed form 36 % to 22% to 42% and back to 28% in two cycles of 1 hour under blue light and 30 minutes in the dark (Figure 4, black). Likewise, the beads with the nMagHigh/pMagHigh pair also switched their aggregation state in repeated blue light/dark cycles (Figure 4, red). Hence, both of the blue light-dependent protein systems can be used to reversibly and repeatedly achieve self-assembly of beads.


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Figure 4. Switching cycles of bead aggregation for mixtures of iLID/Nano (black) and nMagHigh/pMagHigh (red) functionalized beads. Periods when samples were kept under blue light are marked in blue; periods when samples were kept in the dark are marked in grey. Error bars are the standard error from 30 images.

Light-dependent self-assembly is based on specific heterodimerization. To achieve social self-sorting it is important that self-assembly is based on the specific heterodimerization and not unspecific homodimerization. In order to distinguish which of the two interaction modes is responsible for the blue light-dependent self-assembly, we immobilized complementary proteins on green (Nano or pMagHigh) and red (iLID or nMagHigh) fluorescently labeled polystyrene beads through the His-tag-Ni2+-NTA interaction and mixed them under blue light. Red and green labeled beads strongly intermixed in the formed clusters of both photoswitchable protein pairs, demonstrating that heterodimerization is responsible for the blue light-dependent self-assembly (Figure 5a, Figure S9a). Additionally, we quantified the blue light-dependent self-assembly of beads that were functionalized with only one protein at a time (Figure S9 b-e). In all cases the aggregation ratio of the samples kept under blue light was comparable to the aggregation ratio in the dark and there was no blue light-dependent bead aggregation. Another way to show that the aggregation is due to the specific interaction between the proteins under blue light is to add excess protein to the solution and block binding sites. Indeed, when we add 1µM iLID or Nano protein to the iLID/Nano bead mixture and 1µM nMagHigh or pMagHigh to the nMagHigh/pMagHigh bead mixture, we do not observe light dependent aggregation anymore (Figure S10). Therefore, we concluded that the blue light-dependent self-assembly results from the specific heterodimerization of the protein pairs and is not due to the unspecific homodimerization.


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Stoichiometry controlled self-assembly. One of the advantages of social self-sorting over simpler sorting modes is the ability to control the cluster sizes by adjusting the stoichiometry of the building blocks.33 To demonstrate this, we mixed red fluorescent beads functionalized with iLID (or nMagHigh) with green fluorescent beads functionalized with Nano (or pMagHigh) in 1:5 and 5:1 ratios. In all cases, we could observe a corona of 5 to 8 beads of the prevalent bead type surrounding the complementary bead type (Figure 5b-c). Hence, different stoichiometries between the compartments can be used to control the cluster size and their respective arrangement.

Figure 5. Confocal fluorescence microscopy images. (a) 3D clusters formed under blue light with iLID (red) and Nano (green) functionalized beads; different arrangements of (b) iLID


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(red) and Nano (green), (c) nMagHigh (red) and pMagHigh (green) mixed in 1:5 and 5:1 ratios under blue light. The scale bars are 5 µm.

Social self-sorting in a four component mixture. The ultimate goal of this work was to achieve social self-sorting triggered by non-invasive blue light under biocompatible conditions. Social self-sorting is the highest level of self-sorting complexity and requires two orthogonal heterodimerization partners on the colloids, which sort into two families of aggregates.33 To demonstrate that the two photoswitchable protein pairs meet the criteria required for social self-sorting, we began by mixing the four different bead types under blue light for 2 hours (Figure 6a). iLID and Nano, which are expected to sort into one family, were each immobilized on red fluorescent beads while nMagHigh and pMagHigh, which are expected to sort into the second family, were each immobilized on green fluorescent beads. In this four component mixture under blue light, we observed discrete clusters of only red or green beads, but not intermixed clusters (Figure 6b). Subsequently, when these samples were placed in the dark for 1 hour, the clusters disassembled and the social-sorting reversed (Figure 6c). Also, the quantification of these samples shows that the aggregation ratio is much higher after 2 hours under blue light illumination (32%) compared to the dark (16%) and decreases back to 20% upon withdrawal of the blue light for 1 hour (Figure 6b-c). We further confirmed the formation of separate clusters containing only one family of beads (red: iLID/Nano, green: nMagHigh/pMagHigh) in 3D cross-section images (Figure 6d). Overall, these two blue light switchable protein pairs proved to be highly specific and orthogonal to each other. Thus, these interactions can be used as the basis to achieve the social self-sorting of four different compartments into two discrete families with blue light under biocompatible conditions.


