Martin Lehmann

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.

Peroxisomes are essential for cellular homeostasis and human health due to their central metabolic and physiological functions. The peroxisomal membrane protein PEX14 is indispensable for matrix protein import into peroxisomes. Recent studies support a hybrid model in which pre-peroxisomal vesicles originating from both, mitochondria and the endoplasmic reticulum, fuse to generate peroxisomes. PEX14 has repeatedly been observed at mitochondria in biogenesis disease models or upon protein overexpression, but such localization has generally been interpreted as aberrant mislocalization. Whether endogenous PEX14 naturally localizes to mitochondria and contributes to peroxisome biogenesis has remained unknown. Here, using high-content and super-resolution microscopy, we demonstrate that endogenous PEX14 localizes to the mitochondrial outer membrane. We show that ectopically expressed PEX14 accumulates at mitochondria, and PEX14 variants unable to access mitochondria and restricted to peroxisomes fail to rescue the PEX14 knockout phenotype. Vice versa, a mitochondria-targeted PEX14 variant supports peroxisome biogenesis. Our results identify the physiological mitochondrial pool of PEX14 in human cells and establish it as an essential intermediate in de novo peroxisome biogenesis.

Tight junctions (TJs) regulate paracellular permeability, cell polarity and cell mechanics of barrier-forming tissues. This Review explores how TJs adapt their structure and function across tissues, with a focus on the claudin-based strand network, its regulation by zonula occludens scaffold proteins and its dysfunction in human diseases. We discuss recent insights into TJ assembly through biomolecular condensation, highlighting how scaffold self-organization integrates adhesion, actin dynamics and polarity cues. We examine TJ maintenance across timescales, from rapid protein turnover to long-term remodelling during development. Finally, we focus on TJ function in lumen formation and review TJ dysfunctions and strategies to target TJ proteins therapeutically. We close by highlighting emerging approaches to tackle open structural, mechanical and functional questions of TJ physiology and pathology.

ZUSAMMENFASSUNG: Der aktuelle Stand der Forschung zu zellpenetrierenden Peptiden und ihrem Einfluss auf intrazelluläre Proteinaufnahme unterstreicht die entscheidende Rolle einer anhaltenden Membranassoziation für eine hohe Aufnahmeeffizienz. Diese Studie zeigt, wie zelloberflächenreaktive und polyfluoralkylierte Polyarginin‐Peptide mit unterschiedlichem Fluorgehalt als CPP‐Additive die Proteinaufnahme in lebende Säugerzellen signifikant verbessern. Bei niedrigen mikromolaren Konzentrationen (2.5 µM) verbesserten CPP‐Additive mit 11 bis 13 Fluoratomen die intrazelluläre Proteinaufnahme um mehr als das Zweifache im Vergleich zu einer unmarkierten Kontrolle. Hierbei wurde keine Zytotoxizität beobachtet. Zeitaufgelöste Fluoreszenzmikroskopie mit lebenden Zellen zeigte, dass ein CPP‐Additiv mit 13 Fluoratomen im Vergleich zu einem unmarkierten CPP‐Additiv eine längere Membranassoziation (> 5 Minuten) aufwies und gleichzeitig eine schnelle Proteininternalisierung ermöglichte (innerhalb von 10 Minuten). Bemerkenswerterweise zeigten oberflächenverstärkte Infrarotabsorptionsspektroskopie (SEIRAS) Experimente mit POPC‐Modellmembranen, dass fluorhaltige CPP‐Additive zunächst überwiegend als Aggregate mit der Lipid‐Doppelschicht interagierten und anschließend als Monomere und ohne intermolekulare Fluor‐Fluor Wechselwirkungen in das Membraninnere eindrangen. Ergänzende Molekulardynamiksimulationen der initialen Membranassoziation offenbarten die Membranverankerung eines CPP‐Additivs mit 13 Fluoratomen, während für ein unmarkiertes CPP‐Additiv innerhalb desselben Zeitraums keine Membranverankerung beobachtet wurde. Insgesamt zeigen unsere Ergebnisse, dass polyfluoralkylierte CPP‐Additive effiziente und zellverträgliche Hilfsmittel für eine effiziente intrazelluläre Proteinaufnahme sind. Gleichzeitig liefern wir mechanistische Einblicke in die molekularen Grundlagen der Membraninteraktion.

