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Protein photolithography is an invaluable tool for generating protein microchips and regulating interactions between cells and materials. However, the absence of light-responsive molecules that allow for the copatterning of multiple functional proteins with biocompatible visible light poses a significant challenge. Here, a new approach for photopatterning three distinct proteins on a single surface by using green, red, and far-red light is reported. The cofactor of the green light-sensitive protein CarH is engineered such that it also becomes sensitive to red and far-red light. These new cofactors are shown to be compatible with two CarH-based optogenetic tools to regulate bacterial cell-cell adhesions and gene expression in mammalian cells with red and far-red light. Further, by incorporating different CarH variants with varying light sensitivities in layer-by-layer (LbL) multiprotein films, specific layers within the films, along with other protein layers on top are precisely removed by using different colors of light, all with high spatiotemporal accuracy. Notably, with these three distinct colors of visible light, it is possible to incorporate diverse proteins under mild conditions in LbL films based on the reliable interaction between Ni 2+ - nitrilotriacetic acid (NTA) groups and polyhistidine-tags (His-tags)on the proteins and their subsequent photopatterning. This approach has potential applications spanning biofabrication, material engineering, and biotechnology., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

In recent years, cancer immunotherapy has undergone a transformative shift toward personalized and targeted therapeutic strategies. Bacteria-derived outer membrane vesicles (OMVs) have emerged as a promising and adaptable platform for cancer immunotherapy due to their unique properties, including natural immunogenicity and the ability to be engineered for specific therapeutic purposes. In this review, a comprehensive overview is provided of state-of-the-art techniques and methodologies employed in the engineering of versatile OMVs for cancer immunotherapy. Beginning by exploring the biogenesis and composition of OMVs, unveiling their intrinsic immunogenic properties for therapeutic appeal. Subsequently, innovative approaches employed to engineer OMVs are delved into, ranging from the genetic engineering of parent bacteria to the incorporation of functional molecules. The importance of rational design strategies is highlighted to enhance the immunogenicity and specificity of OMVs, allowing tailoring for diverse cancer types. Furthermore, insights into clinical studies and potential challenges utilizing OMVs as cancer vaccines or adjuvants are also provided, offering a comprehensive assessment of the current landscape and future prospects. Overall, this review provides valuable insights for researchers involved in the rapidly evolving field of cancer immunotherapy, offering a roadmap for harnessing the full potential of OMVs as a versatile and adaptable platform for cancer treatment., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Cholesterol is an important lipid playing a crucial role in mediating essential cellular processes as well as maintaining the basic structural integrity of biological membranes. Given its vast biological importance, there is an unabated need for sophisticated strategies to investigate cholesterol-mediated biological processes. Raman-tagged sterol analogs offer the advantage of being visualizable without the need for a bulky dye that potentially affects natural membrane integration and cellular interactions as it is the case for many conventionally used fluorescent analogs. Herein, we report a series of alkyne-tagged imidazolium-based cholesterol analogs (CHIMs) with large Raman scattering cross-sections that readily integrate into HEK cells and primary monocyte-derived macrophages and allow (multiplexed) cellular Raman imaging. We envision Raman-tagged CHIM analogs to be a powerful platform for the investigation of cholesterol-mediated cellular processes complementary to other established methods, such as the use of fluorescent analogs., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The transition of bacteria from an individualistic to a biofilm lifestyle profoundly alters their biology. During biofilm development, the bacterial cell-cell adhesions are a major determinant of initial microcolonies, which serve as kernels for the subsequent microscopic and mesoscopic structure of the biofilm, and determine the resulting functionality. In this study, the significance of bacterial cell-cell adhesion dynamics on bacterial aggregation and biofilm maturation is elucidated. Using photoswitchable adhesins between bacteria, modifying the dynamics of bacterial cell-cell adhesions with periodic dark-light cycles is systematic. Dynamic cell-cell adhesions with liquid-like behavior improve bacterial aggregation and produce more compact microcolonies than static adhesions with solid-like behavior in both experiments and individual-based simulations. Consequently, dynamic cell-cell adhesions give rise to earlier quorum sensing activation, better intermixing of different bacterial populations, improved biofilm maturation, changes in the growth of cocultures, and higher yields in fermentation. The here presented approach of tuning bacterial cell-cell adhesion dynamics opens the door for regulating the structure and function of biofilms and cocultures with potential biotechnological applications., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

