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Scientific Reports Sep 2018Intracellular delivery of functional materials holds great promise in biologic research and therapeutic applications but poses challenges to existing techniques,...
Intracellular delivery of functional materials holds great promise in biologic research and therapeutic applications but poses challenges to existing techniques, including the reliance on exogenous vectors and lack of selectivity. To address these problems, we propose a vector-free approach that utilizes millimeter-sized iron rods or spheres driven by magnetic forces to selectively deform targeted cells, which in turn generates transient disruption in cell membranes and enables the delivery of foreign materials into cytosols. A range of functional materials with the size from a few nanometers to hundreds of nanometers have been successfully delivered into various types of mammalian cells in situ with high efficiency and viability and minimal undesired effects. Mechanistically, material delivery is mediated by force-induced transient membrane disruption and restoration, which depend on actin cytoskeleton and calcium signaling. When used for siRNA delivery, CXCR4 is effectively silenced and cell migration and proliferation are significantly inhibited. Remarkably, cell patterns with various complexities are generated, demonstrating the unique ability of our approach in selectively delivering materials into targeted cells in situ. In summary, we have developed a magnetic force-driven intracellular delivery method with in situ selectivity, which may have tremendous applications in biology and medicine.
Topics: 3T3 Cells; Animals; Biological Transport; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Movement; Cytoplasm; Cytosol; HeLa Cells; Hep G2 Cells; Humans; Magnetics; Mechanical Phenomena; Mice; RNA, Small Interfering; Signal Transduction
PubMed: 30242189
DOI: 10.1038/s41598-018-32605-w -
Environmental Health Perspectives Mar 1990Use of clonal strains of prolactin (PRL)- and growth hormone-producing rat pituitary cells has proven informative in elucidating a number of the early biochemical,... (Review)
Review
Use of clonal strains of prolactin (PRL)- and growth hormone-producing rat pituitary cells has proven informative in elucidating a number of the early biochemical, ionic, and secretory events regulated by the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH). TRH causes biphasic changes in the concentration of cytosolic free calcium [( Ca2+]i) in GH4C1 cells and biphasic changes in hormone secretion. Early changes occur on a msecond to second scale and late changes, on a time scale of minutes. Although increases in [Ca2+]i are essential for enhanced secretion, at least in the case of the rapid initial phase, the TRH-induced increase in [Ca2+]i is necessary, but not sufficient to enhance secretion. A co-mediator with calcium appears to be diacylglycerol. The majority of the calcium involved in the early phase of rise in [Ca2+]i induced by TRH is derived from intracellular sources, while essentially all of the calcium rise observed in the late phase is derived from extracellular calcium entering the cell through both voltage-dependent and voltage-independent conductances. Because TRH causes an elevation of inositol(1,4,5) trisphosphate [Ins(1,4,5)P3] within seconds, but not mseconds, further studies are required before it can be concluded unequivocally that Ins(1,4,5)P3 is the sole mediator of the rapid phase of rise in [Ca2+]i induced by TRH in GH-cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Calcium; Cells, Cultured; Cytosol; Hormones; Humans; Models, Biological
PubMed: 2190815
DOI: 10.1289/ehp.908427 -
ACS Infectious Diseases Nov 2018Studying redox metabolism in malaria parasites is of great interest for understanding parasite biology, parasite-host interactions, and mechanisms of drug action.... (Comparative Study)
Comparative Study
Studying redox metabolism in malaria parasites is of great interest for understanding parasite biology, parasite-host interactions, and mechanisms of drug action. Genetically encoded fluorescent redox sensors have recently been described as powerful tools for determining the glutathione-dependent redox potential in living parasites. In the present study, we genomically integrated and expressed the ratiometric redox sensors hGrx1-roGFP2 (human glutaredoxin 1 fused to reduction-oxidation sensitive green fluorescent protein) and sfroGFP2 (superfolder roGFP2) in the cytosol of NF54- attB blood-stage Plasmodium falciparum parasites. Both sensors were evaluated in vitro and in cell culture with regard to their fluorescence properties and reactivity. As genomic integration allows for the stable expression of redox sensors in parasites, we systematically compared single live-cell imaging with plate reader detection. For these comparisons, short-term effects of redox-active compounds were analyzed along with mid- and long-term effects of selected antimalarial agents. Of note, the single components of the redox probes themselves did not influence the redox balance of the parasites. Our analyses revealed comparable results for both the hGrx1-roGFP2 and sfroGFP2 probes, with sfroGFP2 exhibiting a more pronounced fluorescence intensity in cellulo. Accordingly, the sfroGFP2 probe was employed to monitor the fluorescence signals throughout the parasites' asexual life cycle. Through the use of stable genomic integration, we demonstrate a means of overcoming the limitations of transient transfection, allowing more detailed in-cell studies as well as high-throughput analyses using plate reader-based approaches.