Recent advances in cell‐penetrating peptide (CPP)‐mediated intracellular protein delivery emphasized the critical role of sustained membrane association in enhancing delivery efficiency. Here, we report cell‐surface‐reactive, polyfluoroalkyl‐tagged polyarginine peptides with varying fluorine content as CPP‐additives that significantly enhance protein delivery in living cells. At low micromolar concentrations (2.5 µM), CPP‐additives containing 11–13 fluorine atoms enhanced intracellular protein delivery over 2‐fold relative to a tagless control without observable cytotoxicity. Live‐cell time‐lapse fluorescence imaging revealed that a CPP‐additive with 13 fluorine atoms showed prolonged membrane association (>5 min) relative to a tagless control and facilitated rapid protein internalization within 10 min. Remarkably, surface‐enhanced infrared absorption spectroscopy (SEIRAS) with POPC membranes showed that fluorous CPP‐additives initially interacted with the lipid bilayer predominantly as aggregates but subsequently inserted into the membrane interior as monomers without fluorous tag‐tag association. Complementary molecular dynamics simulations of the initial membrane‐association step provided atomistic insight, showing partial lipid insertion of a monomeric CPP‐additive with 13 fluorine atoms while no insertion was observed for a tagless control within the same time scale. Collectively, our findings establish polyfluoroalkyl‐tagged CPP‐additives as potent, non‐cytotoxic vectors for intracellular protein delivery and provide mechanistic detail regarding the molecular basis of their lipid bilayer interactions.

Cellular adhesion to the extracellular matrix is essential for morphogenesis, tissue integrity and survival signalling. The best understood adhesion structures are focal adhesions (FAs). In spite of their importance, our knowledge of upstream factors that integrate FA dynamics with other cellular processes, such as metabolism, remains fragmentary. Using a genome-wide screen, we identify aldolase A, a key glycolytic enzyme that converts fructose-1,6-bisphosphate (FBP), as a regulatory switch that links metabolic flux to FA assembly and cell morphogenesis. We show that cellular FBP serves as a signalling metabolite, which transmits information about the metabolic cell state to the actin-based machinery for cell adhesion and protrusion. This mechanism involves FBP binding to the Rac1 inhibitor RCC2 and a concomitant elevation of Rac1 activity resulting in actin reorganization, increased FA assembly and elevated protrusive activity. Here we predict this mechanism to be crucial for processes ranging from development to cancer.

Providing immediate access for functional proteins inside living cells would unlock unprecedented control over cellular processes; however, commonly used endocytic delivery suffers from endosomal trapping and degradation. One of the most powerful non-endosomal delivery methods uses cell surface anchored cell penetrating peptide (CPP)-additives that allow proteins to enter cells directly. Nevertheless, the underlying molecular mechanism involved in direct entry via crossing the cell membrane (protein translocation through the cell) and the major driving forces remain controversially discussed. Here, we provide a stepwise molecular picture on how CPP-additives enable uptake of protein cargoes through direct membrane translocation. CPP-additives accumulate on the cell surface in nucleation zones, locally hyperpolarizing the membrane, and induce transient water pores that allow selective CPP-protein entry without compromising membrane integrity. These fundamental mechanistic insights provide a firm basis for rationally optimizing delivery strategies using highly cationic CPPs, ultimately resulting in innovative and smart protein delivery strategies to advance therapeutic protein applications. Abstract Figure:

Cytokinesis critically depends on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P ₂ ]. Synthesis of PI(4,5)P ₂ is crucial for several stages of cytokinesis, including actomyosin ring assembly and constriction, membrane tethering of spindle microtubules, and midbody organization. How these activities of PI(4,5)P ₂ are spatiotemporally controlled is unknown. Here we unravel a crucial function for local PI(4,5)P ₂ synthesis at the ingressed cleavage furrow by septin-binding isoforms of PIPKIγ to control midbody formation. We demonstrate that loss of PIPKIγ isoforms perturbs cytokinesis by impairing septin association with microtubules, and anillin and septin deposition at the intercellular bridge and at the midbody. This mechanism requires the ability of PIPKIγ isoforms to synthesize PI(4,5)P ₂ and to associate with septins. Septins and PIPKIγ further synergize to promote centralspindlin recruitment to the midbody. Our findings establish septin-associated PIPKIγ isoforms as spatiotemporal controllers of midbody organization during cytokinesis that act through generating a local pool of PI4,5P ₂ at the ingressed cleavage furrow.

Visualization of proteins can be achieved by genetically grafting HaloTag Protein (HTP) into the protein of interest followed by incubation with a dye-linked HaloTag Ligand (HTL). This approach allows for use of fluorophores optimized for specific optical techniques or of cell-impermeable dyes to selectively label cell surface proteins. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Here we show that several dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed, using a one-step protocol. We validate such compounds, termed dye-SHTL (dye shuttle), in living cells, and demonstrate exclusive membrane staining. In transduced primary hippocampal neurons, we label a neuromodulatory receptor with dyes optimized for stimulated emission by depletion super-resolution microscopy, allowing accuracy in distinguishing surface versus internal receptors of the presynaptic terminal. This approach offers broad utility for surface-specific protein labelling.