The cell-cell adhesion molecule E-cadherin has been intensively studied due to its prevalence in tissue function and its spatiotemporal regulation during epithelial-to-mesenchymal cell transition. Nonetheless, regulating and studying the dynamics of it has proven challenging. We developed a photoswitchable version of E-cadherin, named opto-E-cadherin, which can be toggled OFF with blue light illumination and back ON in the dark. Herein, we describe easy-to-use methods to test and characterise opto-E- cadherin cell clones for downstream experiments. Key features • This protocol describes how to implement optogenetic cell-cell adhesion molecules effectively (described here on the basis of opto-E-cadherin), while highlighting possible pitfalls. • Utilises equipment commonly found in most laboratories with high ease of use. • Phenotype screening is easy and done within a few hours (comparison of cell clusters in the dark vs. blue light in an aggregation assay). • Three different functionality assay systems are described. • After the cell line is established, all experiments can be performed within three days., Competing Interests: Competing interestsThe authors declare no financial or non-financial competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Bacterial biofilm-associated infection was one of the most serious threats to human health. However, effective drugs for drug-resistance bacteria or biofilms remain rarely reported. Here, we propose an innovative strategy to develop a multifunctional antimicrobial agent with broad-spectrum antibacterial activity by coupling photosensitizers (PSs) with antimicrobial peptides (AMPs). This strategy capitalizes on the ability of PSs to generate reactive oxygen species (ROS) and the membrane-targeting property of AMPs (KRWWKWIRW, a peptide screened by an artificial neural network), synergistically enhancing the antimicrobial activity. In addition, unlike conventional aggregation-caused quenching (ACQ) photosensitizers, aggregation-induced emission (AIE) PSs show stronger fluorescence emission in the aggregated state to help visualize the antibacterial mechanism. In vitro antibacterial experiments demonstrated the excellent killing effects of the developed agent against both Gram-positive (G + ) and Gram-negative (G - ) bacteria. The bacterial-aggregations induced ability enhanced the photoactivatable antibacterial activity against G - bacteria. Notably, it exhibited a significant effect on destroying MRSA biofilms. Moreover, it also showed remarkable efficacy in treating wound infections in mice in vivo . This multifunctional antimicrobial agent holds significant potential in addressing the challenges posed by bacterial biofilm-associated infections and drug-resistant bacteria., (© 2024 The Authors.)

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.

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 H 2 O 2 . 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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

E-cadherin-based cell-cell adhesions are dynamically and locally regulated in many essential processes, including embryogenesis, wound healing and tissue organization, with dysregulation manifesting as tumorigenesis and metastasis. However, the lack of tools that would provide control of the high spatiotemporal precision observed with E-cadherin adhesions hampers investigation of the underlying mechanisms. Here, we present an optogenetic tool, opto-E-cadherin, that allows reversible control of E-cadherin-mediated cell-cell adhesions with blue light. With opto-E-cadherin, functionally essential calcium binding is photoregulated such that cells expressing opto-E-cadherin at their surface adhere to each other in the dark but not upon illumination. Consequently, opto-E-cadherin provides remote control over multicellular aggregation, E-cadherin-associated intracellular signalling and F-actin organization in 2D and 3D cell cultures. Opto-E-cadherin also allows switching of multicellular behaviour between single and collective cell migration, as well as of cell invasiveness in vitro and in vivo. Overall, opto-E-cadherin is a powerful optogenetic tool capable of controlling cell-cell adhesions at the molecular, cellular and behavioural level that opens up perspectives for the study of dynamics and spatiotemporal control of E-cadherin in biological processes., (© 2023. Springer Nature Limited.)

Vitamin B12 (cobalamin) is essential for human health and its deficiency results in anemia and neurological damage. Vitamin B12 exists in different forms with various bioactivity but most sensors are unable to discriminate between them. Here, a whole-cell agglutination assay that is specific for adenosylcobalamin (AboB12), which is one of two bioactive forms, is reported. This biosensor consists of Escherichia coli that express the AdoB12 specific binding domain of CarH at their surface. In the presence of AdoB12, CarH forms tetramers, which leads to specific bacterial cell-cell adhesions and agglutination. These CarH tetramers disassemble upon green light illumination such that reversion of the bacterial aggregation can serve as internal quality control. The agglutination assay has a detection limit of 500 nм AdoB12, works in protein-poor biofluids such as urine, and has high specificity to AdoB12 over other forms of vitamin B12 as also demonstrated with commercially available supplements. This work is a proof of concept for a cheap and easy-to-readout AdoB12 sensor that can be implemented at the point-of-care to monitor high-dose vitamin B12 supplementation., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

The nongenetic modification of cell membranes with proteins is a straightforward way of cellular engineering. In these processes, it is important to specifically address the proteins to liquid-ordered (Lo) or liquid-disordered (Ld) domains as this can largely affect their biological functions. Herein, we report a cholesterol analogue (CHIM) with a nitrilotriacetic acid (NTA) headgroup, named CHIM-NTA. CHIM-NTA integrates into lipid membranes similar to the widely used phospholipid-derived DGS-NTA and, when loaded with Ni 2+ , allows for specific membrane immobilization of any polyhistidine-tagged proteins of choice. Yet, unlike DGS-NTA, it localizes to the Lo phase in phase-separated giant unilamellar vesicles (GUVs) and allows addressing His-tagged proteins to Lo domains. Furthermore, CHIM-NTA readily integrates into the membranes of live cells and thus enables the nongenetic modification of the cell surface with proteins. Overall, CHIM-NTA provides a facile and flexible way to modify biological membranes, in particular Lo domains, with His-tagged proteins and can serve as a broadly applicable molecular tool for cell surface engineering.

