Target Cell Membrane Research Articles

Pore-Forming Protein LIN-24 Enhances Starvation Resilience in Caenorhabditis elegans by Modulating Lipid Metabolism and Mitochondrial Dynamics

The ability to survive starvation is a critical evolutionary adaptation, yet the molecular mechanisms underlying this capability remain incompletely understood. Pore-forming proteins (PFPs) are typically associated with immune defense, where they disturb the membranes of target cells. However, the role of PFPs in non-immune functions, particularly in metabolic and structural adaptations to starvation, is less explored. Here, we investigate the aerolysin-like PFP LIN-24 in Caenorhabditis elegans and uncover its novel function in enhancing starvation resistance. We found that LIN-24 expression is upregulated during starvation, leading to increased expression of the lipase-encoding gene lipl-3. This upregulation accelerates the mobilization and degradation of lipid stores, thereby sustaining energy levels. Additionally, LIN-24 overexpression significantly preserves muscle integrity, as evidenced by the maintenance of muscle structure compared to wild-type worms. Furthermore, we demonstrate that LIN-24 induces the formation of donut-shaped mitochondria, a structural change likely aimed at reducing ATP production to conserve energy during prolonged nutrient deprivation. This mitochondrial remodeling depends on genes involved in mitochondrial dynamics, including mff-1, mff-2, drp-1, and clk-1. Collectively, these findings expand our understanding of PFPs, demonstrating their multifaceted role in stress resistance beyond immune defense. LIN-24’s involvement in regulating metabolism, preserving muscle structure, and remodeling mitochondria highlights its crucial role in the adaptive response to starvation, offering novel insights into the evolution of stress resistance mechanisms and potential therapeutic targets for conditions related to muscle preservation and metabolic regulation.

Cell-membrane targeting sonodynamic therapy combination with FSP1 inhibition for ferroptosis-boosted immunotherapy.

Cell membrane targeting sonodynamic therapy could induce the accumulation of lipid peroxidation (LPO), drive ferroptosis, and further enhances immunogenic cell death (ICD) effects. However, ferroptosis is restrained by the ferroptosis suppressor protein 1 (FSP1) at the plasma membrane, which can catalyze the regeneration of ubiquinone (CoQ10) by using NAD(P)H to suppress the LPO accumulation. This work describes the construction of US-active nanoparticles (TiF NPs), which combinate cell-membrane targeting sonosensitizer TBT-CQi with FSP1 inhibitor (iFSP1), facilitating cell-membrane targeting sonodynamic-triggered ferroptosis. TiF NPs could induce a sonodynamic effect, which promotes lipid peroxidation and drives apoptosis. Furthermore, TiF NPs could suppress FSP1, induce CoQ10 depletion, down-regulate the NADH, enhance LPO accumulation, and finally induce ferroptosis. In vitro results demonstrated that synergetic cell membrane targeting SDT/FSP1 inhibition triggered immunogenic cell death (ICD). Moreover, the as-synthesized TiF NPs-mediated cell membrane targeting SDT/FSP1 inhibition thoroughly inhibited the tumor growth and simultaneously activated antitumor immunity to suppress lung metastasis. This work represents a promising tumor therapeutic strategy combining cell membrane targeting SDT and FSP1 inhibition, potentially inspiring further research in developing logical and effective cancer therapies based on synergistic SDT/ferroptosis.

Melanoma extracellular vesicles membrane coated nanoparticles as targeted delivery carriers for tumor and lungs.

Targeting is the most challenging problem to solve for drug delivery systems. Despite the use of targeting units such as antibodies, peptides and proteins to increase their penetration in tumors the amount of therapeutics that reach the target is very small, even with the use of nanoparticles (NPs). Nature has solved the selectivity problem using a combination of proteins and lipids that are exposed on the cell membranes and are able to recognize specific tissues as demonstrated by cancer metastasis. Extracellular vesicles (EVs) have a similar ability in target only certain organs or to return to their original cells, showing home behavior. Here we report a strategy inspired by nature, using a combination of NPs and the targeting cell membranes of EVs. We implement the EV membranes, extracted by the EVs produced by melanoma B16-BL6 cells, as a coating of organosilica porous particles with the aim of targeting tumors and lung metastasis, while avoiding systemic effects and accumulation of the NPs in undesired organs. The tissue-specific fingerprint provided by the EVs-derived membranes from melanoma cells provides preferential uptake into the tumor and selective targeting of lungs. The ability of the EVs hybrid systems to behave as the natural EVs was demonstrated in vitro and in vivo in two different tumor models. As a proof of concept, the loading and release of doxorubicin, was investigated and its accumulation demonstrated in the expected tissues.

