Intracellular Environment Research Articles

PDZ2-conjugated-PLGA nanoparticles are tiny heroes in the battle against SARS-CoV-2

The COVID-19 pandemic caused by SARS-CoV-2 has highlighted the urgent need for innovative antiviral strategies to fight viral infections. Although a substantial part of the overall effort has been directed at the Spike protein to create an effective global vaccination strategy, other proteins have also been examined and identified as possible therapeutic targets. Among them, although initially underestimated, there is the SARS-CoV-2 E-protein, which turned out to be a key factor in viral pathogenesis due to its role in virus budding, assembly and spreading. The C-terminus of E-protein contains a PDZ-binding motif (PBM) that plays a key role in SARS-CoV-2 virulence as it is recognized and bound by the PDZ2 domain of the human tight junction protein ZO-1. The binding between the PDZ2 domain of ZO-1 and the C-terminal portion of SARS-CoV-2 E-protein has been extensively characterized. Our results prompted us to develop a possible adjuvant therapeutic strategy aimed at slowing down or inhibiting virus-mediated pathogenesis. Such innovation consists in the design and synthesis of externally PDZ2-ZO1 functionalized PLGA-based nanoparticles to be used as intracellular decoy. Contrary to conventional strategies, this innovative approach aims to capitalize on the E protein-PDZ2 interaction to prevent virus assembly and replication. In fact, the conjugation of the PDZ2 domain to polymeric nanoparticles increases the affinity toward the E protein effectively creating a “molecular sponge” able to sequester E proteins within the intracellular environment of infected cells. Our in vitro studies on selected cellular models, show that these nanodevices significantly reduce SARS-CoV-2-mediated virulence, emphasizing the importance of exploiting viral-host interactions for therapeutic benefit.

Programmed Lung Metastasis Immunotherapy via Cascade‐Responsive Cell Membrane‐Mimetic Copolymer‐Wrapped Nanoraspberry‐Mediated Elesclomol‐Copper Delivery

AbstractT lymphocytes play a central role in immunotherapy, utilizing physical interactions to actively inhibit the development of metastases. However, tumor immune privilege and heterogeneity pose challenges by protecting against immune attacks and limiting immune cell infiltration into tumors, particularly in invasive metastatic clusters. Here, a tumor penetrating magnetic particles (TUP) containing the cascade‐responsive cell membrane‐mimetic copolymer (zwitterionic 2‐methacryloyloxyethyl phosphorylcholine‐co‐3‐hydroxypyridin‐4‐one, PH) and cuproptosis molecules (elesclomol‐copper, EsCu) for programming T cell infiltration is developed. The intravenously injected TUP accumulates at the tumor via the charge conversion of PH and hyperthermia effects of TUP. In metastatic clusters, ES and Cu, triggered by intracellular environments and hyperthermia, are readily released. ES and Cu simultaneously induce cuproptosis of cancer cells and stimulate immune responses. This process destroys self‐defense mechanisms and exacerbates cytotoxicity. The therapies facilitate the release of tumor‐associated antigens, including neoantigens and damage‐associated molecular patterns. Following this, the 3‐hydroxypyridin‐4‐one groups on TUP act as antigen reservoirs, transporting autologous tumor‐associated antigens to dendritic cells, thereby inducing prolonged immune activation. TUP acts as an antigen reservoir in copper apoptosis‐mediated lung metastasis, promoting the accumulation of immune cells in metastatic clusters and effectively preventing the progression of metastatic tumors.

Macromolecular Crowding Effect on Chitosan-Hyaluronic Acid Complexation and the Activity of Encapsulated Catalase.