Cell-to-cell communication is not limited to a sender releasing a signaling molecule and a receiver perceiving it but is often self-regulated and bidirectional. Yet, in communities of synthetic cells, such features that render communication efficient and adaptive are missing. Here, we report the design and implementation of adaptive two-way signaling with lipid-vesicle-based synthetic cells. The first layer of self-regulation derives from coupling the temporal dynamics of the signal, H 2 O 2 , production in the sender to adhesions between sender and receiver cells. This way the receiver stays within the signaling range for the duration sender produces the signal and detaches once the signal fades. Specifically, H 2 O 2 acts as both a forward signal and a regulator of the adhesions by activating photoswitchable proteins at the surface for the duration of the chemiluminescence. The second layer of self-regulation arises when the adhesions render the receiver permeable and trigger the release of a backward signal, resulting in bidirectional exchange. These design rules provide a concept for engineering multicellular systems with adaptive communication.

Reversible protein patterning on model membranes is important to reproduce spatiotemporal protein dynamics in vitro . An engineered version of iLID, disiLID, with a disordered domain as a membrane tether improves the recruitment of Nano under blue light and the reversibility in the dark, which enables protein patterning on membranes with higher spatiotemporal precision.

Developing orthogonal chemical communication pathways in diverse synthetic cell communities is a considerable challenge due to the increased crosstalk and interference associated with large numbers of different types of sender-receiver pairs. Herein, the authors control which sender-receiver pairs communicate in a three-membered community of synthetic cells through red and blue light illumination. Semipermeable protein-polymer-based synthetic cells (proteinosomes) with complementary membrane-attached protein adhesion communicate through single-stranded DNA oligomers and synergistically process biochemical information within a community consisting of one sender and two different receiver populations. Different pairs of red and blue light-responsive protein-protein interactions act as membrane adhesion mediators between the sender and receivers such that they self-assemble and socially self-sort into different multicellular structures under red and blue light. Consequently, distinct sender-receiver pairs come into the signaling range depending on the light illumination and are able to communicate specifically without activation of the other receiver population. Overall, this work shows how photoswitchable membrane adhesion gives rise to different self-sorting protocell patterns that mediate member-specific DNA-based communication in ternary populations of synthetic cells and provides a step towards the design of orthogonal chemical communication networks in diverse communities of synthetic cells., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

Morphogens determine cellular differentiation in many developing tissues in a concentration dependent manner. As a central model for gradient formation during animal development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in the Drosophila wing disc. Although heparan sulfate (HS) expression in the disc is essential for this process, it is not known whether HS regulates Hh signaling and spread in a direct or in an indirect manner. To answer this question, we systematically screened two composite Hh binding areas for HS in vitro and expressed mutated proteins in the Drosophila wing disc. We found that selectively impaired HS binding of the second site reduced Hh signaling close to the source and caused striking wing mispatterning phenotypes more distant from the source. These observations suggest that HS constrains Hh to the wing disc epithelium in a direct manner, and that interfering with this constriction converts Hh into freely diffusing forms with altered signaling ranges and impaired gradient robustness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Manikowski, Steffes, Froese, Exner, Ehring, Gude, Di Iorio, Wegner and Grobe.)

Anticancer drug development is important for human health, yet it remains a tremendous challenge. Photodynamic therapy (PDT), which induces cancer cell apoptosis via light-triggered production of reactive oxygen species, is a promising method. However, it has minimal efficacy in subcellular targeting, hypoxic microenvironments, and deep-seated malignancies. Here, we constructed a breast cancer photo-activable theranostic nanosystem through the rational design of a synthetic lysosomal-targeted molecule with multifunctions as aggregation-induced near-infrared (NIR) emission, a photosensitizer (PDT), and organosilver (chemotherapy) for NIR imaging and synergistic cancer therapy. The synthetic molecule could self-assemble into nanoparticles ( TPIMBS NPs) and be stabilized with amphiphilic block copolymers for enhanced accumulation in tumor sites through passive targeting while reducing the leakage in normal tissues. Through photochemical internalization, TPIMBS NPs preferentially concentrated in the lysosomes of cancer cells and generated reactive oxygen species (ROS) upon light irradiation, resulting in lysosomal rupture and release of PSs to the cytosol, which led to cell apoptosis. Further, the photoinduced release of Ag + from TPIMBS NPs could act as chemotherapy, significantly improving the overall therapeutic efficacy by synergistic effects with PDT. This research sheds fresh light on the creation of effective cancer treatments.

The formation of microbial biofilms is acknowledged as a major virulence factor in a range of persistent local infections. Failures to remove biofilms with antibiotics foster the emergence of antibiotic-resistant bacteria and result in chronic infections. As a result, the construction of effective biofilm-inhibiting and biofilm-eradicating chemicals is urgently required. Herein, we designed a water-soluble probe APDIS for membrane-active fluorescence and broad-spectrum antimicrobial actions, particularly against methicillin-resistant Staphylococcus aureus (MRSA), which shows multidrug resistance. In vitro and in vivo experiments demonstrate its high antibacterial effects comparable to vancomycin. Furthermore, it inhibits biofilm formation by effectively killing planktonic bacteria at low inhibitory concentrations, without toxicity to mammalian cells. More importantly, this probe can efficiently penetrate through biofilm barriers and exterminate bacteria that are enclosed within biofilms and startle existing biofilms. In the mouse model of implant-related biofilm infections, this probe exhibits strong antibiofilm activity against MRSA biofilms, thus providing a novel theranostic strategy to disrupt biofilms in vivo effectively. Our results indicate that this probe has the potential to be used for the development of a combinatorial theranostic platform with synergistic enhanced effects for the treatment of multidrug-resistant bacterial infections and antibiofilm medications.