Topics: Antimalarials; Cytosol; Fluorescence; Fluorescent Dyes; Glutaredoxins; Green Fluorescent Proteins; Host-Parasite Interactions; Humans; Oxidation-Reduction; Plasmodium falciparum; Recombinant Proteins; Transfection
PubMed: 30129748
DOI: 10.1021/acsinfecdis.8b00140 -
Biometals : An International Journal on... Apr 2021One candidate for the cytosolic labile iron pool is iron(II)glutathione. There is also a widely held opinion that an equivalent cytosolic labile heme pool exists and...
One candidate for the cytosolic labile iron pool is iron(II)glutathione. There is also a widely held opinion that an equivalent cytosolic labile heme pool exists and that this pool is important for the intracellular transfer of heme. Here we describe a study designed to characterise conjugates that form between heme and glutathione. In contrast to hydrated iron(II), heme reacts with glutathione, under aerobic conditions, to form the stable hematin-glutathione complex, which contains iron(III). Thus, glutathione is clearly not the cytosolic ligand for heme, indeed we demonstrate that the rate of heme degradation is enhanced in the presence of glutathione. We suggest that the concentration of heme in the cytosol is extremely low and that intracellular heme transfer occurs via intracellular membrane structures. Should any heme inadvertently escape into the cytosol, it would be rapidly conjugated to glutathione thereby protecting the cell from the toxic effects of heme.
Topics: Cytosol; Ferric Compounds; Glutathione; Heme; Molecular Structure
PubMed: 33301081
DOI: 10.1007/s10534-020-00274-w -
BioDrugs : Clinical Immunotherapeutics,... Mar 2023Numerous toxins translocate to the cytosol in order to fulfil their function. This demonstrates the existence of routes for proteins from the extracellular space to the... (Review)
Review
Numerous toxins translocate to the cytosol in order to fulfil their function. This demonstrates the existence of routes for proteins from the extracellular space to the cytosol. Understanding these routes is relevant to multiple aspects related to therapeutic applications. These include the development of anti-toxin treatments, the potential use of toxins as shuttles for delivering macromolecular cargo to the cytosol or the use of drugs based on toxins. Compared with other strategies for delivery, such as chemicals as carriers for macromolecular delivery or physical methods like electroporation, toxin routes present paths into the cell that potentially cause less damage and can be specifically targeted. The efficiency of delivery via toxin routes is limited. However, low-delivery efficiencies can be entirely sufficient, if delivered cargoes possess an amplification effect or if very few molecules are sufficient for inducing the desired effects. This is known for example from RNA-based vaccines that have been developed during the coronavirus disease 2019 pandemic as well as for other approved RNA-based drugs, which elicited the desired effect despite their typically low delivery efficiencies. The different mechanisms by which toxins enter cells may have implications for their technological utility. We review the mechanistic principles of the translocation pathway of toxins from the extracellular space to the cytosol, the delivery efficiencies, and therapeutic strategies or applications that exploit toxin routes for intracellular delivery.