The associative phase separation of charged biomacromolecules plays a key role in many biophysical events that take place in crowded intracellular environments. Such natural polyelectrolyte complexation and phase separation often occur at nonstoichiometric charge ratios with the incorporation of bioactive proteins, which is not studied as extensively as those complexations at stoichiometric ratios. In this work, we investigated how the addition of a crowding agent (polyethylene glycol, PEG) affected the complexation between chitosan (CS) and hyaluronic acid (HA), especially at nonstoichiometric ratios, and the encapsulation of enzyme (catalase, CAT) by the colloidal complexes. The crowded environment promoted colloidal phase separation at low charge ratios, forming complexes with increased colloidal and dissolution stability, which resulted in a smaller size and polydispersity (PDI). The binding isotherms revealed that the addition of PEG greatly enhanced the ion-pairing strength (with increased ion-pairing equilibrium constant Ka from 4.92 × 104 without PEG to 1.08 × 106 with 200 g/L PEG) and switched the coacervation from endothermic to exothermic, which explained the promoted complexation and phase separation. At the stoichiometric charge ratio, the enhanced CS-HA interaction in crowded media generated a more solid-like coacervate phase with a denser network, slower chain relaxation, and higher modulus. Moreover, both crowding and complex encapsulation enhanced the activity and catalytic efficiency of CAT, represented by a 2-fold increase in catalytic efficiency (Kcat/Km) under 100 g/L PEG crowding and CS-HA complex encapsulation. This is likely due to the lower polarity in the microenvironment surrounding the enzyme molecules. By a systematic investigation of both nonstoichiometric and stoichiometric charge ratios under macromolecular crowding, this work provided new insights into the complexation between natural polyelectrolytes in a scenario closer to an intracellular environment.

Current updates for hyperuricemia and gout in age-related macular degeneration.

The escalating prevalence of metabolic syndrome poses a significant public health challenge, particularly among aging populations, with metabolic dysfunctions contributing to pro-inflammatory states. In this review, we delved into the less recognized association between hyperuricemia (HUA), a manifestation of metabolic syndrome and a primary risk factor for gout, and age-related macular degeneration (AMD), a sight-threatening ailment predominantly affecting the elderly. In recent years, inflammation, particularly its involvement in complement pathway dysregulation, has gained prominence in AMD pathophysiology. The contradictory role of uric acid (UA) in intercellular and intracellular environments was discussed, highlighting its antioxidant properties in plasma and its pro-oxidant effects intracellularly. Emerging evidence suggests a potential link between elevated serum uric acid levels and choroid neovascularization in AMD, providing insights into the role of HUA in retinal pathologies. Various pathways, including crystal-induced and non-crystal-induced mechanisms, were proposed to indicate the need for further research into the precise molecular interactions. The implication of HUA in AMD underscores its potential involvement in retinal pathologies, which entails interdisciplinary collaboration for a comprehensive understanding of its impact on retina and related clinical manifestations.

Design of diselenide-containing polyprodrug and its pH/redox co-triggered degradation and slow drug release for tumor-specific chemotherapy

Polyprodrugs have attracted intense interest for tumor-specific chemotherapy, by conjugating chemotherapeutic drugs via two dynamic covalent bonds in the polymer main chains. Here, pH/redox co-triggered degradable diselenide-containing polyprodrug with a high doxorubicin (DOX) content of 1.42×10−3 mmol/g was designed by linking diselenide-containing dimer of doxorubicin (D-DOXSeSe) with adipic dihydrazide. The polyprodrug could be triggered to degrade and release drug only when the hydrazone and diselenide conjugations on both sides of the drug units were cleaved, triggered by the higher acidity and higher GSH or ROS levels. Slow drug release was achieved in the simulated tumor intracellular microenvironment due to the lower solubility of the release drugs (DOX derivatives), with cumulative release of 10.3 % and 7.4 % in 96 h co-triggered with 10 mM GSH or 0.5 mM H2O2, without obvious drug mis-release in the simulated intracellular environment of the normal cells. It might provide a safe and efficient tumor-specific chemotherapy avoiding the toxic and side effects on the normal cells.

Revealing the Improved Binding Interaction of Plant Alkaloid Harmaline with Human Hemoglobin in Molecular Crowding Condition.