Synthetic cells, which are assembled anew from well-defined molecular parts, open-up new possibilities for nanotechnological applications due to their reduced complexity and high functionality. In this review, we discuss how synthetic cells are being implemented in different fields ranging from biomedicine to material science. On one hand, synthetic cells can serve as microreactors that house metabolic networks and as therapeutic carriers that directly communicate with living cells. On the other hand, synthetic cells can become active components in a new-generation of materials that process inputs and result in autonomous and adaptive behavior. These early examples highlight the potential impact that synthetic cells will have in future applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Functional microgels provide a versatile basis for synthetic in vitro platforms as alternatives to animal experiments. The tuning of the physical, chemical, and biological properties of synthetic microgels can be achieved by blending suitable polymers and formulating them such to reflect the heterogenous and complex nature of biological tissues. Based on this premise, this paper introduces the development of volume-switchable core-shell microgels as 3D templates to enable cell growth for microtissue applications, using a systematic approach to tune the microgel properties based on a deep conceptual and practical understanding. Microscopic microgel design, such as the tailoring of the microgel size and spherical shape, is achieved by droplet-based microfluidics, while on a nanoscopic scale, a thermoresponsive polymer basis, poly(N-isopropylacrylamide) (PNIPAAm), is used to provide the microgel volume switchability. Since PNIPAAm has only limited cell-growth promoting properties, the cell adhesion on the microgel is further improved by surface modification with polydopamine, which only slightly affects the microgel properties, thereby simplifying the system. To further tune the microgel thermoresponsiveness, different amounts of N-hydroxyethylacrylamide are incorporated into the PNIPAAm network. In a final step, cell growth on the microgel surface is investigated, both at a single microgel platform and in spheroidal cell structures., (© 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

Cells commonly communicate with each other through diffusible molecules but nonchemical communication remains elusive. While bioluminescent organisms communicate through light to find prey or attract mates, it is still under debate if signaling through light is possible at the cellular level. Here, we demonstrate that cell to cell signaling through light is possible in artificial cell communities derived from biomimetic vesicles. In our design, artificial sender cells produce an intracellular light signal, which triggers the adhesion to receiver cells. Unlike soluble molecules, the light signal propagates fast, independent of diffusion and without the need for a transporter across membranes. To obtain a predator-prey relationship, the luminescence predator cells is loaded with a secondary diffusible poison, which is transferred to the prey cell upon adhesion and leads to its lysis. This design provides a blueprint for light based intercellular communication, which can be used for programing artificial and natural cell communities.

Artificial intelligence (AI) is accelerating the development of unconventional computing paradigms inspired by the abilities and energy efficiency of the brain. The human brain excels especially in computationally intensive cognitive tasks, such as pattern recognition and classification. A long-term goal is de-centralized neuromorphic computing, relying on a network of distributed cores to mimic the massive parallelism of the brain, thus rigorously following a nature-inspired approach for information processing. Through the gradual transformation of interconnected computing blocks into continuous computing tissue, the development of advanced forms of matter exhibiting basic features of intelligence can be envisioned, able to learn and process information in a delocalized manner. Such intelligent matter would interact with the environment by receiving and responding to external stimuli, while internally adapting its structure to enable the distribution and storage (as memory) of information. We review progress towards implementations of intelligent matter using molecular systems, soft materials or solid-state materials, with respect to applications in soft robotics, the development of adaptive artificial skins and distributed neuromorphic computing.

The regulation of cell-cell adhesions in space and time plays a crucial role in cell biology, especially in the coordination of multicellular behavior. Therefore, tools that allow for the modulation of cell-cell interactions with high precision are of great interest to a better understanding of their roles and building tissue-like structures. Herein, the green light-responsive protein CarH is expressed at the plasma membrane of cells as an artificial cell adhesion receptor, so that upon addition of its cofactor vitamin B 12 specific cell-cell interactions form and lead to cell clustering in a concentration-dependent manner. Upon green light illumination, the CarH based cell-cell interactions disassemble and allow for their reversion with high spatiotemporal control. Moreover, these artificial cell-cell interactions impact cell migration, as observed in a wound-healing assay. When the cells interact with each other in the presence of vitamin B 12 in the dark, the cells form on a solid front and migrate collectively; however, under green light illumination, individual cells migrate randomly out of the monolayer. Overall, the possibility of precisely controlling cell-cell interactions and regulating multicellular behavior is a potential pathway to gaining more insight into cell-cell interactions in biological processes., (© 2021 The Authors. Advanced Biology published by Wiley-VCH GmbH.)

Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant α-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-α, agalsidase-β and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r 2 = 0.9063 and r 2 = 0.8952, both p < 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-α, agalsidase-β and Moss-AGAL (KD: 1.94 ± 0.11 µM, 2.46 ± 0.21 µM, and 1.33 ± 0.09 µM, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-α, agalsidase-β and Moss-AGAL.

