Biology subjects

Moeglich, A.

Publications and source records attributed to Moeglich, A..

8 recordsLinked to original sources

bioRxiv2025

An Engineered Living Material with pro-angiogenic activity inducible by near-infrared light

Impaired angiogenesis is a central barrier in the treatment of chronic and deep tissue wounds, preventing progression through the normal healing cascade. While the combination of near-infrared (NIR) photobiomodulation and pro-angiogenic growth factors has shown synergistic therapeutic benefit, the clinical translation of growth factor therapy is hindered by high cost, instability and the need for localized dosing to avoid aberrant vasculature. Peptidomimetics such as the VEGF-derived QK peptide offer a more stable and predictable alternative, but still require a means for localized, tunable presentation. Here, we establish an engineered living material based delivery system that responds to clinically relevant NIR light to produce and releases a QK-Fusion protein directly at the target site. The probiotic Escherichia coli Nissle 1917 was engineered with an 800 nm-responsive optogenetic circuit and encapsulated within an optimized alginate core-shell hydrogel that ensures biocontainment while allowing controlled outward diffusion of the secreted peptide. The released peptide remains non-cytotoxic and capable of binding extracellular matrix analogs and promoting the formation of organized, branched capillary-like networks in endothelial cultures. We thus establish a strategy for developing engineered living materials towards remote-controlled angiogenic stimulation.

bioengineering↗

bioRxiv2025

Dimer asymmetry in signaling of blue-light sensor histidine kinases

Photoreceptor sensory histidine kinases (SHKs) couple light absorption to conformational changes regulating two-component signaling. Despite their importance and widespread use in optogenetics, the underlying structural signaling mechanisms remain poorly understood. We here engineered dimeric SHKs based on Pseudomonas putida short light-oxygen-voltage (LOV) proteins and determined their crystal structures. Regardless of illumination, the structures adopted a light-state like LOV-LOV dimer with symmetric/straight kinase modules. In contrast, small-angle X-ray scattering together with functional assays revealed pronounced light-dependent rearrangements in solution and allowed the assignment of the kinase-ON dark state to an asymmetric/kinked conformation, whereas the light state adopts a symmetric/straight structure. Comparative analyses of natural and engineered SHKs identified conserved motifs linking LOV domain rotation to kinase activity. The findings highlight the central role of dimer asymmetry and flexibility in SHK signaling, thereby not least informing the engineering of new light-responsive signaling systems.

biochemistry↗

bioRxiv2025

Engineering NIR-Sighted Bacteria

Spatially and temporally orchestrated gene expression underpins organismal development, physiology, and adaptation. In bacteria, two-component systems (TCS) translate environmental cues into inducible expression outputs. Inducible expression also serves as a versatile instrument in both basic and applied science. Here, we harness the photosensors of rhizobial bathy-phytochromes to construct synthetic TCSs for stringent activation of gene expression by near-infrared (NIR) light in laboratory and probiotic Escherichia coli strains, and in Agrobacterium tumefaciens. Orthogonal TCSs afford the multiplexed expression control of several genes by NIR and visible light. Notwithstanding substantial photochemical activation of bathy-phytochromes by visible radiation, the NIR-light-responsive systems hardly responded to red light. Evidently, light signals can be processed by TCSs into highly nonlinear responses at the physiological relevant level of gene expression. These fundamental aspects likely extend to naturally occurring TCSs. Depending on their photosensor traits and environmental conditions, bathy-phytochromes may thus either be NIR-specific or function as colorblind receptors of light vs. darkness.

biochemistry↗

bioRxiv2024

Potent photoswitch for expression of biotherapeutics in mammalian cells by light

AbstractPrecise temporal and spatial control of gene expression is of great benefit for the study of specific cellular circuits and activities. Compared to chemical inducers, light-dependent control of gene expression by optogenetics achieves a higher spatial and temporal resolution. This could also prove decisive beyond basic research for manufacturing difficult-to-express proteins in pharmaceutical bioproduction. However, current optogenetic gene-expression systems limit this application in mammalian cells as expression levels and fold induction upon light stimulation are not sufficient. To overcome this limitation, we designed a photoswitch by fusing the blue light-activated light-oxygen-voltage receptor EL222 from Erythrobacter litoralis to the three tandem transcriptional activator domains VP64, p65, and Rta. The resultant photoswitch, dubbed DEL-VPR, allows an up to 400-fold induction of target gene expression by blue light, achieving expression levels that surpass those for strong constitutive promoters. Here, we utilized DEL-VPR to enable light-induced expression of complex monoclonal and bispecific antibodies with reduced byproduct expression, increasing the yield of functional protein complexes. Our approach offers temporally controlled yet strong gene expression and applies to both academic and industrial settings.