Topics: Humans; Biological Products; COVID-19; Cytosol; Pharmaceutical Preparations
PubMed: 36729328
DOI: 10.1007/s40259-023-00580-y -
Journal of the American Chemical Society Mar 2021Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction of defined protein quantities into...
Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction of defined protein quantities into the cell. Most applications require that the delivered protein has access to the cytosol, either for protein activity or as a gateway to other organelles such as the nucleus. The vast majority of delivery vehicles employ an endosomal pathway however, and efficient release of entrapped protein cargo from the endosome remains a challenge. Recent research has made significant advances toward efficient cytosolic delivery of proteins using polymers, but the influence of polymer architecture on protein delivery is yet to be investigated. Here, we developed a family of dendronized polymers that enable systematic alterations of charge density and structure. We demonstrate that while modulation of surface functionality has a significant effect on overall delivery efficiency, the endosomal release rate can be highly regulated by manipulating polymer architecture. Notably, we show that large, multivalent structures cause slower sustained release, while rigid spherical structures result in rapid burst release.
Topics: Animals; Cell Line; Cytosol; Humans; Mice; Molecular Structure; Polymers; Protein Engineering; Proteins
PubMed: 33705125
DOI: 10.1021/jacs.1c00258 -
Chemico-biological Interactions Oct 2020This study investigated the enantioselective metabolism of benoxacor, an ingredient of herbicide formulations, in microsomes or cytosol prepared from female or male rat...
This study investigated the enantioselective metabolism of benoxacor, an ingredient of herbicide formulations, in microsomes or cytosol prepared from female or male rat livers. Benoxacor was incubated for ≤30 min with microsomes or cytosol, and its enantioselective depletion was measured using gas chromatographic methods. Benoxacor was depleted in incubations with active microsomes in the presence and absence of NADPH, suggesting its metabolism by hepatic cytochrome P450 enzymes (CYPs) and microsomal carboxylesterases (CESs). Benoxacor was depleted in cytosolic incubations in the presence of glutathione, consistent with its metabolism by glutathione S-transferases (GSTs). The depletion of benoxacor was faster in incubations with cytosol from male than female rats, whereas no statistically significant sex differences were observed in microsomal incubations. The consumption of benoxacor was inhibited by the CYP inhibitor 1-aminobenzotriazole, the CES inhibitor benzil, and the GST inhibitor ethacrynic acid. Estimates of the intrinsic clearance of benoxacor suggest that CYPs are the primary metabolic enzyme responsible for benoxacor metabolism in rats. Microsomal incubations showed an enrichment of the first eluting benoxacor enantiomer (E-benoxacor). A greater enrichment occurred in incubations with microsomes from female (EF = 0.67 ± 0.01) than male rats (EF = 0.60 ± 0.01). Cytosolic incubations from female rats resulted in enrichment of E-benoxacor (EF = 0.54 ± 0.01), while cytosolic incubations from male rats displayed enrichment of the second eluting enantiomer (E-benoxacor; EF = 0.43 ± 0.01). Sex-dependent differences in the metabolism of benoxacor in rats could significantly impact ecological risks and mammalian toxicity. Moreover, changes in the enantiomeric enrichment of benoxacor may be a powerful tool for environmental fate and transport studies.