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a

Blue Dark

b

iLID

nMagHigh

Nano

pMagHigh

Dark

Blue

Blue

c

Reversed

Dark

d

Figure 6. Light-induced reversible social self-sorting. (a) A schematic representation of the social self-sorting of four components using two blue light sensitive protein pairs. (b) 2D fluorescence microscopy images of 4 different beads that reversibly sort into two families. iLID and Nano, were each immobilized on red fluorescent beads and nMagHigh and pMagHigh, were each immobilized on green fluorescent beads. The scale bars are 25 µm. (c) Social sorting aggregation and reversion analysis: samples kept in the dark for 2 hours are depicted in grey; samples kept under blue light for 2 hours are depicted in blue; samples that were reversed for 1 hour in the dark after 2 hours under blue light are depicted in green. OneWay ANOVA test (significance level 0.05) was performed to analyze the statistical


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difference followed by Dunn-Sidak post hoc test (significance level 0.05). Error bars are the standard error from 15 images. (d) Fluorescence microscopy images of the cuts through the exemplary 3D clusters. Left in red is a cluster formed by the iLID/Nano protein pair; right in green is a cluster formed by the nMagHigh/pMagHigh protein pair. The scale bars are 5 µm.

CONCLUSIONS During this study, we employed two pairs of photoswitchable protein interactions as building blocks in order to control social self-sorting with blue light between four different types of colloids, which serve as models for minimal synthetic cells. This was only possible because the iLID/Nano and the nMagHigh/pMagHigh interactions are orthogonal to each other. In addition, these interactions were able to overcome the limitations of current approaches in colloidal self-assembly as they provide high spatiotemporal control, are reversible and respond to biocompatible low intensities of blue light in physiological buffers. To transfer this concept to the assembly of prototissues these photoswitchable proteins can be immobilized on the surface of different compartments such as GUVs, polymersomes and proteinosomes. One possibility for the protein immobilization is also here used interaction between Ni2+-NTA groups on the compartment and His-tags on the proteins. This would allow assembling different minimal synthetic cells that house various life mimetic processes into prototissues and protocell communities with high spatiotemporal precision and complex social sorting behavior. The approach described here can be expanded further as there is a wide variety of photoswitchable proteins that respond to light of different wavelengths that have different interaction modes to achieve self-sorting in even more complex and multicomponent mixtures.43,

46

This makes photoswitchable proteins an important building

block in the synthetic biology toolbox used to assemble minimal synthetic cells that house different functionalities. Ultimately, integrating photoswitchable protein interactions on


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minimal synthetic cells can be used to assemble different types of compartments into multicompartment consortia to mimic more complex processes that occur in nature.

