Johannes Broichhagen

Type 1 diabetes (T1D) is characterized by autoimmune destruction of pancreatic beta cells, leading to insulin dependence. Preclinical models have been instrumental in advancing our understanding of the pathogenesis and treatment of T1D. Chemical agents such as streptozotocin or alloxan are widely used to induce beta cell destruction without the need for genetic alteration. However, streptozotocin and alloxan exhibit variable efficacy, can be taken up non‐specifically into other organs, and possess carcinogenic properties. As such, there is a need for more specific and less toxic models of beta cell stress. Here, we design and evaluate ROXendin668, a glucagon‐like peptide‐1 receptor (GLP1R) agonist coupled to a singlet oxygen ( ¹ O ₂ ) sensitizer. ROXendin668 targets pancreatic beta cells through engagement of GLP1R and potentially GCGR, followed by red light‐controlled generation of ¹ O ₂ . Upon illumination, ROXendin668 selectively ablates MIN6 β‐cells and primary islet β‐cells and induces localized β‐cell stress in vivo after systemic delivery. Thus, ROXendin668 represents a precise, controllable, and potentially safer alternative to existing chemical models of beta cell stress.

Type 1 diabetes (T1D) is characterized by autoimmune destruction of pancreatic beta cells, leading to insulin dependence. Preclinical models have been instrumental in advancing our understanding of the pathogenesis and treatment of T1D. Chemical agents such as streptozotocin or alloxan are widely used to induce beta cell destruction without the need for genetic alteration. However, streptozotocin and alloxan exhibit variable efficacy, can be taken up non‐specifically into other organs, and possess carcinogenic properties. As such, there is a need for more specific and less toxic models of beta cell stress. Here, we design and evaluate ROXendin668, a glucagon‐like peptide‐1 receptor (GLP1R) agonist coupled to a singlet oxygen ( ¹ O ₂ ) sensitizer. ROXendin668 targets pancreatic beta cells through engagement of GLP1R and potentially GCGR, followed by red light‐controlled generation of ¹ O ₂ . Upon illumination, ROXendin668 selectively ablates MIN6 β‐cells and primary islet β‐cells and induces localized β‐cell stress in vivo after systemic delivery. Thus, ROXendin668 represents a precise, controllable, and potentially safer alternative to existing chemical models of beta cell stress.

Aims: Patient and public involvement (PPI) is increasingly recognised as an important component of research, while public engagement provides opportunities to communicate research and stimulate dialogue with wider audiences. In preclinical type 1 diabetes (T1D) research, complex scientific concepts can present particular barriers to engagement across different ages and experiences. Methods: We describe two creative public engagement initiatives—a colouring book and a glass art project—and discuss how PPI and lived‐experience perspectives helped shape their development. Conclusions: These examples illustrate how artistic approaches can make basic science more accessible and create opportunities for dialogue between researchers and people affected by T1D.

Beta-arrestins (β-arrs) are cytosolic proteins which mediate G protein-coupled receptor (GPCR) desensitization, endocytosis, and signaling. Despite the widespread physiological roles of β-arr coupling, the molecular basis of GPCR/β-arr interaction has been studied primarily in monomeric family A GPCRs. Here we develop a single molecule pulldown (SiMPull) assay that reports on both relative GPCR:β-arr interaction strength and stoichiometry to uncover molecular diversity in β-arr coupling to the neuromodulatory metabotropic glutamate receptors (mGluRs), prototypical, dimeric family C GPCRs. We find that mGluRs couple to β-arrs with variable apparent strength and a 2:1 or 2:2 stoichiometry. Focusing on the mGluR8/β-arr1 interaction, we map the contributions of “tail” and “core” interactions with residues in the receptor C-terminal domain and intracellular loops, respectively. Combinatorial mutagenesis enables the identification of a landscape of β-arr complexes with homo- and hetero-dimeric mGluR8, including both “cis” and “trans” interactions. Finally, we use our SiMPull assay in conjunction with live cell imaging and transcriptomic analysis to reveal that mGluRs can form megacomplexes either by simultaneously binding β-arr1 and β-arr2 or β-arr and G proteins. Together, this study provides a comprehensive framework for mGluR/β-arr complex diversity, expanding the known range of GPCR/transducer coupling modes.