Harmaline and harmine are two structurally similar β-carboline alkaloids with several therapeutic activities, such as anti-inflammatory, antioxidant, neuroprotective, nephroprotective, antidiabetic, and antitumor activities. It has been previously reported that the interaction between harmaline and hemoglobin (Hb) is weak in buffer media compared to harmine. Crowding agents induce a molecular crowding environment in the ex vivo condition, which is almost similar to the intracellular environment. In this present study, we have investigated the nature of the interactions of harmaline and harmine with Hb by increasing the percentage of the crowding agent in buffer solution. The results of the UV-vis and fluorescence spectroscopy analysis have showed that with an increasing proportion of crowding agents, the interaction between harmaline and Hb is steadily improving in comparison to harmine. It has been found that the binding constant of Hb-harmaline reaches 6.82 × 105 M-1 in the 40% polyethylene glycol 200-mediated crowding condition, indicating high affinity compared to very low interaction in buffer media. Steady-state fluorescence anisotropy along with fluorescence lifetime measurements further revealed that the rotational movement of harmaline is maximally restricted by Hb in high crowding environments. Stoichiometry results represent that Hb and harmaline interacts in a 1:1 ratio in different percentages of the crowding agent. The circular dichroism spectroscopic results predict stronger interaction of harmaline with Hb (secondary structure alterations) in a higher crowding environment. From the melting study, it was found that the reactions between Hb and harmaline in crowding environments are endothermic (ΔH > 0) and disordering (ΔS > 0) in nature, indicating that hydrogen bonding and van der Waals interactions are the main interacting forces between Hb and harmaline. Harmaline molecules are more reactive in molecular crowding conditions than in normal buffer condition. This study represents that the interaction between harmaline and Hb is stronger compared to the structurally similar harmine in a molecular crowding environment, which may enlighten the drug discovery process in cell-mimicking conditions.

Morphometric evaluation of mitochondrial networks in regenerating myofibers using photothermal microscopy

Background: Mitochondria constitute their interconnected network through dynamic mitochondrial processes such as fission and fusion in response to changes in their intracellular environment, such as energy demand. Transmission electron microscopy is the standard method for evaluating mitochondrial morphology. We have developed a simple method to evaluate mitochondrial morphology using tissue sections for bright-field observation. In this study, we attempted to image light-absorbing molecules (cytochrome c) in mitochondria using photothermal (PT) microscopy. Purpose: This method can observe an entire myocyte, so this study attempted to evaluate the entire mitochondrial network of immature (regenerating) myocytes during the skeletal muscle regeneration process. Methods: The gastrocnemius muscle of anesthetized male Wistar rats (13 weeks old) was subjected to 300 controlled eccentric contractions (ECC) using electrical stimulation. 7 days after ECC loading, in situ perfusion fixation was performed, and transverse sections (1 μm) from red (deep regions) and white (superficial regions) were prepared after Epon embedding. The sections were observed by PT microscopy, and quantitative analysis of mitochondrial morphology was performed. Mitochondrial content was expressed as a percentage of the fiber total area in transverse sections. The PT imaging system uses lasers with wavelengths of 515 nm and 638 nm constituting the PT pump and the probe beams, respectively. Results: After 7 days of ECC, many myofibers with small fiber diameters and central nuclei, which are characteristic of the recovery process after muscle damage, were observed. The cross-sectional area of the regenerating muscle (superficial region 389.9 ± 15.2 μm2, deep region 366.2 ± 17.5 μm2) was significantly smaller than that of the normal muscle (superficial region 2211.1 ± 127.3 μm2, deep region 1396.1 ± 72.9 μm2). Mitochondrial content of the regenerating muscle (superficial region 26.1 ± 1.1%, deep region 22.7 ± 0.8%) was significantly lower than that of the normal muscle (superficial region 31.8 ± 1.9%, deep region 34.9 ± 1.8%). In normal muscle, a linear network of mitochondria along myofibrils was observed. On the other hand, no spatially distributed mitochondria were observed in the cytoplasm of the regenerating muscle. Furthermore, a mitochondrial network was formed around the periphery of the central nucleus. Conclusion: In muscle tissue imaging using PT microscopy, myofibers in the regenerative process have less mitochondrial content than mature muscle fibers and lack the mitochondrial network along myofibrils that characterizes mature muscle. On the other hand, the central nucleus of immature muscle was surrounded by highly developed mitochondria. The mitochondrial morphological characteristics of regenerating muscle fibers may offer a unique window through which to better understand mitochondrial design and function and their plasticity during maturation. This study was supported in part by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) KAKENHI Grant (No. JP20H04074, 21K19703). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TROP2 promotes PINK1-mediated mitophagy and apoptosis to accelerate the progression of senile chronic obstructive pulmonary disease by up-regulating DRP1 expression

Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease characterised by irreversible airflow limitation. The elderly are a vulnerable population for developing COPD. With the growth of age, physiological degenerative changes occur in the thorax, bronchus, lung and vascular wall, which can lead to age-related physiological attenuation of lung function in the elderly, so the prevalence of COPD increases with age. Its pathogenesis has not yet been truly clarified. Mitophagy plays an important role in maintaining the stability of mitochondrial function and intracellular environment by scavenging damaged mitochondria. Currently, studies have shown that trophoblast antigen 2 (TROP2) expression is up-regulated in airway basal cells of patients with COPD, suggesting that TROP2 is involved in the progression of COPD. However, whether it is involved in disease progression by regulating mitochondrial function remains unclear. In this study, compared with non-smoking non-COPD patients, the expression of TROP2 in lung tissues of smoking non-COPD patients and patients with COPD increased, and TROP2 expression in patients with COPD was higher than that in smoking non-COPD patients. To further explore the role of TROP2, we stimulated BEAS-2B with cigarette smoke to construct an in vitro model. We found that TROP2 expression increased, whereas TROP2 silencing reversed the cigarette smoke extract-induced decrease in mitochondrial membrane potential, increased reactive oxygen species content, decreased adenosine triphosphate (ATP) production, increased inflammatory factor secretion and increased apoptosis. In addition, we searched online bioinformatics and screened the gene dynamin-related protein 1 (DRP1) related to mitophagy as the research object. Co-IP assay verified the binding relationship between DRP1 and TROP2. Further study found that TROP2 promoted mitophagy and apoptosis of BEAS-2B cells by up-regulating the expression of DRP1. In addition, PTEN-induced putative kinase 1 (PINK1) is a potential binding protein of DRP1, and DRP1 accelerated mitophagy and apoptosis of BEAS-2B cells by promoting the expression of PINK1. We established a COPD SD rat model by cigarette smoke exposure and LPS instillation and treated it by intraperitoneal injection of si-TROP2. The results showed that TROP2 silencing restored lung function and reduced the secretion of inflammatory factors in bronchoalveolar lavage fluid. In conclusion, TROP2 can be used as a new reference for COPD treatment.

Deciphering Staphylococcus aureus-host dynamics using dual activity-based protein profiling of ATP-interacting proteins.

This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections.

Acute exposure to tris(2,4-di-tert-butylphenyl)phosphate elicits cardiotoxicity in zebrafish (Danio rerio) larvae via inducing ferroptosis

Tris(2,4-di-tert-butylphenyl)phosphate (AO168 =O), a novel organophosphate ester, is prevalent and abundant in the environment, posing great exposure risks to ecological and public health. Nevertheless, the toxicological effects of AO168 =O remain entirely unknown to date. The results in this study indicated that acute exposure to AO168 =O at 10 and 100 μg/L for 5 days obviously impaired cardiac morphology and function of zebrafish larvae, as proofed by decreased heartbeat, stroke volume, and cardiac output and the occurrence of pericardial edema and ventricular hypertrophy. Transcriptomics, polymerase chain reaction, and molecular docking revealed that the strong interaction of AO168 =O and transferrin receptor 1 activated the transportation of ferric iron into intracellular environment. The release of free ferrous ion to cytoplasmic iron pool also contributed to the iron overload in heart region, thus inducing ferroptosis in larvae via generation of excessive reactive oxygen species, glutathione peroxidase 4 inhibition, glutathione depletion and lipid peroxidation. Ferroptosis inhibitor (Fer-1) co-exposure effectively relieved the cardiac dysfunctions of zebrafish, verifying the dominant role of ferroptosis in the cardiotoxicity caused by AO168 =O. This research firstly reported the adverse impact and associated mechanisms of AO168 =O in cardiomyogenesis of vertebrates, underlining the urgency of concerning the health risks of AO168 =O.

Quantification of MicroRNA in a Single Living Cell via Ionic Current Rectification-Based Nanopore for Triple Negative Breast Cancer Diagnosis.

Accurate analysis of microRNAs (miRNAs) at the single-cell level is extremely important for deeply understanding their multiple and intricate biological functions. Despite some advancements in analyzing single-cell miRNAs, challenges such as intracellular interferences and insufficient detection limits still remain. In this work, an ultrasensitive nanopore sensor for quantitative single-cell miRNA-155 detection is constructed based on ionic current rectification (ICR) coupled with enzyme-free catalytic hairpin assembly (CHA). Benefiting from the enzyme-free CHA amplification strategy, the detection limit of the nanopore sensor for miRNA-155 reaches 10 fM and the nanopore sensor is more adaptable to complex intracellular environments. With the nanopore sensor, the concentration of miRNA-155 in living single cells is quantified to realize the early diagnosis of triple-negative breast cancer (TNBC). Furthermore, the nanopore sensor can be applied in screening anticancer drugs by tracking the expression level of miRNA-155. This work provides an adaptive and universal method for quantitatively analyzing intracellular miRNAs, which will greatly improve our understanding of cell heterogeneity and provide a more reliable scientific basis for exploring major diseases at the single-cell level.

Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

MEMO1 binds iron and modulates iron homeostasis in cancer cells.

Mediator of ERBB2-driven cell motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high-MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.

Excited State Rotational Freedom Impacts Viscosity Sensitivity in Arylcyanoamide Fluorescent Molecular Rotor Dyes.

The microviscosity of intracellular environments plays an important role in monitoring cellular function. Thus, the capability of detecting changes in viscosity can be utilized for the detection of different disease states. Viscosity-sensitive fluorescent molecular rotors are potentially excellent probes for these applications; however, the predictable relationships between chemical structural features and viscosity sensitivity are poorly understood. Here, we investigate a set of arylcyanoamide-based fluorescent probes and the effect of small aliphatic substituents on their viscosity sensitivity. We found that the location of the substituents and the type of π-network of the fluorophore can significantly affect the viscosity sensitivity of these fluorophores. Computational analysis supported the notion that the excited state rotational energy barrier plays a dominant role in the relative viscosity sensitivity of these fluorophores. These findings provide valuable insight into the design of molecular rotor-based fluorophores for viscosity measurement.

Scaling up of a Self-Confined Catalytic Hybridization Circuit for Robust microRNA Imaging.

The precise regulation of cellular behaviors within a confined, crowded intracellular environment is highly amenable in diagnostics and therapeutics. While synthetic circuitry system through a concatenated chemical reaction network has rarely been reported to mimic dynamic self-assembly system. Herein, a catalytic self-defined circuit (CSC) for the hierarchically concatenated assembly of DNA domino nanostructures is engineered. By incorporating pre-sealed symmetrical fragments into the preying hairpin reactants, the CSC system allows the hierarchical DNA self-assembly via a microRNA (miRNA)-powered self-sorting catalytic hybridization reaction. With minimal strand complexity, this self-sustainable CSC system streamlined the circuit component and achieved localization-intensified cascaded signal amplification. Profiting from the self-adaptively concatenated hybridization reaction, a reliable and robust method has been achieved for discriminating carcinoma tissues from the corresponding para-carcinoma tissues. The CSC-sustained self-assembly strategy provides a comprehensive and smart toolbox for organizing various hierarchical DNA nanostructures, which may facilitate more insights for clinical diagnosis and therapeutic assessment.

Polyethylene Glycol Impacts Conformation and Dynamics of Escherichia coli Prolyl-tRNA Synthetase Via Crowding and Confinement Effects.

Polyethylene glycol (PEG) is a flexible, nontoxic polymer commonly used in biological and medical research, and it is generally regarded as biologically inert. PEG molecules of variable sizes are also used as crowding agents to mimic intracellular environments. A recent study with PEG crowders revealed decreased catalytic activity of Escherichia coli prolyl-tRNA synthetase (Ec ProRS), where the smaller molecular weight PEGs had the maximum impact. The molecular mechanism of the crowding effects of PEGs is not clearly understood. PEG may impact protein conformation and dynamics, thus its function. In the present study, the effects of PEG molecules of various molecular weights and concentrations on the conformation and dynamics of Ec ProRS were investigated using a combined experimental and computational approach including intrinsic tryptophan fluorescence spectroscopy, atomic force microscopy, and atomistic molecular dynamic simulations. Results of the present study suggest that lower molecular weight PEGs in the dilute regime have modest effects on the conformational dynamics of Ec ProRS but impact the catalytic function primarily via the excluded volume effect; they form large clusters blocking the active site pocket. In contrast, the larger molecular weight PEGs in dilute to semidilute regimes have a significant impact on the protein's conformational dynamics; they wrap on the protein surface through noncovalent interactions. Thus, lower-molecular-weight PEG molecules impact protein dynamics and function via crowding effects, whereas larger PEGs induce confinement effects. These results have implications for the development of inhibitors for protein targets in a crowded cellular environment.

Intraphagosomal Free Ca2+ Changes during Phagocytosis.

Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca2+ ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca2+ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca2+ ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca2+ channels on the phagosome that allow Ca2+ ions to "drain" into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca2+ influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca2+ signalling events, such as peri-phagosomal Ca2+ hotspots, and other phenomena.

Maturation and Conformational Switching of a De Novo Designed Phase-Separating Polypeptide.

Cellular compartments formed by biomolecular condensation are widespread features of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arises in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using a droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely β conformations, analogous to models of pathological phase separation.

Single-Molecule Diffusivity Quantification Unveils Ubiquitous Net Charge-Driven Protein-Protein Interaction.

Recent microscopy and nuclear magnetic resonance (NMR) studies have noticed substantial suppression of intracellular diffusion for positively charged proteins, suggesting an overlooked role of electrostatic attraction in nonspecific protein interactions in a predominantly negatively charged intracellular environment. Utilizing single-molecule detection and statistics, here, we quantify in aqueous solutions how protein diffusion, in the limit of low diffuser concentration to avoid aggregate/coacervate formation, is modulated by differently charged interactor proteins over wide concentration ranges. We thus report substantially suppressed diffusion when oppositely charged interactors are added at parts per million levels, yet unvaried diffusivities when same-charge interactors are added beyond 1%. The electrostatic attraction-driven suppression of diffusion is sensitive to the protein net charge states, as probed by varying the solution pH and ionic strength or chemically modifying the proteins and is robust across different diffuser-interactor pairs. By converting the measured diffusivities to diffuser diameters, we further show that in the limit of excess interactors, a positively charged diffuser molecule effectively drags along just one monolayer of negatively charged interactors, where further interactions stop. We thus unveil ubiquitous, net charge-driven protein-protein interactions and shed new light on the mechanism of charge-based diffusion suppression in living cells.

Fluorescence lifetime imaging and phasor analysis of intracellular porphyrinic photosensitizers applied with different polymeric formulations

The fluorescence lifetime of a porphyrinic photosensitizer (PS) is an important parameter to assess the aggregation state of the PS even in complex biological environments. Aggregation-induced quenching of the PS can significantly reduce the yield of singlet oxygen generation and thus its efficiency as a medical drug in photodynamic therapy (PDT) of diseased tissues. Hydrophobicity and the tendency to form aggregates pose challenges on the development of efficient PSs and often require carrier systems. A systematic study was performed to probe the impact of PS structure and encapsulation into polymeric carriers on the fluorescence lifetime in solution and in the intracellular environment. Five different porphyrinic PSs including chlorin e6 (Ce6) derivatives and tetrakis(m-hydroxyphenyl)-porphyrin and -chlorin were studied in free form and combined with polyvinylpyrrolidone (PVP) or micelles composed of triblock-copolymers or Cremophor. Following incubation of HeLa cells with these systems, fluorescence lifetime imaging combined with phasor analysis and image segmentation was applied to study the lifetime distribution in the intracellular surrounding. The data suggest that for free PSs, the structure-dependent cell uptake pathways determine their state and emission lifetimes. PS localization in the plasma membrane yielded mostly monomers with long fluorescence lifetimes whereas the endocytic pathway with subsequent lysosomal deposition adds a short-lived component for hydrophilic anionic PSs. Prolonged incubation times led to increasing contributions from short-lived components that derive from aggregates mainly localized in the cytoplasm. Encapsulation of PSs into polymeric carriers led to monomerization and mostly fluorescence emission decays with long fluorescence lifetimes in solution. However, the efficiency depended on the binding strength that was most pronounced for PVP. In the cellular environment, PVP was able to maintain monomeric long-lived species over prolonged incubation times. This was most pronounced for Ce6 derivatives with a logP value around 4.5. Micellar encapsulation led to faster release of the PSs resulting in multiple components with long and short fluorescence lifetimes. The hydrophilic hardly aggregating PS exhibited a mostly stable invariant lifetime distribution over time with both carriers. The presented data are expected to contribute to optimized PDT treatment protocols and improved PS-carrier design for preventing intracellular fluorescence quenching. In conclusion, amphiphilic and concurrent hydrophobic PSs with high membrane affinity as well as strong binding to the carrier have best prospects to maintain their photophysical properties in vivo and serve thus as efficient photodynamic diagnosis and PDT drugs.