EXPERIMENTAL METHODS Plasmids. pQE-80L iLID (C530M) and pQE-80L MBP-SspB Nano were gifts from Brian Kuhlman (Addgene plasmids # 60408 and # 60409 respectively). The sequence for nMagHigh was synthesized by the GeneScript and inserted into pET21b plasmid between the NdeI and Xho1 cutting sites to include a C-terminal His6-tag. The pMagHigh plasmid was obtained through site-directed mutagenesis of nMagHigh (D52R, G55R).48 pET His6 GFP TEV LIC cloning vector (1GFP) was a gift from Scott Gradia (Addgene plasmid # 29663). Protein expression and purification. Each plasmid was transformed into E. coli BL21 (DE3) and a 10 ml overnight culture was grown in LB medium with 50 µg/ml ampicillin at 37 °C at 200 rpm. The culture was then transferred into a 1 L LB medium with 50 µg/ml ampicillin and grown at 37 °C at 200 rpm until the OD600=0.4-0.6, before inducing protein expression with 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside). The culture was incubated at 16 °C overnight while continuously being shaken at 200 rpm. The pelleted bacteria (6000 rpm, 4 °C, 8 minutes) were resuspended in Buffer A (300 mM NaCl, 50 mM Tris, pH=7.4) and lysed by sonication. The lysate was spun down (12000 rpm, 4 °C, 20 minutes) and the supernatant was purified using an Ni2+-NTA affinity column. The protein purity was verified by SDS-PAGE (Figure S3a). Protein immobilization on the beads. The Ni2+-NTA functionalized polystyrene beads with a 2 µm diameter with and without fluorescent labels were purchased from Micromod Partikeltechnologie GmbH as a water suspension (50 mg/ml, 1.2x1010 beads/ml, stable in aqueous solutions, methanol, ethanol and DMSO). Fluorescein (excitation/emission: 485/510 nm) and rhodamine B (excitation/emission: 572/590 nm) derivatives were used as labels to


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obtain green and red labeled beads, respectively. The proteins were immobilized on the beads through His-tag-Ni2+-NTA interaction by incubating 5 mg/ml of Ni2+-NTA-functionalized beads in Buffer A with 1 µM His-tagged protein at 4 °C for 1 hour. Then the excess protein was washed away by twice spinning the beads down (13000 rpm, 2 minutes) and resuspending them in the Buffer A. Before each experiment the protein functionalized beads were sonicated for 1 minute to disperse them. The number of His-tagged proteins per bead was estimated by incubating an excess of 1 µM His6-tagged GFP (green fluorescent protein) with 5 mg/ml beads for 1 hour at 4 °C. After spinning down the beads the fluorescence of the remaining in the supernatant GFP was measured using the microplate reader and compared to a calibration curve. The number of His6-tagged GFPs was calculated to be ≈ 200 proteins per bead (Figure S2a). The functionalization efficiency was estimated using fluorescence-activated cell sorting (FACS). For this an excess of 5 µM His6-tagged GPF was incubated with 5 mg/ml beads for 1 hour at 4 °C followed by two washings with Buffer A. The samples were then diluted 1:500 in Buffer A and analysed with Attune NxT Acoustic Focusing Cytometer (Figure S2b). Bead fluorescence was compared to the fluorescence of an unfunctionalized beads at the same dilution. Bead aggregation assay. 25 µl (5 mg/ml) of beads functionalized with one of the complementary interaction partners (iLID and Nano or nMagHigh and pMagHigh) were mixed and diluted to a total volume of 300 µl in Buffer A. In the homodimerization experiments 50 µl (5 mg/ml) of protein functionalized beads were used to keep the bead concentration constant.

The samples were either kept in the dark or under blue light

(460 nm) for 2 hours in LoBind Eppendorf tubes, while being gently agitated at 50 rpm in an orbital shaker. For the reversibility experiments the samples were first incubated under blue light for 2 hours and then kept in the dark for 1 hour. Subsequently, the samples were


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removed from the shaker, fixed with 300 µl of 10 % (w/v) paraformaldehyde (PFA) for 20 minutes, 300 µl of the sample were transferred into an imaging chamber (Lab-tek) and allowed to settle for 30 minutes before acquiring 15 bright field images (250x250 µm) for each sample through a 40x air objective. To study the aggregation dynamics, the samples were kept in the orbital shaker at 50 rpm under blue light or in the dark for 30 minutes, 1 hour or 2 hours before fixation, transfer into the imaging chambers and imaging as described above. For t=0 time point the samples were fixed immediately after mixing in an Eppendorf tube. For the reversion dynamics study, the samples were first kept under the blue light for 2 hours on the orbital shaker at 50 rpm. Then the samples were moved into the dark for 15 minutes, 30 minutes or 1 hour before fixation and imaging as described above. The effect of the buffer salt concentration on the bead aggregation was studied by preparing the samples in a buffer with a lower salt concentration (low salt buffer: 100 mM NaCl, 10 mM Tris, pH 7.4) (Figure S11). Light sources and light intensity dependence. A LED light panel (Albrillo LL-GL003, 225 LEDs, 460 nm, 14 W) was used for the blue light illumination. White polycarbonate plates with 30 % transmission (from alt-intech) were used as neutral density filters to control the light intensity. Light intensity dependence was studied by keeping the samples under the blue light LED panel with 1, 2 (as in all other experiments) or 3 neutral density filters (Figure S6a-b). Imaging. All images were either acquired on an inverted fluorescent microscope (DMi8, Leica) through the 40x air objective, or on a confocal laser scanning fluorescence microscope (SP5 Leica) through the 63x water objective. Data analysis. All the images were analyzed using ImageJ software. First, the intensity threshold was applied to the images to select the beads in the acquired images. Then the area


