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The trimethylguanosine (TMG) cap is a motif present inter alia at the 5' end of small nuclear RNAs, which are involved in RNA splicing. The TMG cap plays a crucial role in RNA processing and stability as it protects the RNA molecule from degradation by exonucleases and facilitates its export from the nucleus. Additionally, the TMG cap plays a role in the recognition of snRNA by snurportin, a protein that facilitates nuclear import. TMG cap analogs are used in biochemical experiments as molecular tools to substitute the natural TMG cap. To expand the range of available TMG-based tools, here we conjugated the TMG cap to Fluorescent Molecular Rotors (FMRs) to open the possibility of detecting protein-ligand interactions in vitro and, potentially, in vivo, particularly visualizing interactions with snurportin. Consequently, we report the synthesis of 34 differently modified TMG cap-FMR conjugates and their evaluation as molecular probes for snurportin. As FMRs we selected three GFP-like chromophores (derived from green fluorescent protein) and one julolidine derivative. The evaluation of binding affinities for snurportin showed unexpectedly a strong stabilizing effect for TMGpppG-derived dinucleotides containing the FMR at the 2'-O-position of guanosine. These newly discovered compounds are potent snurportin ligands with nanomolar KD (dissociation constant) values, which are two orders of magnitude lower than that of natural TMGpppG. The effect is diminished by â¼50-fold for the corresponding 3'-regioisomers. To deepen the understanding of the structure-activity relationship, we synthesized and tested FMR conjugates lacking the TMG cap moiety. These studies, supported by molecular docking, suggested that the enhanced affinity arises from additional hydrophobic contacts provided by the FMR moiety. The strongest snurportin ligand, which also gave the greatest fluorescence enhancement (Fm/F0) when saturated with the protein, were tested in living cells to detect interactions and visualize complexes by fluorescence lifetime monitoring. This approach has potential applications in the study of RNA processing and RNA-protein interactions.
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Colorantes Fluorescentes , Guanosina , Ligandos , Humanos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/síntesis química , Análogos de Caperuza de ARN/metabolismo , Células HeLa , Estructura MolecularRESUMEN
The Malus sylvestris L. (LE1), Pinus sylvestris L. (LE2), and Sorbus aucuparia L. (LE3) leaves` extracts were used for the synthesis of silver (Ag) nanocomposites containing different amounts of silver chloride (AgCl), silver metal (Agmet), and silver phosphate (Ag3PO4). These nanocomposites were capped with the organic functional groups in the leaf extract. Notably, the nanocomposites caused biphasic cytotoxic response on cells; first attributed to the inhibition of cell growth and second to cell death. The nanocomposites were biocompatible with normal embryonic kidney (HEK293) cells in the cytotoxic range for cancer cells. [25(±1) °C synthesis] nanocomposites exhibited the highest cytotoxicity towards HeLa (lethal concentration- LC50 value of 11.4 µg mL-1) and A549 (LC50 value of 14.7 µg mL-1) after 24-h incubation and its efficiency was shown also for the more resistant MCF-7 and MDA-MB-231, however, their respective LC50 values were larger. For the HeLa cell line, this designed nanocomposite exhibited an LC50 value similar to the effective concentration (EC50) value of Cisplatin and about 3 times larger than Doxorubicin. nanocomposite contained Ag3PO4 in the composite and P on the surface, higher AgCl content, smaller crystallite size of all nanoparticle phases, and carbon-rich oxygen-deficient surface compared to all other nanocomposites.
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Nanocompuestos , Extractos Vegetales , Hojas de la Planta , Humanos , Nanocompuestos/química , Extractos Vegetales/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Células HeLa , Plata/química , Células HEK293 , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Supervivencia Celular/efectos de los fármacos , Propiedades de Superficie , Línea Celular TumoralRESUMEN
The vast majority of chemical processes that govern our lives occur within living cells. At the core of every life process, such as gene expression or metabolism, are chemical reactions that follow the fundamental laws of chemical kinetics and thermodynamics. Understanding these reactions and the factors that govern them is particularly important for the life sciences. The physicochemical environment inside cells, which can vary between cells and organisms, significantly impacts various biochemical reactions and increases the extent of population heterogeneity. This paper discusses using physical chemistry approaches for biological studies, including methods for studying reactions inside cells and monitoring their conditions. The potential for development in this field and possible new research areas are highlighted. By applying physical chemistry methodology to biochemistry in vivo, we may gain new insights into biology, potentially leading to new ways of controlling biochemical reactions.
