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.

Cold acclimation is a crucial physiological process that enables plants to adapt to low temperatures. A key aspect of this acclimation is lipid remodeling, which preserves membrane fluidity and integrity under cold stress. Proteins of the chloroplast envelope membranes are increasingly recognized for their role in acclimation to changing environmental conditions. While lipid synthesis occurs at the inner envelope membrane, little is known about specific proteins involved in lipid remodeling during cold acclimation. In this study, we investigated the role of CHLOROPLAST LIPID REMODELING PROTEIN 23 (CLRP23) as a component of the inner chloroplast envelope membrane. Subcellular fractionation combined with protease protection assays provided evidence for its orientation toward the intermembrane space. To explore its function, we analyzed the physiological performance and lipid composition in CLRP23-deficient mutant plants. Under cold stress, we observed significant impairments in photosynthesis and increases in the galactolipid response, suggesting CLRP23 is involved in lipid remodeling. Lipid overlay assays, supported by in silico docking analyses, demonstrated that CLRP23 can directly interact with chloroplast lipids, including galactolipids. Complementary transcriptomic and proteomic analyses revealed broader effects on cold-responsive pathways, supporting the view that CLRP23 contributes to the integration of membrane and metabolic responses during acclimation. These findings expand our understanding of protein-mediated processes during cold acclimation.

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.

Oxygenic photosynthesis generates ATP and NADPH via linear electron flow from water to NADP+, a process thought to require photosystem I (PSI) for reductant formation. Here we demonstrate that oxygenic photosynthesis can operate without PSI in the cyanobacterium Synechocystis sp. PCC 6803. Using genetic engineering and adaptive laboratory evolution, we obtained PSI-deficient lineages capable of photoautotrophic growth, inorganic carbon fixation, and light-driven oxygen evolution. PSI-independent photoautotrophy arose from co-mutations in at least two proteins, including the translation elongation factor G (FusA), and required a functional NDH-1 complex. We propose that the light-driven electron transport in the evolved strains is reorganised in two branches: one involving terminal oxidases to generate proton motive force, and a second in which reverse NDH-1 activity exploits this gradient to produce reductant. These findings uncover unexpected plasticity in the thylakoid electron transport network and prompt a reassessment of the canonical requirement for PSI in oxygenic photosynthesis.

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.

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.

Plants trigger specific changes in gene transcription to acclimate to low-oxygen concentrations. Plastid-localized STEAROYL-ACYL CARRIER PROTEIN ∆9-DESATURASE 6 (SAD6) converts C18:0- into C18:1-fatty acids and belongs to the core hypoxia-responsive genes. SAD6 expression under hypoxia is activated by RAP2.12, which is a group-VII ethylene-response factor (ERF-VII) transcription factor regulated by sequestering to ACYL-CoA BINDING PROTEIN (ACBP) at the plasma membrane or proteasomal degradation via the PLANT CYSTEINE-OXIDASE N-degron pathway in response to molecular oxygen. Besides its role as hypoxia-specific marker gene, the biological function of SAD6 remains unclear. Here, we show that SAD6 overexpression mostly phenocopies plants overexpressing an N-degron insensitive Δ13RAP2.12 protein, which fails to be targeted to proteasomal degradation, while silencing of SAD6 in Δ13RAP2.12 -overexpressor plants largely restores the wildtype phenotype, indicating SAD6 is crucial to shape the growth phenotype of plants with deregulated RAP2.12. However, silencing of SAD6 also attenuated the expression of other important hypoxia-responsive genes, both, in Δ13RAP2.12 -overexpressor and wildtype backgrounds in response to hypoxic treatment, indicating a signaling role of SAD6 in activating RAP2.12. By using green-fluorescent protein-reporter constructs we found that this is due to SAD6 promoting RAP2.12 relocation from the plasma membrane to the nucleus, most likely by its role to increase C18:1-acyl-CoA, which is bound by ACBP as ligand. These results indicate SAD6 to be crucial to trigger relocation of sequestered RAP2.12 protein to the nucleus, showing an involvement of plastid function in hypoxia signaling, which links plastid fatty-acid metabolism with plastid-to-nucleus retrograde signaling via ERF-VII factors to improve hypoxic-stress resistance.

Cytokinesis critically depends on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Synthesis of PI(4,5)P2 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)P2 are spatiotemporally controlled is unknown. Here we unravel a crucial function for local PI(4,5)P2 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)P2 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 PI4,5P2 at the ingressed cleavage furrow.

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.

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.