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occupied by bead clusters (> 10 beads) and all beads (including single beads, small clusters (10 beads)) in an image was measured using the analyze particle tool in ImageJ. From this analysis the aggregation ratio was calculated by dividing the area occupied by the clusters by the area occupied by all the beads in the same image.50 The number of clusters and their sizes were also quantified using the analyze particles tool. The data was plotted and statistically analyzed (either Mann-Whitney test or One-Way ANOVA test) using OriginLab. Stoichiometry experiments. iLID or nMagHigh were immobilized on red fluorescent beads (5 mg/ml) and Nano or pMagHigh were immobilized on green fluorescent beads (5 mg/ml) as described above. 5 µl of one bead type were mixed with 25 µl of the bead type with the complementary interaction partner (1:5 or 5:1 ratios) and diluted to the total volume of 300 µl in Buffer A. The mixture was incubated at 25 rpm for 2 hours directly in the imaging chamber. Afterwards, the samples were fixed and fluorescent images were acquired in the FITC and TRITC channels. Social self-sorting experiments. iLID and Nano were each immobilized on red fluorescent beads and nMagHigh and pMagHigh were each immobilized on green fluorescent beads as described above. Then all four types of beads were mixed together in equal proportions (5 mg/ml, 12.5 µl each, total volume 300 µl). The samples were kept in the dark/under blue light for 2 hours or first for 2 hours under the blue light and then for 1 hour in the dark (reversed) at 50 rpm agitation directly in the imaging chamber before being fixed with 10 % (w/v) PFA and imaged. Supporting Information: Exemplary bright field images of unfunctionalized beads and protein functionalized beads (iLID/Nano or nMagHigh/pMagHigh) in the dark, under blue light and reversed, analysis of protein functionalization for beads with fluorescence and FACS, SDS-PAGE gel images of purified proteins, cluster number and size analysis,


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aggregation analysis in dependence of light intensity, salt concentrations and for homo interactions, reversion dynamics and confocal images of nMagHigh/pMagHigh clusters, aggregation analysis in presence of blocking agents and in presence of soluble protein interaction partners. AUTHOR CONTRIBUTIONS E.C. and S.V.W. designed experiments, E.C. performed experiments, E.C. and S.V.W analyzed the data and wrote the manuscript. ACKNOWLEDGMENTS This work is part of the MaxSynBio consortium, which is jointly funded by the Federal Ministry of Education and Research (BMBF) of Germany (FKZ 031A359L) and the Max Planck Society (MPG). E.C. would like to thank the MPG for a doctoral fellowship. The pQE-80L iLID (C530M) (Addgene plasmid # 60408) and pQE-80L MBP-SspB Nano (Addgene plasmid # 60409) plasmids were gifts from Brian Kuhlman. The pET His6 GFP TEV LIC cloning vector (1GFP) was a gift from Scott Gradia (Addgene plasmid # 29663). Our thanks go to Simon Bretschneider for the light intensity measurements, and to Stefan Schumacher for the illustrations.