Cells have the ability to sense different environmental signals and position themselves accordingly in order to support their survival. Introducing analogous capabilities to the bottom-up assembled minimal synthetic cells is an important step for their autonomy. Here, a minimal synthetic cell which combines a multistimuli sensitive adhesion unit with an energy conversion module is reported, such that it can adhere to places that have the right environmental parameters for ATP production. The multistimuli sensitive adhesion unit senses light, pH, oxidative stress, and the presence of metal ions and can regulate the adhesion of synthetic cells to substrates in response to these stimuli following a chemically coded logic. The adhesion unit is composed of the light and redox responsive protein interaction of iLID and Nano and the pH sensitive and metal ion mediated binding of protein His-tags to Ni 2+ -NTA complexes. Integration of the adhesion unit with a light to ATP conversion module into one synthetic cell allows it to adhere to places under blue light illumination, non-oxidative conditions, at neutral pH and in the presence of metal ions, which are the right conditions to synthesize ATP. Thus, the multistimuli responsive adhesion unit allows synthetic cells to self-position and execute their functions., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

The preparation of precise macromolecules with multiple functionalities remains a challenge in drug delivery. Here, a method to prepare stoichiometrically precise tetrafunctional streptavidin conjugates is presented with an exemplary structure combining exactly one fluorescent label, one cell targeting group, one nucleus penetrating peptide and one drug molecule.

The self-assembly of different cell types into multicellular structures and their organization into spatiotemporally controlled patterns are both challenging and extremely powerful to understand how cells function within tissues and for bottom-up tissue engineering. Here, we not only independently control the self-assembly of two cell types into multicellular architectures with blue and red light, but also achieve their self-sorting into distinct assemblies. This required developing two cell types that form selective and homophilic cell-cell interactions either under blue or red light using photoswitchable proteins as artificial adhesion molecules. The interactions were individually triggerable with different colors of light, reversible in the dark, and provide noninvasive and temporal control over the cell-cell adhesions. In mixtures of the two cells, each cell type self-assembled independently upon orthogonal photoactivation, and cells sorted out into separate assemblies based on specific self-recognition. These self-sorted multicellular architectures provide us with a powerful tool for producing tissue-like structures from multiple cell types and investigate principles that govern them.

Spatiotemporal control of integrin-mediated cell adhesions to extracellular matrix regulates cell behavior with has numerous implications for biotechnological applications. In this work, two approaches for regulating cell adhesions in space and time with high precision are reported, both of which utilize green light. In the first design, CarH, which is a tetramer in the dark, is used to mask cRGD adhesion-peptides on a surface. Upon green light illumination, the CarH tetramer dissociates into its monomers, revealing the adhesion peptide so that cells can adhere. In the second design, the RGD motif is incorporated into the CarH protein tetramer such that cells can adhere to surfaces functionalized with this protein. The cell adhesions can be disrupted with green light, due to the disassembly of the CarH-RGD protein. Both designs allow for photoregulation with noninvasive visible light and open new possibilities to investigate the dynamical regulation of cell adhesions in cell biology., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

We present a new concept for whole-cell biosensors that couples the response to Hg 2+ with bioluminescence and bacterial aggregation. This allows us to use the bacterial aggregation to preconcentrate the bioluminescent bacteria at the substrate surface and increase the sensitivity of Hg 2+ detection. This whole-cell biosensor combines a Hg 2+ -sensitive bioluminescence reporter and light-responsive bacterial cell-cell adhesions. We demonstrate that the blue luminescence in response to Hg 2+ is able to photoactivate bacterial aggregation, which provides a second readout for Hg 2+ detection. In return, the Hg 2+ -triggered bacterial aggregation leads to faster sedimentation and more efficient formation of biofilms. At low Hg 2+ concentrations, the enrichment of the bacteria in biofilms leads to an up to 10-fold increase in the signal. The activation of photoswitchable proteins with biological light is a new concept in optogenetics, and the presented bacterial biosensor design is transferable to other bioluminescent reporters with particular interest for environmental monitoring.

Although the fundamental importance and biotechnological potential of multibacterial communities, also called biofilms, are well-known, our ability to control them is limited. We present a new way of dynamically controlling bacteria-bacteria adhesions by using blue light and how these photoswitchable adhesions can be used to regulate multicellularity and associated bacterial behavior. To achieve this, the photoswitchable proteins nMagHigh and pMagHigh were expressed on bacterial surfaces as adhesins to allow multicellular clusters to assemble under blue light and reversibly disassemble in the dark. Regulation of the bacterial cell-cell adhesions with visible light provides unique advantages including high spatiotemporal control, tunability, and noninvasive remote regulation. Moreover, these photoswitchable adhesions make it possible to regulate collective bacterial functions including aggregation, quorum sensing, biofilm formation, and metabolic cross-feeding between auxotrophic bacteria with light. Overall, the photoregulation of bacteria-bacteria adhesions provides a new way of studying bacterial cell biology and will enable the design of biofilms for biotechnological applications.