Mechanistic insights on lycopene usage against diabetes and associated complications.

Lycopene is a tetraterpene compound belonging to carotenoids that are widely present in tomatoes and similar products. It is known as a powerful anti-oxidant and a non-provitamin A carotenoid. Lycopene has been found to effectively improve diabetes mellitus and its complications, such as cardiac complications, disorders caused by oxidative stress, and liver and neurological disorders. Furthermore, free radicals have been shown to disrupt the action of insulin by changing the physical state of the target cell membrane, while carotenoids improve insulin secretion and function in blood sugar regulation by neutralizing free radicals. It, therefore, seems that targeted clinical studies are needed to investigate the therapeutic effect of lycopene against metabolic disorders induced by diabetes. This review aims to summarize information on the sources and potential uses of lycopene and the possible mechanisms involved in the reduction of the above diseases. Its protective effects, in terms of toxicity and safety, are also discussed. The literature sources used in this review were PubMed, Google Scholar, Scopus, and Web of Science databases.

Targeting heparan sulfate proteoglycans as an effective strategy for inhibiting cancer cell migration and invasiveness compared to heparin.

By virtue of their ability to bind different growth factors, morphogens and extracellular matrix proteins, heparan sulfate proteoglycans (HSPGs) play a determinant role in cancer cell differentiation and migration. Despite a strong conceptual basis and promising preclinical results, clinical trials have failed to demonstrate any significant advantage of administering heparin to oncology patients. We exploited our anti-heparan sulfate branched peptide NT4 to test the opposite approach, namely, targeting HSPGs to interfere with their functions, instead of using heparin as a soluble competitor in human cell lines from pancreas adenocarcinoma, colon adenocarcinoma, rhabdomyosarcoma and two different breast cancers. We found that the anti-heparan sulfate peptide NT4 is more effective than heparin for inhibiting cancer cell adhesion, directional migration, colony formation and even cell growth, suggesting that targeting cell membrane HSPGs may be a more effective anti-metastatic strategy than using soluble heparin. Analysis of NT4 effects on cancer cell directional migration, associated to cellular distribution of HSPGs and cadherins in different migrating cancer cell lines, provided further indications on the molecular basis of HSPG functions, which may explain the efficiency of the HSPG targeting peptide.

Fluorination of Aza-BODIPY for Cancer Cell Plasma Membrane-Targeted Imaging and Therapy.

Photodynamic therapy (PDT) holds great potential in cancer treatment, leveraging photosensitizers (PSs) to deliver targeted therapy. Fluorination can optimize the physicochemical and biological properties of PSs for better PDT performance. Here, we report some high-performance multifunctional PSs specifically designed for cancer PDT by fluorinating aza-BODIPY with perfluoro-tert-butoxymethyl (PFBM) groups. Fluorination plays several roles, including enhancing selective cancer cell uptake, plasma membrane (PM) targeting, and inducing pyroptosis. It also enables fluorescence imaging (FLI) and fluorine-19 magnetic resonance imaging (19F MRI) as well as facilitates oxygen delivery and oxygen partial pressure (pO2) measurements. Comparative physicochemical and biological studies, along with molecular dynamics simulations, reveal that fluorinated PSs selectively eradicate cancer cells by oxidizing PM phospholipids with singlet oxygen (1O2) and inducing pyroptosis, which enables effectively suppressed tumor growth by self-oxygenated 19F MRI-FLI-guided PDT in mice. This study demonstrates a fluorination strategy for tailoring high-performance multifunctional cancer PM-targeting materials for cancer therapy and beyond.

Neuronal Tracing and Visualization of Nerve Injury by a Membrane-Anchoring Aggregation-Induced Emission Probe.