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Diffusive motion accompanies many physical and biological processes. The Stokes-Sutherland-Einstein relation for the translational diffusion coefficient, DT, agrees with experiments done in simple fluids but fails for complex fluids. Moreover, the interdependence between DT and rotational diffusion coefficient, DR, also deviates in complex fluids from the classical relation of DT/DR = 4r2/3 known in simple fluids. Makuch et al. Soft Matter, 2020, 16, 114-124 presented a generalization of the classical translational and rotational diffusion theory for complex fluids. In this work, we empirically verify this model based on simultaneous translational and rotational diffusion measurements. We use fluorescently stained cowpea chlorotic mottle virus (CCMV) particles as monodisperse probes and aqueous polyethylene glycol (PEG) solutions as a model complex fluid. The theory and experimental data obtained from fluorescence correlation spectroscopy (FCS) measurements agreed. Finally, we used the same model and analyzed the diffusion of Yo-Pro-1 stained large ribosomal subunits (LSU) in the cytoplasm and nucleus of living HeLa cells.
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Polietilenglicoles , Células HeLa , Humanos , Difusión , Polietilenglicoles/química , Rotación , Bromovirus/química , Bromovirus/metabolismo , Espectrometría de FluorescenciaRESUMEN
Thermal fluctuations power all processes inside living cells. Therefore, these processes are inherently random. However, myriad multistep chemical reactions act in concerto inside a cell, finally leading to this chemical reactor's self-replication. We speculate that an underlying mechanism in nature must exist that allows all of these reactions to synchronize at multiple time and length scales, overcoming in this way the random nature of any single process in a cell. This Perspective discusses what type of research is needed to understand this undiscovered synchronization law.
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In the paper, "Fluorimetric sensing of ATP in water by an imidazolium hydrazone based sensor," Farshbaf and Anzenbacher presented the application of bisantrene as a fluorescent ATP sensor in organic-inorganic mixtures of solvents. Encouraged by the results presented in the parent study, we aimed to apply this strategy for physiologically relevant water-based buffers and - preferably - intracellular application. Here we present the results of our investigations and point to the limitations of bisantrene applications in vivo as the ATP sensor.
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Bevacizumab is a biological drug that is now extensively studied in clinics against various types of cancer. Although bevacizumab's action is preferably extracellular, there are reports suggesting its internalization into cancer cells, consequently decreasing its therapeutic potential. Here we are solving this issue by applying fluorescence correlation spectroscopy in living cells. We tracked single molecules of fluorescent bevacizumab in MDA-MB-231 and HeLa cells and proved that mobility measurements bring significant added value to standard imaging techniques. We confirmed the presence of the drug in intracellular vesicles. Additionally, we explicitly excluded the presence of a free cytosolic fraction of bevacizumab in both studied cell types. Extracellular and intracellular concentrations of the drug were measured, giving a partition coefficient on the order of 10-5, comparable with the spontaneous uptake of biologically inert nanoparticles. Our work presents how techniques and models developed for physics can answer biological questions.
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Inhibidores de la Angiogénesis , Neoplasias de la Mama , Humanos , Femenino , Bevacizumab , Inhibidores de la Angiogénesis/uso terapéutico , Células HeLa , Imagen Individual de Molécula , Neoplasias de la Mama/tratamiento farmacológicoRESUMEN
Biocompatible polyacrylamide gel and core-shell nanoparticles (NPs) were synthesized using a one-step electrochemically initiated gelation. Constant-potential electrochemical decomposing of ammonium persulfate initiated the copolymerization of N-isopropyl acrylamide, methacrylic acid, and N,N'-methylenebisacrylamide monomers. This decomposing potential and monomers' concentrations were optimized to prepare gel NPs and thin gel film-grafted core-shell NPs. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging confirmed the gel NP formation. The lyophilized gel NPs and core-shell NPs were applied to support the three-dimensional (3D) cell culture. In all, core-shell NPs provided superior support for complex 3D tissue structures.
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HYPOTHESIS: Most experimental procedures applied in modern biology involve cargo delivering into cells. One of the ways to cargo introduction is osmotic-mediated intracellular vesicle swelling. However, its widespread use was hindered due to cargo size (<10 nm) and cell-type-related restrictions. We addressed the issue of the composition of colloidal loading solution to enhance the efficiency of cellular delivery. EXPERIMENTS: We examined the effectiveness of colloidal loading solutions of varied compositions, including various types and sizes of polymers building osmotic pressure. We used confocal imaging coupled with fluorescence correlation spectroscopy to evaluate the introduction of polymers, proteins, nanoparticles, and DNA plasmids (cargos of sizes 1-175 nm) to cells representing eight cell lines: cancer, normal, epithelial, and mesenchymal ones. FINDINGS: We found that cellular delivery effectiveness strongly correlates with the size and concentration of osmotic pressure building polymers and not with the high value of the osmotic pressure itself. We show that polymer solutions at the entangled regime of concentrations enhance the delivery of large biomacromolecules even of size 200 nm (DNA plasmids) into cells, including MDA-MB-231 cells - so far resistant to the osmotic procedure. We show that the colloid loading medium based on entangled polymer chains is a versatile cargo delivery tool for molecular biology.