Building tissue from cells as the basic building block based on principles of self-assembly is a challenging and promising approach. Understanding how far principles of self-assembly and self-sorting known for colloidal particles apply to cells remains unanswered. In this study, we demonstrate that not just controlling the cell-cell interactions but also their dynamics is a crucial factor that determines the formed multicellular structure, using photoswitchable interactions between cells that are activated with blue light and reverse in the dark. Tuning dynamics of the cell-cell interactions by pulsed light activation results in multicellular architectures with different sizes and shapes. When the interactions between cells are dynamic, compact and round multicellular clusters under thermodynamic control form, while otherwise branched and loose aggregates under kinetic control assemble. These structures parallel what is known for colloidal assemblies under reaction- and diffusion-limited cluster aggregation, respectively. Similarly, dynamic interactions between cells are essential for cells to self-sort into distinct groups. Using four different cell types, which expressed two orthogonal cell-cell interaction pairs, the cells sorted into two separate assemblies. Bringing concepts of colloidal self-assembly to bottom-up tissue engineering provides a new theoretical framework and will help in the design of more predictable tissue-like structures.

In bacteria-driven microswimmers, i.e., bacteriabots, artificial cargos are attached to flagellated chemotactic bacteria for active delivery with potential applications in biomedical technology. Controlling when and where bacteria bind and release their cargo is a critical step for bacteriabot fabrication and efficient cargo delivery/deposition at the target site. Toward this goal, photoregulating the cargo integration and release in bacteriabots using red and far-red light, which are noninvasive stimuli with good tissue penetration and provide high spatiotemporal control, is proposed. In the bacteriabot design, the surfaces of E. coli and microsized model cargo particles with the proteins PhyB and PIF6, which bind to each other under red light and dissociate from each other under far-red light are functionalized. Consequently, the engineered bacteria adhere and transport the model cargo under red light and release it on-demand upon far-red light illumination due to the photoswitchable PhyB-PIF6 protein interaction. Overall, the proof-of-concept for red/far-red light switchable bacteriabots, which opens new possibilities in the photoregulation in biohybrid systems for bioengineering, targeted drug delivery, and lab-on-a-chip devices, is demonstrated., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Decorating GUVs, used as minimal synthetic cell models, with photoswitchable proteins allows controlling the adhesion between them and their assembly into multicellular structures with light. Thereby, the chemical communication between a sender and a receiver GUV, which strongly depends on their spatial proximity, can also be photoregulated.

Although assembly of recombinant proteins by SpyTag/SpyCatcher chemistry has proven to be a versatile approach for creating bioactive hydrogels, the resulting Spy networks often exhibit weak mechanics due to the poor efficiency of interchain cross-linking. Here we leverage metal/ligand (i.e., cobalt/His6-tag) coordination interactions to modulate the bulk mechanics of the protein networks. The drastic difference between the Co 2+ and Co 3+ complexes in thermodynamic and kinetic properties enabled us to regulate the materials' properties and to immobilize and release recombinant proteins in a redox-dependent manner. The resulting hydrogels are capable of not only supporting cell growth and proliferation, but also influencing specific cell signaling via immobilized growth factors such as leukemia inhibitory factor (LIF). The integrated use of stimuli-responsive metal coordination and SpyTag/SpyCatcher chemistry opens up a new dimension for designing bioactive protein materials.