REFERENCES 1. Dzieciol, A. J.; Mann, S., Designs for life: protocell models in the laboratory. Chem. Soc. Rev. 2012, 41, 79-85. 2. Mann, S., Life as a nanoscale phenomenon. Angew. Chem. Int. Ed. 2008, 47, 53065320. 3. Xu, C.; Hu, S.; Chen, X., Artificial cells: from basic science to applications. Mater. Today 2016, 19, 516-532. 4. Adamala, K. P.; Martin-Alarcon, D. A.; Guthrie-Honea, K. R.; Boyden, E. S., Engineering genetic circuit interactions within and between synthetic minimal cells. Nat. Chem. 2017, 9, 431-439. 5. Weiss, M.; Frohnmayer, J. P.; Benk, L. T.; Haller, B.; Janiesch, J. W.; Heitkamp, T.; Borsch, M.; Lira, R. B.; Dimova, R.; Lipowsky, R.; Bodenschatz, E.; Baret, J. C.; VidakovicKoch, T.; Sundmacher, K.; Platzman, I.; Spatz, J. P., Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics. Nat. Mater. 2018, 17, 89-96. 6. Deng, N. N.; Yelleswarapu, M.; Zheng, L.; Huck, W. T. S., Microfluidic Assembly of Monodisperse Vesosomes as Artificial Cell Models. J. Am. Chem. Soc. 2017, 139, 587-590.


21


Page 22 of 25

7. Grosberg, R. K.; Strathmann, R. R., The Evolution of Multicellularity: A Minor Major Transition? Annu. Rev. Ecol. Evol. Syst. 2007, 38, 621-654. 8. Mantri, S.; Sapra, K. T., Evolving protocells to prototissues: rational design of a missing link. Biochem. Soc. Trans. 2013, 41, 1159-65. 9. Qiao, Y.; Li, M.; Booth, R.; Mann, S., Predatory behaviour in synthetic protocell communities. Nat. Chem. 2017, 9, 110-119. 10. Dewey, D. C.; Strulson, C. A.; Cacace, D. N.; Bevilacqua, P. C.; Keating, C. D., Bioreactor droplets from liposome-stabilized all-aqueous emulsions. Nat. Commun. 2014, 5, 4670. 11. Tang, T. D.; Cecchi, D.; Fracasso, G.; Accardi, D.; Coutable-Pennarun, A.; Mansy, S. S.; Perriman, A. W.; Anderson, J. L. R.; Mann, S., Gene-Mediated Chemical Communication in Synthetic Protocell Communities. ACS Synth. Biol. 2018, 7, 339-346. 12. Huang, X.; Li, M.; Green, D. C.; Williams, D. S.; Patil, A. J.; Mann, S., Interfacial assembly of protein-polymer nano-conjugates into stimulus-responsive biomimetic protocells. Nat. Commun. 2013, 4, 2239. 13. Villringer, S.; Madl, J.; Sych, T.; Manner, C.; Imberty, A.; Romer, W., Lectinmediated protocell crosslinking to mimic cell-cell junctions and adhesion. Sci. Rep. 2018, 8, 1932. 14. Li, F.; Josephson, D. P.; Stein, A., Colloidal assembly: the road from particles to colloidal molecules and crystals. Angew. Chem. Int. Ed. 2011, 50, 360-388. 15. Merindol, R.; Walther, A., Materials learning from life: concepts for active, adaptive and autonomous molecular systems. Chem. Soc. Rev. 2017, 46, 5588-5619. 16. Higashi, N.; Ochiai, T.; Kanazawa, C.; Koga, T., Site-specific adsorption of gold nanoparticles coated with thermo-responsive peptides. Polym. J. 2012, 45, 523-528. 17. Schoen, A. P.; Hommersom, B.; Heilshorn, S. C.; Leunissen, M. E., Tuning colloidal association with specific peptide interactions. Soft Matter 2013, 9, 6781-6785. 18. Hamner, K. L.; Maye, M. M., Thermal aggregation properties of nanoparticles modified with temperature sensitive copolymers. Langmuir 2013, 29, 15217-15223. 19. Di Michele, L.; Eiser, E., Developments in understanding and controlling self assembly of DNA-functionalized colloids. Phys. Chem. Chem. Phys. 2013, 15, 3115-3129. 20. Di Michele, L.; Varrato, F.; Kotar, J.; Nathan, S. H.; Foffi, G.; Eiser, E., Multistep kinetic self-assembly of DNA-coated colloids. Nat. Commun. 2013, 4, 1-7. 21. Macfarlane, R. J.; Lee, B.; Jones, M. R.; Harris, N.; Schatz, G. C.; Mirkin, C. A., Nanoparticle Superlattice Engineering with DNA. Science 2011, 334, 204-208. 22. Valignat, M. P.; Theodoly, O.; Crocker, J. C.; Russel, W. B.; Chaikin, P. M., Reversible self-assembly and directed assembly of DNA-linked micrometer-sized colloids. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4225-4229. 23. Van der Meulen, S. A.; Leunissen, M. E., Solid colloids with surface-mobile DNA linkers. J. Am. Chem. Soc. 2013, 135, 15129-15134. 24. Wang, Y.; Wang, Y.; Zheng, X.; Ducrot, E.; Lee, M. G.; Yi, G. R.; Weck, M.; Pine, D. J., Synthetic Strategies Toward DNA-Coated Colloids that Crystallize. J. Am. Chem. Soc. 2015, 137, 10760-10766. 25. Wang, Y.; Wang, Y.; Zheng, X.; Ducrot, E.; Yodh, J. S.; Weck, M.; Pine, D. J., Crystallization of DNA-coated colloids. Nat. Commun. 2015, 6, 7253-7261. 26. Kundu, P. K.; Samanta, D.; Leizrowice, R.; Margulis, B.; Zhao, H.; Borner, M.; Udayabhaskararao, T.; Manna, D.; Klajn, R., Light-controlled self-assembly of nonphotoresponsive nanoparticles. Nat. Chem. 2015, 7, 646-652. 27. Klajn, R.; Bishop, K. J. M.; Grzybowski, B. A., Light-controlled self-assembly of reversible and irreversible nanoparticle suprastructures. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10305-10309.