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Nanopartículas , Polímeros , Coloides , ADN , Nanopartículas/química , Plásmidos , Polímeros/químicaRESUMEN
Nitrophenols (NPs) are hazardous pollutants found in various environmental matrices, including ambient fine particulate matter (PM2.5), agricultural residues, rainwater, wildfires, and industrial wastes. This study showed for the first time the effect of three pure nitrophenols and their mixture on human lung cells to provide basic understanding of the NP influence on cell elements and processes. We identified NPs in ambient PM2.5 and secondary organic aerosol (SOA) particles generated from the photooxidation of monocyclic aromatic hydrocarbons in the U.S. EPA smog chamber. We assessed the toxicity of identified NPs and their equimolar mixture in normal bronchial epithelial (BEAS-2B) and alveolar epithelial cancer (A549) lung cell lines. The inhibitory concentration-50 (IC50) values were highest and lowest in BEAS-2B cells treated with 2-nitrophenol (2NP) and 4-nitrophenol (4NP), respectively, at 24 h of exposure. The lactate dehydrogenase (LDH) assay showed that 4NP, the most abundant NP we identified in PM2.5, was the most cytotoxic NP examined in both cell lines. The annexin-V/fluorescein isothiocyanate (FITC) analysis showed that the populations of late apoptotic/necrotic BEAS-2B and A549 cells exposed to 3NP, 4NP, and NP equimolar mixture increased between 24 and 48 h. Cellular reactive oxygen species (ROS) buildup led to cellular death post exposure to 3NP, 4NP and the NP mixtures, while 2NP induced the lowest ROS buildup. An increased mitochondrial ROS signal following NP exposure occurred only in BEAS-2B cells. The tetramethylrhodamine, methyl ester, perchlorate (TMRM) assay showed that exposed cells exhibited collapse of the mitochondrial membrane potential. TMRM signals decreased significantly only in BEAS-2B cells, and most strongly with 4NP exposures. Our results suggest that acute atmospheric exposures to NPs may be toxic at high concentrations, but not at ambient PM2.5 concentrations. Further chronic studies with NP and NP-containing PM2.5 are warranted to assess their contribution to lung pathologies.
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Contaminantes Atmosféricos , Células Epiteliales , Contaminantes Atmosféricos/análisis , Humanos , Pulmón , Nitrofenoles/metabolismo , Estrés Oxidativo , Material Particulado/análisisRESUMEN
Quantitative description of biochemical processes inside living cells and at single-molecule levels remains a challenge at the forefront of modern instrumentation and spectroscopy. This paper demonstrates such single-cell, single-molecule analyses performed to study the mechanism of action of olaparib - an up-to-date, FDA-approved drug for germline-BRCA mutated metastatic breast cancer. We characterized complexes formed with PARPi-FL - fluorescent analog of olaparib in vitro and in cancer cells using the advanced fluorescent-based method: Fluorescence Correlation Spectroscopy (FCS) combined with a length-scale dependent cytoplasmic/nucleoplasmic viscosity model. We determined in vitro olaparib-PARP1 equilibrium constant (6.06 × 108 mol L-1). In the cell nucleus, we distinguished three states of olaparib: freely diffusing drug (24%), olaparib-PARP1 complex (50%), and olaparib-PARP1-RNA complex (26%). We show olaparib accumulation in 3D spheroids, where intracellular concentration is twofold higher than in 2D cells. Moreover, olaparib concentration was tenfold higher (506 nmol L-1vs. 57 nmol L-1) in cervical cancer (BRCA1 high abundance) than in breast cancer cells (BRCA1 low abundance) but with a lower toxic effect. Thus we confirmed that the amount of BRCA1 protein in the cells is a better predictor of the therapeutic effect of olaparib than its penetration into cancer tissue. Our single-molecule and single-cell approach give a new perspective of drug action in living cells. FCS provides a detailed in vivo insight, valuable in drug development and targeting.