Deciphering neuronal circuits is pivotal for deepening our understanding of neuronal functions and advancing treatments for neurological disorders. Conventional neuronal tracers suffer from restrictions such as limited penetration depth, high immunogenicity, and inadequacy for long-term and in vivo imaging. In this context, we introduce an aggregation-induced emission luminogen (AIEgen), MeOTFVP, engineered for enhanced neuronal tracing and imaging. MeOTFVP is strategically designed to target cell membranes by integrating into the phospholipid bilayer through its amphipathy. The donor-acceptor molecular skeleton facilitates a red shift of its photoluminescence into the near-infrared (NIR) spectrum, significantly improving tissue penetration. The affinity of MeOTFVP for cell membranes, coupled with its deep tissue penetration, allows precise tracing in the paw-dorsal root ganglia (DRG) circuit and detailed imaging of the sciatic nerve. This study showcases the application of MeOTFVP as a dual-function neuronal tracer, propelling forward the possibilities for advanced neuronal tracing and imaging using AIEgens.

Anti-Candida albicans effect and mechanism of Pachysandra axillaris Franch.

Pachysandra axillaris Franch., a traditional herbal medicine in Yunnan, has been used to treat traumatic injuries and stomach ailments, some of which were related to microbial infections in conventional applications, but, to the best of our knowledge, the antifungal bioactivity of this plant and its main antifungal components have not been previously reported. To identify the antifungal compounds of P. axillaris against fluconazole-resistant C. albicans in vitro and in vivo, and then elucidate the underlying mechanism of action. The antifungal compounds were obtained by bioguided isolation, and then they were investigated in vitro by MIC, growth curves, time-kill assay, and drug resistance induction. The antifungal mechanism was explored using combined network pharmacology and metabolomic analysis, and further supported by analyzing sterol composition using LC-MS/MS, scanning and transmission electron microscopy observation of fungal cell morphology, examining its effects on cell membranes using the fluorescent probes and RT-qPCR. Additionally, the antifungal effect in vivo was evaluated by a murine C. albicans skin infection model. Three bioactive compounds from P. axillaris efficiently inhibited fluconazole-resistant C. albicans (MIC=4μg/mL), in which the major compound, pachysamine M, affected the ergosterol biosynthesis pathway by inhibiting ERG genes (ERG1, ERG4, ERG7, ERG9, and ERG24), leading to the accumulation of squalene, lanosterol, and zymosterol. So, pachysamine M targeted cell membranes in vitro by reducing the ergosterol level, to avoid drug resistance. In addition, it promoted wound healing, reduced fungal load, and alleviated inflammation in vivo. Pachysamine M, an antifungal compound without reported before, inhibited fluconazole-resistant C. albicans efficiently in vitro and in vivo, and its mechanism targeted cell membranes, reducing the risk of drug resistance, which validated the traditional use of P. axillaris for the treatment of fungal skin infections.

Dengue Virus Structural Proteins Are Expressed on the Surface of DENV-Infected Cells and Are a Target for Antibody-Dependent Cellular Phagocytosis.

Dengue virus (DENV) is an arboviral pathogen found in >100 countries and a source of significant morbidity and mortality. While the mechanisms underpinning the pathophysiology of severe Dengue are incompletely understood, it has been hypothesized that antibodies directed against the DENV envelope (E) protein can facilitate antibody-dependent enhancement (ADE) of the infection, increasing the number of infected cells and the severity of disease in an exposed individual. Accordingly, there is interest in defining mechanisms for directly targeting DENV-infected cells for immunologic clearance, an approach that bypasses the risk of ADE. We have previously demonstrated that antibodies specific to DENV nonstructural protein 1 (NS1) can opsonize and facilitate the phagocytic clearance of DENV-infected cells. However, it is currently unclear if other DENV antigens are expressed on the surface of infected cells and if these antigens can be targeted by antibody-dependent clearance mechanisms. In this study, we demonstrate that DENV structural proteins are expressed on the surface of DENV-infected cells and that these antigens can be opsonized by both DENV-immune sera and monoclonal antibodies. In addition, DENV E-specific antibodies can facilitate phagocytic uptake of material from DENV-infected cells, resulting in the target-cell membrane localizing to endosomes of the engulfing phagocyte. Notably, there was no selective enrichment of DENV genomic material in monocytes that had phagocytosed DENV-infected cell material compared with nonphagocytic monocytes. In their totality, these data reinforce the concept that DENV E-reactive antibodies have a multifaceted role in DENV immunity and pathogenesis beyond neutralization and/or infection enhancement.

Repurposing TAK-285 as An Antibacterial Agent against Multidrug-Resistant Staphylococcus aureus by Targeting Cell Membrane.