22

Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


28. Manna, D.; Udayabhaskararao, T.; Zhao, H.; Klajn, R., Orthogonal light-induced selfassembly of nanoparticles using differently substituted azobenzenes. Angew. Chem. Int. Ed. 2015, 54, 12394-12397. 29. Zhang, L.; Dai, L.; Rong, Y.; Liu, Z.; Tong, D.; Huang, Y.; Chen, T., Light-triggered reversible self-assembly of gold nanoparticle oligomers for tunable SERS. Langmuir 2015, 31, 1164-1171. 30. Zhang, Q.; Dong, R.; Chang, X.; Ren, B.; Tong, Z., Spiropyran-Decorated SiO(2)-Pt Janus Micromotor: Preparation and Light-Induced Dynamic Self-Assembly and Disassembly. ACS Appl. Mater. Interfaces 2015, 7, 24585-24591. 31. He, H.; Feng, M.; Chen, Q., ; ; Zhang, X.; Zhan, H., Light-Induced Reversible SelfAssembly of Gold Nanoparticles Surface-Immobilized with Coumarin Ligands. Angew. Chem. Int. Ed. 2016, 55, 936-940. 32. Li, S.; Moosa, B. A.; Croissant, J. G.; Khashab, N. M., Electrostatic assembly/disassembly of nanoscaled colloidosomes for light-triggered cargo release. Angew. Chem. Int. Ed. 2015, 54, 6804-6808. 33. Han, K.; Go, D.; Tigges, T.; Rahimi, K.; Kuehne, A. J. C.; Walther, A., Social SelfSorting of Colloidal Families in Co-Assembling Microgel Systems. Angew. Chem. Int. Ed. 2017, 56, 2176-2182. 34. Han, K.; Go, D.; Hoenders, D.; Kuehne, A. J. C.; Walther, A., Switchable Supracolloidal Coassembly of Microgels Mediated by Host/Guest Interactions. ACS Macro Lett. 2017, 6, 310-314. 35. Zhou, Y.; Wang, D.; Huang, S.; Auernhammer, G.; He, Y.; Butt, H. J.; Wu, S., Reversible Janus particle assembly via responsive host-guest interactions. Chem. Commun. 2015, 51, 2725-2727. 36. Lan, Y.; Wu, Y.; Karas, A.; Scherman, O. A., Photoresponsive hybrid raspberry-like colloids based on cucurbit[8]uril host-guest interactions. Angew. Chem. Int. Ed. 2014, 53, 2166-2169. 37. Stoffelen, C.; Voskuhl, J.; Jonkheijm, P.; Huskens, J., Dual stimuli-responsive selfassembled supramolecular nanoparticles. Angew. Chem. Int. Ed. 2014, 53, 3400-3404. 38. Zhang, Q.; Qu, D. H.; Wang, Q. C.; Tian, H., Dual-Mode Controlled Self-Assembly of TiO2 Nanoparticles Through a Cucurbit[8]uril-Enhanced Radical Cation Dimerization Interaction. Angew. Chem. Int. Ed. 2015, 54 (52), 15789-15793. 39. Morris, K. L.; Chen, L.; Raeburn, J.; Sellick, O. R.; Cotanda, P.; Paul, A.; Griffiths, P. C.; King, S. M.; O'Reilly, R. K.; Serpell, L. C.; Adams, D. J., Chemically programmed selfsorting of gelator networks. Nat. Commun. 2013, 4, 1480-1486. 40. Mukhopadhyay, P.; Wu, A.; Isaacs, L., Social Self-Sorting in Aqueous Solution. J. Org. Chem. 2004, 69, 6157-6164. 41. Vilanova, N.; Feijter, I.; Teunissen, A. J. P.; Voets, I. K., Light induced assembly and self-sorting of silica microparticles. Sci. Rep. 2018, 8, 1271. 42. Furuya, A.; Kawano, F.; Nakajima, T.; Ueda, Y.; Sato, M., Assembly Domain-Based Optogenetic System for the Efficient Control of Cellular Signaling. ACS Synth. Biol. 2017, 6, 1086-1095. 43. Guglielmi, G.; Falk, H. J.; De Renzis, S., Optogenetic Control of Protein Function: From Intracellular Processes to Tissue Morphogenesis. Trends Cell. Biol. 2016, 26, 864-874. 44. Guntas, G.; Hallett, R. A.; Zimmerman, S. P.; Williams, T.; Yumerefendi, H.; Bear, J. E.; Kuhlman, B., Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 112117. 45. Levskaya, A.; Weiner, O. D.; Lim, W. A.; Voigt, C. A., Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 2009, 461, 997-1001.