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Fenómenos Bioquímicos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Imagen Individual de Molécula , Línea Celular Tumoral , Reparación del ADN , Humanos , Ftalazinas , Piperazinas , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacologíaRESUMEN
Targeting cap-dependent translation initiation is one of the experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5',5'-triphosphates cap analogs are potent antagonists of eukaryotic translation initiation factor 4E (eIF4E) in vitro and could counteract elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic agents remains challenging - they do not easily penetrate into cells and are susceptible to enzymatic cleavage. Here, we tested the potential of several small molecule ligands - folic acid, biotin, glucose, and cholesterol - to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad structure-activity relationship (SAR) study using model fluorescent probes and cap-ligand conjugates showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity towards cancer cells.
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Secondary organic aerosol (SOA) is a major component of airborne fine particulate matter (PM2.5) that contributes to adverse human health effects upon inhalation. Atmospheric ozonolysis of α-pinene, an abundantly emitted monoterpene from terrestrial vegetation, leads to significant global SOA formation; however, its impact on pulmonary pathophysiology remains uncertain. In this study, we quantified an increasing concentration response of three well-established α-pinene SOA tracers (pinic, pinonic, and 3-methyl-1,2,3-butanetricarboxylic acids) and a full mixture of α-pinene SOA in A549 (alveolar epithelial carcinoma) and BEAS-2B (bronchial epithelial normal) lung cell lines. The three aforementioned tracers contributed â¼57% of the α-pinene SOA mass under our experimental conditions. Cellular proliferation, cell viability, and oxidative stress were assessed as toxicological end points. The three α-pinene SOA molecular tracers had insignificant responses in both cell types when compared with the α-pinene SOA (up to 200 µg mL-1). BEAS-2B cells exposed to 200 µg mL-1 of α-pinene SOA decreased cellular proliferation to â¼70% and 44% at 24- and 48-h post exposure, respectively; no changes in A549 cells were observed. The inhibitory concentration-50 (IC50) in BEAS-2B cells was found to be 912 and 230 µg mL-1 at 24 and 48 h, respectively. An approximate 4-fold increase in cellular oxidative stress was observed in BEAS-2B cells when compared with untreated cells, suggesting that reactive oxygen species (ROS) buildup resulted in the downstream cytotoxicity following 24 h of exposure to α-pinene SOA. Organic hydroperoxides that were identified in the α-pinene SOA samples likely contributed to the ROS and cytotoxicity. This study identifies the potential components of α-pinene SOA that likely modulate the oxidative stress response within lung cells and highlights the need to carry out chronic exposure studies on α-pinene SOA to elucidate its long-term inhalation exposure effects.
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Monoterpenos Bicíclicos/efectos adversos , Aerosoles/efectos adversos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Estrés Oxidativo/efectos de los fármacosRESUMEN
Understanding the mobility of nano-objects in the eukaryotic cell nucleus, at multiple length-scales, is essential for dissecting nuclear structure-function relationships both in space and in time. Here, we demonstrate, using single-molecule fluorescent correlation spectroscopies, that motion of inert probes (proteins, polymers, or nanoparticles) with diameters ranging from 2.6 to 150 nm is mostly unobstructed in a nucleus. Supported by the analysis of electron tomography images, these results advocate the â¼150 nm-wide interchromosomal channels filled with the aqueous diluted protein solution. The nucleus is percolated by these channels to allow various cargos to migrate freely at the nanoscale. We determined the volume of interchromosomal channels in the HeLa cell nucleus to 237 ± 61 fL, which constitutes 34% of the cell nucleus volume. The volume fraction of mobile proteins in channels equals 16% ± 4%, and the concentration is 1 mM.
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Núcleo Celular/química , Nanoestructuras/química , Supervivencia Celular , Células HeLa , Humanos , Espectrometría de Fluorescencia , ViscosidadRESUMEN
The efficient delivery of drugs to cells depends on their diffusion through the extracellular matrix (ECM) of tissues. Here we present a study on the diffusion of nanoprobes of radius from 1 nm to over 100 nm in the ECM of spheroids of three cell types (HeLa, MCF-7 and fibroblasts). We quantified the nanoparticle transport in the spheroids' proliferating zone. We determined the size-dependent viscosity of the ECM. We revealed that nanoobjects up to 10 nm in radius exhibited unobstructed diffusion in the ECM, regardless of the spheroid type. The presented length-scale dependent viscosity profiles for spheroids pave the way for advanced modelling of drug administration through tissues.