Infections and antimicrobial resistance are becoming serious global public health crises. Multidrug-resistant Staphylococcus aureus (S. aureus) infections necessitate novel antimicrobial development. In this study, we demonstrated TAK-285, a novel dual HER2/EGFR inhibitor, exerted antibacterial activity against 17 clinical methicillin-resistant S. aureus (MRSA) and 15 methicillin sensitive S. aureus (MSSA) isolates in vitro, with a minimum inhibitory concentration (MIC) of 13.7μg/mL. At 1 × MIC, TAK-285 completely inhibited the growth of S. aureus bacterial planktonic cells, and at 2 × MIC, it exhibited a superior inhibitory effect on intracellular S. aureus SA113-GFP compared to linezolid. Moreover, TAK-285 effectively inhibited biofilm formation at sub-MIC, eradicated mature biofilm and eliminated bacteria within biofilms, as confirmed by CLSM. Furthermore, the disruption of cell membrane permeability and potential was found by TAK-285 on S. aureus, suggesting its targeting of cell membrane integrity. Global proteomic analysis demonstrated that TAK-285 disturbed the metabolic processes of S. aureus, interfered with biofilm-related gene expression, and disrupted membrane-associated proteins. Conclusively, we repurposed TAK-285 as an antimicrobial with anti-biofilm properties against S. aureus by targeting cell membrane. This study provided strong evidence for the potential of TAK-285 as a promising antimicrobial agent against S. aureus.

C-terminal tagging enhances the detection sensitivity of interlekin receptor type 1.

Substances released outside of the cells during cell necrosis are collectively called danger-associated molecular patterns (DAMPS) or alarmins. A pro-inflammatory cytokine, interleukin-1α (IL-1α) is known as a typical alarmin. IL-1α transmits signals by binding to IL-1 receptor 1 (IL-1R1), type I protein, expressed on the cell membrane of target cells, but detection of IL-1R1 at the protein and mRNA levels is difficult. Although the reasons are not elucidated, we attempted to add the HiBiT-tag to the N-terminus (N'-R1) or C-terminus (C'-R1) of IL-1R1 to examine whether the detection sensitivity can be augmented. Increase in detection sensitivity will allow the investigation of its function and subcellular localization much further. Using uterine cervical cancer-derived HeLa cells and its derivative CR-R1-4 cells lacking IL-1R1, C'-R1 was demonstrated to significantly increase the detection sensitivity of IL-1R1. Furthermore, the signal transduction function of neither N'-R1 nor C'-R1 was affected. Immunofluorescence cell staining revealed that wild-type IL-1R1 is mainly localized in the nucleus, whereas C'-R1 is localized both in the nucleus and the cytoplasm. The above results showed that adding a tag to the C-terminus of IL-1R1 increases detection sensitivity while maintaining its function. In the future, we would like to further investigate the relationship between changes in the intracellular localization of C'-R1 and increases in detection sensitivity.

New near-infrared fluorescent probe for imaging superoxide anion of cell membrane

Selective imaging of superoxide anion is important for understanding its role in cell membrane biology, but is often a challenging task because of the lack of an effective fluorescence probe. In this study, a new near-infrared fluorescent probe (SHX-O) that can target cell membrane was developed for imaging superoxide anion. SHX-O was designed by simultaneously incorporating a sulfonated bis-indole and a diphenylphosphinyl recognition group into the hemicyanine moiety. The probe itself showed a rather weak fluorescence due to the hemicyanine's hydroxyl substitution; however, its reaction with superoxide anion caused a large enhancement of near-infrared fluorescence at 790nm. Moreover, SHX-O exhibited not only high selectivity for superoxide anion over other reactive oxygen species, but also specific cell membrane localization, which may be attributed to the probe's amphiphilic structure. Using the probe, fluorescence imaging of cell membrane superoxide anion produced in the presence of xanthine oxidase and xanthine has been achieved in living cells. We believe that SHX-O may serve as a potential tool for imaging and investigating superoxide anion of cell membrane.