Cell adhesions to the extracellular matrix and to neighboring cells are fundamental to cell behavior and have also been implemented into minimal synthetic cells, which are assembled from molecular building blocks from the bottom-up. Investigating adhesion in cell mimetic models with reduced complexity provides a better understanding of biochemical and biophysical concepts underlying the cell adhesion machinery. In return, implementing cell-matrix and cell-cell adhesions into minimal synthetic cells allows reconstructing cell functions associated with cell adhesions including cell motility, multicellular prototissues, fusion of vesicles, and the self-sorting of different cell types. Cell adhesions have been mimicked using both the native cell receptors and reductionist mimetics providing a variety of specific, reversible, dynamic, and spatiotemporally controlled interactions. This review gives an overview of different minimal adhesion modules integrated into different minimal synthetic cells drawing inspiration from cell and colloidal science., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The ability of living systems to self-sort different cells into separate assemblies and the ability to independently regulate different structures are one ingredient that gives rise to their spatiotemporal complexity. Here, this self-sorting behavior is replicated in a synthetic system with two types of colloidal particles; where each particle type independently self-assembles either under blue or red light into distinct clusters, known as narcissistic self-sorting. For this purpose, each particle type is functionalized either with the light-switchable protein VVDHigh or Cph1, which homodimerize under blue and red light, respectively. The response to different wavelengths of light and the high specificity of the protein interactions allows for the independent self-assembly of each particle type with blue or red light and narcissistic self-sorting. Moreover, as both of the photoswitchable protein interactions are reversible in the dark; also, the self-sorting is reversible and dynamic. Overall, the independent blue and red light controlled self-sorting in a synthetic system opens new possibilities to assemble adaptable, smart, and advanced materials similar to the complexity observed in tissues., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Controlling cell-cell interactions is central for understanding key cellular processes and bottom-up tissue assembly from single cells. The challenge is to control cell-cell interactions dynamically and reversibly with high spatiotemporal precision noninvasively and sustainably. In this study, cell-cell interactions are controlled with visible light using an optogenetic approach by expressing the blue light switchable proteins CRY2 or CIBN on the surfaces of cells. CRY2 and CIBN expressing cells form specific heterophilic interactions under blue light providing precise control in space and time. Further, these interactions are reversible in the dark and can be repeatedly and dynamically switched on and off. Unlike previous approaches, these genetically encoded proteins allow for long-term expression of the interaction domains and respond to nontoxic low intensity blue light. In addition, these interactions are suitable to assemble cells into 3D multicellular architectures. Overall, this approach captures the dynamic and reversible nature of cell-cell interactions and controls them noninvasively and sustainably both in space and time. This provides a new way of studying cell-cell interactions and assembling cellular building blocks into tissues with unmatched flexibility., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The dynamic and spatiotemporal control of integrin-mediated cell adhesion to RGD motifs in its extracellular matrix (ECM) is important for understating cell biology and biomedical applications because cell adhesion fundamentally regulates cellular behavior. Herein, the first photoswitchable synthetic ECM protein, Photo-ECM, based on the blue light switchable protein LOV2 is engineered. The Photo-ECM protein includes a RGD sequence, which is hidden in the folded LOV2 protein structure in the dark and is exposed under blue light so that integrins can bind and cells can adhere. The switchable presentation of the RGD motif allows to reversibly mediate and modulate integrin-based cell adhesions using noninvasive blue light. With this protein cell adhesions in live cells could be reversed and the dynamics at the cellular level is observed. Hence, the Photo-ECM opens a new possibility to investigate the spatiotemporal regulation of cell adhesions in cell biology and is the first step toward a genetically encoded and light-responsive ECM., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biofilms are not only a leading cause of chronic infections and biofouling, but they also have a tremendous positive potential in biotechnology for biocatalysis and waste treatment. Biofilms are spatially structured communities of microbes, which exchange chemicals and communicate with each other. By spatially controlling bacterial adhesion to surfaces, and therefore the microstructure of biofilms, a promising method of understanding social interactions between bacteria and designed biofilms is developed. The bacterial photolithography approach described here allows to photopattern specific bacteria adhesion molecules, to control surface adhesion, and to guide the formation of biofilms. To do this, α-D-mannoside, which is recognized by the Escherichia coli FimH receptor, is linked to a nonadhesive polyethylene glycol surface through a photocleavable 2-nitrobenzyl linker. When a pattern of UV light in a specific shape is projected onto these surfaces, the light-exposed areas become nonadhesive and bacteria only adhere to the dark, unexposed areas in the photopattern. Bacterial photolithography enables bacterial patterning with high spatial resolution down to 10 µm without mechanical interference. Additionally, patterning biofilms with complicated geometries allows studying the importance of microscale spatial organization on the collective behavior of bacteria such as quorum sensing., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Single-particle plasmon spectroscopy has become a standard technique to detect and quantify the presence of unlabeled macromolecules. Here, we extend this method to determine their exact distance from the plasmon sensors with sub-nanometer resolution by systematically varying the sensing range into the surrounding by adjusting the size of the plasmonic nanoparticles. We improved current single-particle plasmon spectroscopy to record continuously for hours the scattering spectra of thousands of nanoparticles of different sizes simultaneously with 1.8 s time resolution. We apply this technique to study the interaction dynamics of bacterial Min proteins with supported lipid membranes of different composition. Our experiments reveal a surprisingly flexible operating mode of the Min proteins: In the presence of cardiolipin and membrane curvature induced by nanoparticles, the protein oscillation occurs on top of a stationary MinD patch. Our results reveal the need to consider membrane composition and local curvature as important parameters to quantitatively understand the Min protein system and could be extrapolated to other macromolecular systems. Our label-free method is generally easily implementable and well suited to measure distances of interacting biological macromolecules.

Cell motility is an important but complex process; as cells move, new adhesions form at the front and adhesions disassemble at the back. To replicate this dynamic and spatiotemporally controlled asymmetry of adhesions and achieve motility in a minimal synthetic cell, we controlled the adhesion of a model giant unilamellar vesicle (GUV) to the substrate with light. For this purpose, we immobilized the proteins iLID and Micro, which interact under blue light and dissociate from each other in the dark, on a substrate and a GUV, respectively. Under blue light, the protein interaction leads to adhesion of the vesicle to the substrate, which is reversible in the dark. The high spatiotemporal control provided by light, allowed partly illuminating the GUV and generating an asymmetry in adhesions. Consequently, the GUV moves into the illuminated area, a process that can be repeated over multiple cycles. Thus, our system reproduces the dynamic spatiotemporal distribution of adhesions and establishes mimetic motility of a synthetic cell.

We use plasmon rulers to follow the conformational dynamics of a single protein for up to 24 h at a video rate. The plasmon ruler consists of two gold nanospheres connected by a single protein linker. In our experiment, we follow the dynamics of the molecular chaperone heat shock protein 90 (Hsp90), which is known to show "open" and "closed" conformations. Our measurements confirm the previously known conformational dynamics with transition times in the second to minute time scale and reveals new dynamics on the time scale of minutes to hours. Plasmon rulers thus extend the observation bandwidth 3-4 orders of magnitude with respect to single-molecule fluorescence resonance energy transfer and enable the study of molecular dynamics with unprecedented precision.

Toward 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 self-sorting 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 noninvasive, 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 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.