23


Page 24 of 25

46. Muller, K.; Weber, W., Optogenetic tools for mammalian systems. Mol. Biosyst. 2013, 9, 596-608. 47. Lungu, O. I.; Hallett, R. A.; Choi, E. J.; Aiken, M. J.; Hahn, K. M.; Kuhlman, B., Designing photoswitchable peptides using the AsLOV2 domain. Chem. Biol. 2012, 19, 507517. 48. Kawano, F.; Suzuki, H.; Furuya, A.; Sato, M., Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat. Commun. 2015, 6, 6256-6264. 49. Yu, G.; Onodera, H.; Aono, Y.; Kawano, F.; Ueda, Y.; Furuya, A.; Suzuki, H.; Sato, M., Optical manipulation of the alpha subunits of heterotrimeric G proteins using photoswitchable dimerization systems. Sci. Rep. 2016, 6, 35777-35786. 50. Chavda, A. P.; Prole, D. L.; Taylor, C. W., A bead aggregation assay for detection of low-affinity protein-protein interactions reveals interactions between N-terminal domains of inositol 1,4,5-trisphosphate receptors. PLoS One 2013, 8, e60609. 51. Bartelt, S. M.; Chervyachkova, E.; Steinkuhler, J.; Ricken, J.; Wieneke, R.; Tampe, R.; Dimova, R.; Wegner, S. V., Dynamic blue light-switchable protein patterns on giant unilamellar vesicles. Chem. Commun. (Camb.) 2018, 54, 948-951.


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For Table of Contents Use Only: Reversible Social Self-Sorting of Colloidal Cell-Mimics with Blue Light Switchable Proteins Elizaveta Chervyachkovaa and Seraphine V. Wegnera*


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Reversible Social Self-Sorting of Colloidal Cell-Mimics with Blue Light

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