Mechanistic study of SCOOPs recognition by MIK2–BAK1 complex reveals the role of N-glycans in plant ligand–receptor–coreceptor complex formation

Ligand-induced receptor and co-receptor heterodimerization is a common mechanism in receptor kinase (RK) signalling activation. SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) mediate the complex formation of Arabidopsis RK MIK2 and co-receptor BAK1, triggering immune responses. Through structural, biochemical and genetic analyses, we demonstrate that SCOOPs use their SxS motif and adjacent residues to bind MIK2 and the carboxy-terminal GGR residues to link MIK2 to BAK1. While N-glycosylation of plant RKs is typically associated with protein maturation, plasma membrane targeting and conformation maintenance, a surprising revelation emerges from our crystal structural analysis of MIK2–SCOOP–BAK1 complexes. Specific N-glycans on MIK2 directly interact with BAK1 upon SCOOP sensing. The absence of N-glycosylation at the specific site in MIK2 neither affects its subcellular localization and protein accumulation in plant cells nor alters its structural conformation, but markedly reduces its affinity for BAK1, abolishing SCOOP-triggered immune responses. This N-glycan-mediated receptor and co-receptor heterodimerization occurs in both Arabidopsis and Brassica napus. Our findings elucidate the molecular basis of SCOOP perception by the MIK2–BAK1 immune complex and underscore the crucial role of N-glycans in plant receptor–coreceptor interactions and signalling activation, shaping immune responses.

Wheat TaPYL9-involved signalling pathway impacts plant drought response through regulating distinct osmotic stress-associated physiological indices.

The abscisic acid (ABA) signalling pathway plays a crucial role in plants' response to drought stress. In this study, we aimed to characterize the impact of an ABA signalling module, which consisted of TaPYL9 and its downstream partners in Triticum aestivum, on plant drought adaptation. Our results showed that TaPYL9 protein contains conserved motifs and targets plasma membrane and nucleus after being sorted by the endoplasmic reticulum. In addition, TaPYL9 transcripts in both roots and leaves were significantly upregulated in response to drought stress. We conducted glucuronidase (GUS) histochemical staining analysis for transgenic plants carrying a truncated TaPYL9 promoter, which suggested that cis-elements associate with ABA and drought response, such as ABRE, DRE and recognition sites MYB and MYC, regulating the gene transcription under drought conditions. Using protein interaction assays (i.e., yeast two-hybrid, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP) and in vitro pull-down), we demonstrated interactions between the intermediate segment of TaPYL9, the intermediate segment of TaPP2C6, the N-terminus of TaSnRK2.8 and the C-terminus of the transcription factor TabZIP1 in wheat, indicating the involvement of TaPYL9 in the constitution of an ABA signalling module, namely TaPYL9/TaPP2C6/TaSnRK2.8/TabZIP1. Transgene analysis revealed that TaPYL9, TaSnRK2.8 and TabZIP1 positively regulated drought response, while TaPP2C6 negatively regulated it, and that these genes were closely associated with the regulation of stomata movement, osmolyte accumulation and ROS homeostasis. Electrophoretic mobility shift (EMSA) and transcriptioal activation assays indicated that TabZIP1 interacted promoters of TaP5CS2, TaSLAC1-1 and TaCAT2 and activated transcription of these genes, which regulated proline biosynthesis, stomata movement and ROS scavenging upon drought signalling, respectively. Furthermore, we found that the transcripts of TaPYL9 and stress-responsive genes were positively correlated with yields in wheat cultivars under field drought conditions. Altogether, our findings suggest that the TaPYL9-involved signalling pathway significantly regulates drought response by modulating osmotic stress-associated physiological processes in T. aestivum.

In Situ Bioorthogonal Repair of the Vascular Endothelium Glycocalyx to Treat Acute Lung Injury.

In acute lung injury, destruction of the lung endothelial glycocalyx leads to vessel permeabilization and contributes to pulmonary edema and inflammation. Heparan sulfate, which accounts for >70% of glycosaminoglycans in the endothelial glycocalyx, plays a crucial physiological anti-inflammatory role. To treat acute lung injury, it is explored whether a two-step in vivo bioorthogonal chemistry strategy can covalently link intravenously administered heparan sulfate to the lung vascular endothelium and the damaged glycocalyx. First, fusogenic liposomes (EBP-Tz-FLs) carrying the reactive group tetrazine (Tz), and an E-selectin-binding peptide (EBP) to target the lung inflammatory endothelium are administered intravenously. This step aimed to anchor the tetrazine group to the membrane of inflammatory endothelial cells. Second, heparan sulfate (HS-TCO) conjugated to the trans-cyclooctene (TCO) group, which spontaneously reacts with Tz, is injected intravenously, leading to covalent heparan sulfate addition to the vascular endothelium. In a mouse model of acute lung injury, this approach substantially reduced vascular permeability and attenuated lung tissue infiltration. The EBP-Tz-FLs and HS-TCO showed favorable biocompatibility and safety both in vitro and in vivo. The proposed strategy shows good promise in acute lung injury therapy and covalently anchoring functional molecules onto the membrane of target cells.