Control of bacterial adhesions to a substrate with high precision in space and time is important to form a well-defined biofilm. Here, we present a method to engineer bacteria such that they adhere specifically to substrates under blue light through the photoswitchable proteins nMag and pMag. This provides exquisite spatiotemporal remote control over these interactions. The engineered bacteria express pMag protein on the surface so that they can adhere to substrates with nMag protein immobilization under blue light, and reversibly detach in the dark. This process can be repeatedly turned on and off. In addition, the bacterial adhesion property can be adjusted by expressing different pMag proteins on the bacterial surface and altering light intensity. This protocol provides light switchable, reversible and tunable control of bacteria adhesion with high spatial and temporal resolution, which enables us to pattern bacteria on substrates with great flexibility., (Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC.)

Independent control over multiple cell-material interactions with high spatiotemporal resolution is a key for many biomedical applications and understanding cell biology, as different cell types can perform different tasks in a multicellular context. In this study, the binding of two different cell types to materials is orthogonally controlled with blue and red light providing independent regulation in space and time. Cells expressing the photoswitchable protein cryptochrome 2 (CRY2) on cell surface bind to N-truncated CRY-interacting basic helix-loop-helix protein 1 (CIBN)-immobilized substrates under blue light and cells expressing the photoswitchable protein phytochrome B (PhyB ) on cell surface bind to phytochrome interaction factor 6 (PIF6)-immobilized substrates under red light, respectively. These light-switchable cell interactions provide orthogonal and noninvasive control using two wavelengths of visible light. Moreover, both cell-material interactions are dynamically switched on under light and reversible in the dark. The specificity of the CRY2/CIBN and PhyB/PIF6 interactions and their response to different wavelengths of light allow selectively activating the binding of one cell type with blue and the other cell type with red light in the presence of the other cell type.

Collagen, a strong platelet activator, is recognized by integrin α2β1 and GPVI. It induces aggregation, if added to suspended platelets, or platelet adhesion if immobilized to a surface. The recombinant non-prolylhydroxylated mini-collagen FC3 triple helix containing one α2β1 integrin binding site is a tool to specifically study how α2β1 integrin activates platelet. Whereas soluble FC3 monomers antagonistically block collagen-induced platelet activation, immobilization of several FC3 molecules to an interface or to colloidal nanobeads determines the agonistic action of FC3. Nanopatterning of FC3 reveals that intermolecular distances below 64 nm between α2β1 integrin binding sites trigger signaling through dot-like clusters of α2β1 integrin, which are visible in high resolution microscopy with dSTORM. Upon signaling, these integrin clusters increase in numbers per platelet, but retain their individual size. Immobilization of several FC3 to 100 nm-sized nanobeads identifies α2β1 integrin-triggered signaling in platelets to occur at a twentyfold slower rate than collagen, which activates platelet in a fast integrative signaling via different platelet receptors. As compared to collagen stimulation, FC3-nanobead-triggered signaling cause a significant stronger activation of the protein kinase BTK, a weak and dispensable activation of PDK1, as well as a distinct phosphorylation pattern of PDB/Akt., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The blue light-dependent interaction between the proteins iLID and Nano allows recruiting and patterning proteins on GUV membranes, which thereby capture key features of patterns observed in nature. This photoswitchable protein interaction provides non-invasive, reversible and dynamic control over protein patterns of different sizes with high specificity and spatiotemporal resolution.

The control of where and when bacteria adhere to a substrate is a key step toward controlling the formation and organization in biofilms. This study shows how we engineer bacteria to adhere specifically to substrates with high spatial and temporal control under blue light, but not in the dark, by using photoswitchable interaction between nMag and pMag proteins. For this, we express pMag proteins on the surface of E. coli so that the bacteria can adhere to substrates with immobilized nMag protein under blue light. These adhesions are reversible in the dark and can be repeatedly turned on and off. Further, the number of bacteria that can adhere to the substrate as well as the attachment and detachment dynamics are adjustable by using different point mutants of pMag and altering light intensity. Overall, the blue light switchable bacteria adhesions offer reversible, tunable and bioorthogonal control with exceptional spatial and temporal resolution. This enables us to pattern bacteria on substrates with great flexibility.

Over the last decade, there has been a growing interest in the development of new materials to improve bone morphogenetic protein-2 (BMP-2) delivery for tissue regeneration. This study reports the development and application of model surfaces that present BMP-2 via heparan sulfate (HS), a ubiquitous component of the extracellular matrix (ECM). On these surfaces, HS is grafted by its reducing end, to mimic the natural arrangement of HS proteoglycans in the ECM. The binding of each component on these biomimetic surfaces is highly controlled, in terms of stoichiometry of molecules and BMP-2/grafted-HS affinity, as determined by surface-sensitive techniques. For comparison, this study also uses surfaces presenting immobilized BMP-2 alone. Functional validations of the surfaces are performed using a murine myoblast cell line (C2C12) and primary human mesenchymal stromal cells. In both cell types, HS-bound BMP-2 and surface-immobilized BMP-2 significantly prolong SMAD 1/5 phosphorylation, compared to BMP-2 added to the culture media. Moreover, HS-bound BMP-2 enhances p-SMAD 1/5 levels in C2C12 cells and reduces noggin antagonistic activity. Thus, grafted HS positively affects BMP-2 cellular activity. This innovative surface design, which mimics natural interactions of growth factors with ECM components, constitutes a promising candidate for future regenerative medicine applications., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)