Metabotropic glutamate receptors (mGluRs) are prototypical, dimeric family C G protein-coupled receptors (GPCR) that perform crucial modulatory roles throughout the nervous system. While mGluR activation and signaling through G proteins has been studied extensively, how these receptors interact with and are desensitized by β-arrestins (β-arrs) is not well understood. Here, we use an integrative biophysical and structural approach to probe the coupling of mGluR8 and β-arrs. Using negative stain electron microscopy (EM), we identify tail- and core-bound orientations and stoichiometries of mGluR8/β-arr complexes. Cryo-EM structures of mGluR8 alone or bound to either G proteins or β-arr1 reveal mGluR8 active states with transducer-specific differences. The mGluR8/β-arr structure shows a distinct complex orientation compared to other GPCR/β-arr structures which supports a steric mechanism of mGluR desensitization involving interactions with both subunits and the lipid bilayer. Coupling of mGluR8 to β-arr1 in an active-like conformation is verified by live-cell and single molecule FRET analysis. Finally, molecular dynamics simulations further define the positioning and dynamics of mGluR8-bound β-arr1 and the importance of critical mGluR8 residues for stabilizing β-arr1 complexes. Together, our data provide a framework for agonist-driven family C GPCR/β-arr coupling.

Fluorescence lifetime (FL) is a robust strategy for multiplexed imaging; however, variability in fluorophores’ local microenvironment when used on affinity probes broadens lifetime distributions, limiting its practical applicability. We create a modular platform for FL encoding with nanobody–HaloTag variants, enabling universal FL multiplexing. By characterizing dozens of fluorophores across four HaloTag variants, we generate an FL landscape that reveals tunable, consistent FL signatures within a single spectrum. Leveraging this physicochemically defined encoding, we achieve multiplexed imaging of up to eight targets in a single staining step while preserving high spatial fidelity across cellular and tissue samples. Unlike conventional antibody labeling, our approach decouples probe identity from species constraints and eliminates batch-to-batch variability in FL. These results confirm nanobody-guided FL engineering as a scalable strategy for multiplexed imaging (NanoFLex), offering a standard and straightforward framework for FL multiplexing and expanding the accessible dimensions of optical readout.

Intrinsically disordered regions (IDRs) are major drivers of protein functional diversification, yet the molecular features that enable the emergence of new functions within disordered sequences remain poorly understood. FCHO1 and FCHO2 are paralogous pioneer proteins of clathrin-mediated endocytosis that share a conserved domain architecture but perform distinct cellular functions and cannot compensate for each other's loss. Here we show that functional divergence between these proteins is associated with the acquisition of transient structure within their disordered regions. Using nuclear magnetic resonance spectroscopy, we identify two highly populated α-helical elements in the IDR of FCHO1 that are absent from FCHO2. One of these helices mediates FCHO1 self-association and drives intracellular assembly, whereas FCHO2 lacks this behavior. Introduction of the FCHO1 helix into FCHO2 is sufficient to confer self-association and cellular assembly, demonstrating that a transient structural element embedded within an IDR can act as a transferable functional module. Evolutionary analysis reveals that this helical propensity emerged following duplication of the ancestral FCHO gene and became progressively reinforced during evolution. Despite this divergence, the same region retains a conserved membrane-binding activity in both paralogs. Together, our findings show how acquisition of transient secondary structure within an intrinsically disordered region can generate new molecular behaviors while preserving ancestral functions, providing a mechanism for the functional specialization of paralogous proteins.

Transmembrane‐spanning cell surface proteins undergo maturation from the site of synthesis in the endoplasmic reticulum to the plasma membrane, and may be endocytosed back into the cell. Microscopy allows the visual separation of such populations at the cell surface and in intracellular compartments and to monitor trafficking and dissect the relative abundance of such pools. Protein tags, like SNAP‐tag, HTP, and FAST, combined with their respective (im)permeable ligands fused to fluorescent dyes, open up new possibilities to distinguish between intra‐ and extracellular protein pools. Introducing cleavable groups in dye‐ligand molecules allows interrogation of endocytosed pools by stripping the remaining surface pools. Latest developments, for instance, fluorescence lifetime imaging microscopy and brightness demixing, give further opportunities in microscopy.

The presence of intrinsically disordered proteins (IDPs) in the extracellular environment of the brain suggests that protein disorder may serve functions beyond those confined to the intracellular space. Tau, a prototypical IDP linked to tauopathies, a class of neurodegenerative diseases, is continuously released and spreads between cells in the brain, yet the biological significance of this extracellular phase remains unresolved. Here, we show that spreading tau coordinates neutral lipid homeostasis by promoting neuronal lipid efflux and enhancing resilience to lipid toxicity. Integrating transcriptomics and lipidomics, we demonstrate that tau spreading reprograms lipid transport pathways and triggers redistribution of triacylglycerol pools, remodeling neutral lipid metabolism. Mechanistically, cellular uptake of spreading tau drives its accumulation within neutral lipid-rich compartments and promotes neuronal lipid efflux. The exported lipids are enriched in peroxidized species that are subsequently transferred to astrocytes, reducing neuronal lipid stress and revealing a pathway through which tau regulates lipid homeostasis. Our findings reframe spreading tau as a regulator of intercellular lipid flux and position protein disorder as an active mediator of cell-to-cell communication in coordinating tissue metabolism. Loss of this homeostatic function is likely to contribute to the early metabolic dysfunction associated with tauopathy.