ABSTRACT Fluorescent dyes are critical to visualizing nucleic acids in many applications. SYTOX Orange and SYTOX Green are cyanine dyes, used in dead cell staining and increasingly in single-molecule assays to probe DNA supercoiling and processing. However, their structures and effects on DNA mechanics are not or only partially known. We determine the structure of SYTOX Orange to be (E)-2-((2-(4 ((diethyl(methyl)ammonio)methyl)phenyl)-6-methoxy-1-methylquinolin-4(1H)-ylidene)methyl)-4-methyloxazolo[4,5-b]pyridin-4-ium, identical to SYBR Gold except for an aza-benzoxazol core that is fundamentally different from other dyes in the SYTOX and SYBR families. We report SYTOX Green to be (Z)-2-(bis(3-(trimethylammonio)propyl)amino)-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-1-phenylquinolin-1-ium, similar to PicoGreen. Using magnetic tweezers, we characterize the effect of SYTOX Orange and SYTOX Green on DNA mechanics. They lengthen and unwind DNA consistent with intercalation and the DNA unwinding angles per dye are 21.1(1)° and 20.5(1)° for SYTOX Orange and Green, respectively. Both dyes leave the DNA bending persistence length and plectoneme size almost unaltered (<10% change up to 1 µM), which is advantageous in assays probing DNA supercoiling. Their photophysical properties reveal close agreement between single-molecule manipulation and optical absorbance and fluorescence spectroscopy. Our comprehensive set of complementary measurements relates mechanical and optical properties to the molecular structures and provides recommendations for their use in applications.

Cross-linking mass spectrometry (XL-MS) enables the mapping of protein-protein interactions on the cellular level. When applied to all compartments of mitochondria, the sheer number of cross-links and connections can be overwhelming, rendering simple cluster analyses convoluted and uninformative. To address this limitation, we integrate the XL-MS data, 3D electron microscopy data, and localization annotations with a supra coarse-grained molecular dynamics simulation to sort all data, making clusters more accessible and interpretable. In the context of mitochondria, this method, through a total of 6.9 milliseconds of simulations, successfully identifies known, suggests unknown protein clusters, and reveals the distribution of inner mitochondrial membrane proteins allowing a more precise localization within compartments. Our integrative approach suggests, that two so-far ambigiously placed proteins FAM162A and TMEM126A are localized in the cristae, which is validated through super resolution microscopy. Together, this demonstrates the strong potential of the presented approach.

Cold acclimation is a crucial physiological process that enables plants to adapt to low temperatures. A key aspect of this adaptation is lipid remodeling, which preserves membrane fluidity and integrity under cold stress. Proteins of the chloroplast envelope membranes are increasingly recognized for their role in acclimation to changing environmental conditions. While lipid synthesis occurs at the inner envelope membrane, little is known about specific proteins involved in lipid remodeling during cold acclimation. In this study, we investigate the role of Chloroplast Lipid Remodeling Protein 23 (CLRP23) as a component of the inner chloroplast envelope membrane. Subcellular fractionation combined with protease protection assays provided evidence for its orientation toward the intermembrane space. To explore its function, we analyzed the physiological performance and lipid composition in CLRP23-deficient mutant plants. Under cold stress, we observed significant impairments in photosynthesis and exaggerations in galactolipid response, suggesting CLRP23 is involved in lipid remodeling. Lipid overlay assays, supported by in silico docking analyses, demonstrate that CLRP23 can directly interact with chloroplast lipids, including galactolipids. Complementary transcriptomic and proteomic analyses reveal broader effects on cold-responsive pathways, supporting the view that CLRP23 contributes to the integration of membrane and metabolic responses during acclimation. These findings expand our understanding of protein-mediated processes during cold acclimation. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, TRR175

Claudin-2 (Cldn2) is a typical tight junction protein of leaky epithelia that forms paracellular channels for small cations and water. Claudin-3 (Cldn3) and claudin-1 (Cldn1) are barrier formers and may interact with Cldn2. We aimed to investigate whether this interaction affects the permeability of Cldn2 channels to ions and/or water. To achieve this, two knockout kidney cell lines (MDCK C7/Cldn3KO and MDCK II/quinKO) were used to express Cldn2 and Cldn2/Cldn3. Furthermore, MDCK II/quinKO/Cldn2/Cldn1 cells were generated for comparison. Electrophysiological assays were performed to evaluate the function and properties of Cldn2 channels in these cell models. Cis- and trans-interaction of Cldn2 with Cldn1 or Cldn3 was assessed in MDCK II/quinKO cells by FRET and enrichment assays, respectively. At the tight junction, Cldn2 had a closer cis-proximity to Cldn1 than to Cldn3, but a stronger trans-interaction with the latter. In comparison to cells expressing Cldn2 alone, co-expression with Cldn3 (in both cell models) or Cldn1 (in MDCK II/quinKO cells) resulted in lower cation permeabilities without altering the Eisenman sequences. Other than ion permeability, water flux showed no differences between MDCK C7/Cldn3KO cells expressing Cldn2 and those co-expressing Cldn2/Cldn3. Based on these results, we propose a model in which Cldn2-Cldn1 cis- and Cldn2-Cldn3 trans-interaction leads to a mixture of homo-oligomeric Cldn2 and hetero-oligomeric Cldn2/Cldn1 or Cldn2/Cldn3 channels. The latter would have a pore center where charges are neutralized, by this impairing cation permeability while still allowing water to pass.

Bright near-infrared fluorophores are in demand for microscopy. We showcase a deuterated oxazine being 23% brighter vs. ATTO700. With a longer lifetime of 1.85 nanoseconds, we find the best-in-class SulfoOxazine700-d10 to stain mitochondria for confocal microscopy, and demonstrate unaffected diffusion properties in single molecule fluorescence correlation spectroscopy.