Direct cytosolic delivery of siRNA via cell membrane fusion using cholesterol-enriched exosomes.

Efficient cytosolic delivery is a significant hurdle when using short interfering RNA (siRNA) in therapeutic applications. Here we show that cholesterol-rich exosomes are prone to entering cancer cells through membrane fusion, achieving direct cytosolic delivery of siRNA. Molecular dynamics simulations suggest that deformation and increased contact with the target cell membrane facilitate membrane fusion. In vitro we show that cholesterol-enriched milk-derived exosomes (MEs) achieve a significantly higher gene silencing effect of siRNA, inducing superior cancer cell apoptosis compared with the native and cholesterol-depleted MEs, as well as conventional transfection agents. When administered orally or intravenously to mice bearing orthotopic or subcutaneous tumours, the cholesterol-enriched MEs/siRNA exhibit antitumour activity superior to that of lipid nanoparticles. Collectively, by modulating the cholesterol content of exosome membranes to facilitate cell entry via membrane fusion, we provide a promising approach for siRNA-based gene therapy, paving the way for effective, safe and simple gene therapy strategies.

Probing Gag-Env dynamics at HIV-1 assembly sites using live-cell microscopy.

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.

A Kinesin-Like Protein, KAC, is Required for Light-Induced and Actin-Based Chloroplast Movement in Marchantia polymorpha.

Chloroplasts accumulate on the cell surface under weak light conditions to efficiently capture light but avoid strong light to minimize photodamage. The blue light receptor phototropin regulates the chloroplast movement in various plant species. In Arabidopsis thaliana, phototropin mediates the light-induced chloroplast movement and positioning via specialized actin filaments on the chloroplasts, chloroplast-actin filaments. KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT (KAC) and CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) are pivotal for actin-based chloroplast movement and positioning in land plants. However, the mechanisms by which KAC and CHUP1 regulate chloroplast movement and positioning remain unclear. In this study, we characterized KAC and CHUP1 orthologs in the liverwort Marchantia polymorpha, MpKAC and MpCHUP1, respectively. Their knockout mutants, Mpkacko and Mpchup1ko, impaired the light-induced chloroplast movement. Although Mpchup1ko showed mild chloroplast aggregation, Mpkacko displayed severe chloroplast aggregation, suggesting the greater contribution of MpKAC to the chloroplast anchorage to the plasma membrane. Analysis of the subcellular localization of the functional MpKAC-Citrine indicated that MpKAC-Citrine formed a punctate structure on the plasma membrane. Structure-function analysis of MpKAC revealed that the deletion of the conserved C-terminal domain abrogates its targeting to the plasma membrane and its function. The deletion of the N-terminal motor domain retains the plasma membrane targeting but abrogates the formation of punctate structure and shows a severe defect in the light-induced chloroplast movement. Our findings suggest that the formation of the punctate structure on the plasma membrane of MpKAC is essential for chloroplast movement.

Proximity-driven site-specific cyclization of phage-displayed peptides

Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvβ3.

Leveraging Chlorination-Based Mechanism for Resolving Subcellular Hypochlorous Acid.

Hypochlorous acid (HOCl) is crucial for pathogen defense, but an imbalance in HOCl levels can lead to tissue damage and inflammation. Existing HOCl indicators employ an oxidation approach, which may not truly reveal the chlorinative stress environment. We designed a suite of indicators with a new chlorination-based mechanism, termed HOClSense dyes, to resolve HOCl in sub-cellular compartments. HOClSense dyes allow the visualization of HOCl with both switch-on and switch-off detection modes with diverse emission colors, as well as a unique redshift in emission. HOClSense features a minimalistic design with impressive sensing performance in terms of HOCl selectivity, and our design also facilitates functionalization through click chemistry for resolving subcellular HOCl. As a proof of concept, we targeted plasma membrane and lysosomes with HOClSense for subcellular HOCl mapping. With utilizing HOClSense, we discovered the STING pathway-induced HOCl production and the abnormal HOCl production in Niemann-Pick diseases. To the best of our knowledge, this is the first chlorination-based HOCl indicator series for resolving subcellular HOCl.