The mechanical properties of the extracellular environment govern key cellular decision-making processes such as proliferation, differentiation, or migration. [1] Thus, analyzing how cells gauge and interact with their mechanical environment is critical not only for understanding physiological and pathological processes but also for engineering cell and tissue growth and differentiation in regenerative medicine. [2] Although studies using passive elastic or viscoelastic materials have revealed valuable information about cell-matrix interactions, matrices with adjustable mechanical properties more closely reflect the dynamic environments many cells are exposed to in a living organism. [3] In order to recapitulate these dynamic environments, several materials have been developed, which enable the Interrogation and control of cellular fate and function using optogenetics is providing revolutionary insights into biology. Optogenetic control of cells is achieved by coupling genetically encoded photoreceptors to cellular effectors and enables unprecedented spatiotemporal control of signaling processes. Here, a fast and reversibly switchable photoreceptor is used to tune the mechanical properties of polymer materials in a fully reversible, wavelengthspecific, and dose-and space-controlled manner. By integrating engineered cyanobacterial phytochrome 1 into a poly(ethylene glycol) matrix, hydrogel materials responsive to light in the cell-compatible red/far-red spectrum are synthesized. These materials are applied to study in human mesenchymal stem cells how different mechanosignaling pathways respond to changing mechanical environments and to control the migration of primary immune cells in 3D. This optogenetics-inspired matrix allows fundamental questions of how cells react to dynamic mechanical environments to be addressed. Further, remote control of such matrices can create new opportunities for tissue engineering or provide a basis for optically stimulated drug depots. BiomaterialsThe ORCID identification number(s) for the author(s) of this article can be found under https://doi.

The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.

Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

Apoptosis occurs through a tightly regulated cascade of caspase activation. In the context of extrinsic apoptosis, caspase-8 is activated by dimerization inside a death receptor complex, cleaved by auto-proteolysis and subsequently released into the cytosol. This fully processed form of caspase-8 is thought to cleave its substrates BID and caspase-3. To test if the release is required for substrate cleavage, we developed a novel approach based on localization probes to quantitatively characterize the spatial-temporal activity of caspases in living single cells. Our study reveals that caspase-8 is significantly more active at the plasma membrane than within the cytosol upon CD95 activation. This differential activity is controlled by the cleavage of caspase-8 prodomain. As a consequence, targeting of caspase-8 substrates to the plasma membrane can significantly accelerate cell death. Subcellular compartmentalization of caspase-8 activity may serve to restrict enzymatic activity before mitochondrial pathway activation and offers new possibilities to interfere with apoptotic sensitivity of the cells. Apoptosis is coordinated by the activity of initiator and effector caspases.1-4 While effector caspases are dimeric zymogens that become activated by cleavage, initiator caspases are normally expressed as monomeric zymogens and their activity is initiated by dimerization in a multimeric complex. 5,6 The initiator caspase procaspase-8 dimerizes in the death-inducing signaling complex (DISC) formed around activated death receptors. 7 In the context of CD95, the DISC contains clustered receptors bound to the adaptor protein FADD. FADD can recruit several proteins, 8,9 including procaspase-8 and -10 through their prodomain. In type I cells, active caspase-8/10 directly cleaves and activates effector caspase-3/-7, thus inducing apoptosis. In type II cells, this activation is blocked by XIAP, but cleavage of BID by active caspase-8/10 induces mitochondrial outer membrane permeabilization (MOMP), followed by initiator caspase-9 activation and release of XIAP-inhibitor SMAC, leading to massive caspase-3/7 activity. 10,11 Biochemical and structural studies showed that dimerization but also cleavage in the catalytic subunit of caspase-8 is required for efficient cleavage of caspase-3 and BID and for apoptosis.12-14 Supporting this, the non-cleavable D387A caspase-8 mutant compromises apoptosis in mice. 15Although caspase-8 dimerization generates some activity, cleavage in the catalytic units likely stabilizes the active form and increases activity. 13,16 Caspase-8 is also cleaved between the prodomain and the catalytic unit of the enzyme, at D210 and D216, 17,18 and subsequently released from the DISC. Active caspase-8 can be detected on the plasma membrane, before release, with fluorescent inhibitors. 19 In contrast, by designing a procaspase-8 artificially dimerized on the plasma membrane, Martin et al.18 suggested that the cytosolic release is necessary to cleave caspase-3 and BID. Other studies proposed that fully processed ...