The light‐driven formation of a proton‐motive force ( pmf ) across thylakoid membranes is crucial for ATP synthesis and photosynthesis in chloroplasts and cyanobacteria. Cyclic electron flow (CEF) around photosystem (PS) I is hypothesized to be a key contributor to pmf formation, but direct observation of CEF in vivo remains a major challenge. As one possible proxy, pmf formation can be measured on a millisecond scale using electrochromic shifts (ECS) of thylakoid pigments conventionally observed in plants through absorbance changes at a wavelength of 515 nm (P515). In this study, we describe a new P515 signal in the model cyanobacterium Synechocystis sp. PCC 6803, which can be observed on a time‐scale of seconds to minutes upon red actinic light treatment. Treatments with uncouplers of electrochemical gradients and inhibitors of the photosynthetic electron transport chain indicate that the signal primarily traces proton gradient formation across the thylakoid membrane and suggest a major ECS contribution, but its precise origin remains to be deciphered. Still, the measuring routine allowed for phenotypic distinction between mutants with altered capacities for NDH‐ and PGR5‐dependent CEF around PSI, thus enabling future research on both CEF pathways and photosynthetic trans‐thylakoid pmf formation.

The Band-7 proteins, known as FLOTILLINs (FLOT), are present at the plasma membranes of most land plants. They function in clathrin-independent endocytosis and contribute to nodule formation following symbiotic infections. This study reveals that the single FLOT variant in Physcomitrium patens is located at the thylakoid membranes in chloroplasts, serving an unanticipated function. Phenotypic analysis of knockout and overexpression lines demonstrates that PpFLOT overexpression significantly impairs the high salinity tolerance of P. patens . Additionally, liquid protonema cultures of PpFLOT- OEX lines exhibited a distinct color change due to necrotic events and developed brachycyte-like cells. These changes correlate with the strength of PpFLOT expression and do not occur when these lines are cultivated on solid medium. Our study found that PpFLOT -OEX lines display increased chlorophyll and H2O2 production. We also discovered that PpFLOT is regulated by ABA and light, and its high expression can potentially affect retrograde signaling. Metabolomics and proteomics analyses revealed changes in the pigment and lipid composition as well as differentially accumulated proteins in PpFLOT mutant lines. We also observed changes in the expression of ion-transport related genes, accumulation of lipids crucial during pathogen defense, and differentially accumulated proteins taking part in multiple metabolomic pathways. Consequently, our study suggests a novel role for chloroplastic PpFLOT in plant terrestrialization, as it is putatively involved in Ca2+ and reactive oxygen species (ROS) signaling in response to abiotic and biotic stress, along with the light-dependent regulation of chlorophyll biosynthesis. ### Competing Interest Statement The authors have declared no competing interest.

Synaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine dynamic changes in synaptic protein nanoarchitecture typically rely on immunolabeling or on the expression of fluorescent proteins. The former employs chemical fluorophores and signal amplification but requires fixation. The latter enables monitoring of proteins by live microscopy but uses suboptimal fluorophores. Self-labeling tags have been introduced to combine the advantages of these two approaches, and here we introduce a knock-in mouse line where the essential presynaptic protein Munc13-1 is endogenously fused to the self-labeling SNAP tag. We demonstrate efficient Munc13-1-SNAP labeling in fixed cultured neurons and in brain sections by various SNAP dyes, as well as by a novel far-red and cell impermeable compound, SBG-SiR-d12. We introduce and characterize SBG-SiR-d12 as a highly efficient dye for SNAP-tag labeling of extracellular epitopes and of intracellular proteins such as Munc13-1 in fixed and permeabilized tissue. Finally, we show that Munc13-1-SNAP can be labeled in living neurons and monitored through live-cell imaging using confocal and super resolution microscopy. We conclude that the Unc13aSNAP mouse line is a useful tool for labeling the presynaptic compartment and for the analysis of presynaptic nanoarchitectural dynamics, with potential for wide adoption.

Mitochondria feature a sophisticated membrane architecture, with a planar mitochondrial outer membrane (MOM) and a folded inner membrane (MIM). Due to the remarkable adaptability of mitochondria, a proteinaceous network in the intermembrane space (IMS) was proposed to confer both stability and flexibility. However, components of such scaffolds, tentatively termed the 'mitoskeleton', have remained largely elusive. The mitochondrial contact site and organizing system (MICOS), a central organizer of mitochondrial membrane architecture, was suggested to participate in 'mitoskeleton' formation. Here, we structurally characterize the coiled-coil domain-containing 127 (CCDC127) protein, a putative interactor of MICOS. We show that CCDC127's amino-terminal transmembrane region is anchored in the MOM and the bulk soluble part exposed to the IMS. A crystal structure of CCDC127's central coiled-coil displays a parallel dimer which further oligomerizes into tetramers. We demonstrate that the carboxy-terminal helical bundle (CHB) domain dimerizes to create a peripheral membrane-binding site. Supported by electron microscopy data, we propose a structural model of CCDC127 as intramitochondrial membrane contact site protein mediating the structural organization of the IMS as part of the 'mitoskeleton'.