Topics: Animals; Cytochrome P-450 Enzyme System; Cytosol; Female; Herbicides; Liver; Male; Microsomes, Liver; Oxazines; Rats; Sex Factors; Stereoisomerism; Subcellular Fractions
PubMed: 32866466
DOI: 10.1016/j.cbi.2020.109247 -
International Journal of Molecular... Dec 2019VGF gene encodes for a neuropeptide precursor of 68 kDa composed by 615 (human) and 617 (rat, mice) residues, expressed prevalently in the central nervous system (CNS),... (Review)
Review
VGF gene encodes for a neuropeptide precursor of 68 kDa composed by 615 (human) and 617 (rat, mice) residues, expressed prevalently in the central nervous system (CNS), but also in the peripheral nervous system (PNS) and in various endocrine cells. This precursor undergoes proteolytic cleavage, generating a family of peptides different in length and biological activity. Among them, TLQP-21, a peptide of 21 amino acids, has been widely investigated for its relevant endocrine and extraendocrine activities. The complement complement C3a receptor-1 (C3aR1) has been suggested as the TLQP-21 receptor and, in different cell lines, its activation by TLQP-21 induces an increase of intracellular Ca. This effect relies both on Ca release from the endoplasmic reticulum (ER) and extracellular Ca entry. The latter depends on stromal interaction molecules (STIM)-Orai1 interaction or transient receptor potential channel (TRPC) involvement. After Ca entry, the activation of outward K-Ca-dependent currents, mainly the K currents, provides a membrane polarizing influence which offset the depolarizing action of Ca elevation and indirectly maintains the driving force for optimal Ca increase in the cytosol. In this review, we address the main endocrine and extraendocrine actions displayed by TLQP-21, highlighting recent findings on its mechanism of action and its potential in different pathological conditions.
Topics: Animals; Calcium Signaling; Cytosol; Endoplasmic Reticulum; Humans; Neuropeptides; Peptide Fragments; Stromal Interaction Molecules; Transient Receptor Potential Channels
PubMed: 31878142
DOI: 10.3390/ijms21010130 -
PLoS Computational Biology Sep 2020Hydrogen peroxide (H2O2) promotes a range of phenotypes depending on its intracellular concentration and dosing kinetics, including cell death. While this qualitative...
Hydrogen peroxide (H2O2) promotes a range of phenotypes depending on its intracellular concentration and dosing kinetics, including cell death. While this qualitative relationship has been well established, the quantitative and mechanistic aspects of H2O2 signaling are still being elucidated. Mitochondria, a putative source of intracellular H2O2, have recently been demonstrated to be particularly vulnerable to localized H2O2 perturbations, eliciting a dramatic cell death response in comparison to similar cytosolic perturbations. We sought to improve our dynamic and mechanistic understanding of the mitochondrial H2O2 reaction network in HeLa cells by creating a kinetic model of this system and using it to explore basal and perturbed conditions. The model uses the most current quantitative proteomic and kinetic data available to predict reaction rates and steady-state concentrations of H2O2 and its reaction partners within individual mitochondria. Time scales ranging from milliseconds to one hour were simulated. We predict that basal, steady-state mitochondrial H2O2 will be in the low nM range (2-4 nM) and will be inversely dependent on the total pool of peroxiredoxin-3 (Prx3). Neglecting efflux of H2O2 to the cytosol, the mitochondrial reaction network is expected to control perturbations well up to H2O2 generation rates ~50 μM/s (0.25 nmol/mg-protein/s), above which point the Prx3 system would be expected to collapse. Comparison of these results with redox Western blots of Prx3 and Prx2 oxidation states demonstrated reasonable trend agreement at short times (≤ 15 min) for a range of experimentally perturbed H2O2 generation rates. At longer times, substantial efflux of H2O2 from the mitochondria to the cytosol was evidenced by peroxiredoxin-2 (Prx2) oxidation, and Prx3 collapse was not observed. A refined model using Monte Carlo parameter sampling was used to explore rates of H2O2 efflux that could reconcile model predictions of Prx3 oxidation states with the experimental observations.
Topics: Computational Biology; Cytosol; HeLa Cells; Humans; Hydrogen Peroxide; Kinetics; Mitochondria; Models, Biological; Neoplasms; Reactive Oxygen Species; Signal Transduction
PubMed: 32925922
DOI: 10.1371/journal.pcbi.1008202 -
EMBO Reports May 2021In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins...
In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain-of-cytosolic functions-a phenomenon we name ER to Cytosol Signaling (ERCYS).
Topics: Animals; Cytosol; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Endoplasmic Reticulum-Associated Degradation; Mice; Proteins
PubMed: 33710763
DOI: 10.15252/embr.202051412