molecular biology↗

bioRxiv2024

Reduction Midpoint Potential of a Paradigm Light-Oxygen-Voltage Receptor and its Modulation by Methionine Residues

Light-dependent adaptations of organismal physiology, development, and behavior abound in nature and depend on sensory photoreceptors. As one class, light-oxygen-voltage (LOV) photoreceptors harness flavin-nucleotide chromophores to sense blue light. Photon absorption drives the LOV receptor to its signaling state, characterized by a metastable thioadduct between the flavin and a conserved cysteine residue. With this cysteine absent, LOV receptors instead undergo photoreduction to the flavin semiquinone which however can still elicit downstream physiological responses. Irrespective of the cysteine presence, the LOV photochemical response thus entails a formal reduction of the flavin. Against this backdrop, we here investigate the reduction midpoint potential E0 in the paradigm LOV2 domain from Avena sativa phototropin 1 (AsLOV2), and how it can be deliberately varied. Replacements of residues at different sites near the flavin by methionine consistently increase E0 from its value of around -280 mV by up to 40 mV. Moreover, methionine introduction invariably impairs photoactivation efficiency and thus renders the resultant AsLOV2 variants less light-sensitive. Although individual methionine substitutions also affect the stability of the signaling state and downstream allosteric responses, no clear-cut correlation with the redox properties emerges. With a reduction midpoint potential near -280 mV, AsLOV2 and, by inference, other LOV receptors may be partially reduced inside cells which directly affects their light responsiveness. The targeted modification of the chromophore environment, as presently demonstrated, may mitigate this effect and enables the design of LOV receptors with stratified redox sensitivities.

biochemistry↗

bioRxiv2022

Light-dependent Control of Bacterial Expression at the mRNA Level

Sensory photoreceptors mediate numerous light-dependent adaptations across organisms. In optogenetics, photoreceptors achieve the reversible, non-invasive, and spatiotemporally precise control by light of gene expression and other cellular processes. The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination. By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest, their expression can be downregulated by light. We developed the pCrepusculo and pAurora optogenetic systems that are based on PAL and allow to down- and upregulate, respectively, bacterial gene expression using blue light. Both systems are realized as compact, single plasmids that exhibit stringent blue-light responses with low basal activity and up to several ten-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that generally control transcription initiation. By integrating regulatory mechanisms operating at the DNA and mRNA levels, optogenetic circuits with emergent properties can thus be devised. As a case in point, the pEnumbra setup permits to upregulate gene expression under moderate blue light whereas strong blue light shuts off expression again. Beyond providing novel signal-responsive expression systems for diverse applications in biotechnology and synthetic biology, our work also illustrates how the light-dependent PAL-aptamer interaction can be harnessed for the control and interrogation of RNA-based processes.

biochemistry↗

bioRxiv2020

Optoribogenetic control of regulatory RNA molecules

Short regulatory RNA molecules underpin gene expression and govern cellular state and physiology. To establish a novel layer of control over these processes, we generated chimeric regulatory RNAs that interact reversibly and light-dependently with the light-oxygen-voltage photoreceptor PAL. By harnessing this interaction, the function of micro RNAs (miRs) and short hairpin (sh) RNAs in mammalian cells can be regulated in spatiotemporally precise manner. The underlying strategy is generic and can be adapted to near-arbitrary target sequences. Owing to full genetic encodability, it establishes unprecedented optoribogenetic control of cell state and physiology. The method stands to facilitate the non-invasive, reversible and spatiotemporally resolved study of regulatory RNAs and protein function in cellular and organismal environments.

molecular biology↗

bioRxiv2020

Nanobody-directed targeting of optogenetic tools reveals differential regulation of cilia length

Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling and function has been challenging due to the lack of tools to temporarily manipulate and analyze ciliary signaling. Here, we describe a nanobodybased targeting approach for optogenetic tools that is applicable in vitro and in vivo and allows to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after direct fusion to a ciliary targeting sequence. We functionally localized modifiers of cAMP signaling, i.e. the photo-activated adenylate cyclase bPAC and the light-activated phosphodiesterase LAPD, as well as the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

cell biology↗