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Nanopartículas , Esferoides Celulares , Difusión , Matriz Extracelular , FibroblastosRESUMEN
Metabolic reactions in living cells are limited by diffusion of reagents in the cytoplasm. Any attempt to quantify the kinetics of biochemical reactions in the cytosol should be preceded by careful measurements of the physical properties of the cellular interior. The cytoplasm is a complex, crowded fluid characterized by effective viscosity dependent on its structure at a nanoscopic length scale. In this work, we present and validate the model describing the cytoplasmic nanoviscosity, based on measurements in seven human cell lines, for nanoprobes ranging in diameters from 1 to 150 nm. Irrespective of cell line origin (epithelial-mesenchymal, cancerous-noncancerous, male-female, young-adult), we obtained a similar dependence of the viscosity on the size of the nanoprobes, with characteristic length-scales of 20 ± 11 nm (hydrodynamic radii of major crowders in the cytoplasm) and 4.6 ± 0.7 nm (radii of intercrowder gaps). Moreover, we revealed that the cytoplasm behaves as a liquid for length scales smaller than 100 nm and as a physical gel for larger length scales.
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Citoplasma/química , Línea Celular Tumoral , Citoplasma/ultraestructura , Dextranos/química , Difusión , Fluoresceínas/química , Colorantes Fluorescentes/química , Humanos , Nanopartículas/química , Tamaño de la Partícula , Rodaminas/química , Dióxido de Silicio/química , ViscosidadRESUMEN
Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and - as a consequence - rates of biochemical reactions in a cell. Nanoviscosity is an outcome of variable chemical and structural factors, which can temporarily change with cell-cycle associated changes of intracellular architecture. Thus, the question arises, whether rates of biochemical reactions depend on the point of cell cycle. In this paper we address this topic by constant observation of nanoviscosity of HeLa cells cytoplasm during S, G2 and G1 phases after Aphidicolin synchronization. For this purpose we measured diffusion rates of EGFP molecules using fluorescence correlation spectroscopy (FCS). To our surprise, a counter-intuitive stability of cytoplasmic viscosity was observed during the cell cycle. Our results hint at possible existence of robust mechanism maintaining stable physiological viscosity of the cytoplasm, despite huge structural changes during cell cycle.
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Afidicolina/farmacología , Fenómenos Biofísicos/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Tamaño de la Célula , Citoplasma/metabolismo , Citometría de Flujo , Células HeLa , Humanos , Viscosidad/efectos de los fármacosRESUMEN
Biochemistry in living cells is an emerging field of science. Current quantitative bioassays are performed ex vivo, thus equilibrium constants and reaction rates of reactions occurring in human cells are still unknown. To address this issue, we present a non-invasive method to quantitatively characterize interactions (equilibrium constants, KD) directly within the cytosol of living cells. We reveal that cytosolic hydrodynamic drag depends exponentially on a probe's size, and provide a model for its determination for different protein sizes (1-70 nm). We analysed oligomerization of dynamin-related protein 1 (Drp1, wild type and mutants: K668E, G363D, C505A) in HeLa cells. We detected the coexistence of wt-Drp1 dimers and tetramers in cytosol, and determined that KD for tetramers was 0.7 ± 0.5 µM. Drp1 kinetics was modelled by independent simulations, giving computational results which matched experimental data. This robust method can be applied to in vivo determination of KD for other protein-protein complexes, or drug-target interactions.
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Citosol/metabolismo , Dinaminas/química , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Multimerización de Proteína , Dinaminas/genética , Dinaminas/metabolismo , Humanos , Dinámicas Mitocondriales , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Transporte de ProteínasRESUMEN
One of the main players in the process of mitochondrial fragmentation is dynamin-related protein 1 (Drp1), which assembles into a helical ring-like structure on the mitochondria and facilitates fission. The fission mechanism is still poorly understood and detailed information concerning oligomeric form of Drp1, its cellular distribution and the size of the fission complex is missing. To estimate oligomeric forms of Drp1 in the cytoplasm and on the mitochondria, we performed a quantitative analysis of Drp1 diffusion and distribution in gene-edited HeLa cell lines. This paper provides an insight into the fission mechanism based on the quantitative description of Drp1 cellular distribution. We found that approximately half of the endogenous GFP-Drp1 pool remained in the cytoplasm, predominantly in a tetrameric form, at a concentration of 28 ± 9 nM. The Drp1 mitochondrial pool included many different oligomeric states with equilibrium distributions that could be described by isodesmic supramolecular polymerization with a Kd of 31 ± 10 nM. We estimated the average number of Drp1 molecules forming the functional fission complex to be approximately 100, representing not more than 14% of all Drp1 oligomers. We showed that the upregulated fission induced by niclosamide is accompanied by an increase in the number of large Drp1 oligomers.
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GTP Fosfohidrolasas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/metabolismo , Supervivencia Celular , Citoplasma/metabolismo , Dinaminas , GTP Fosfohidrolasas/química , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/química , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Transporte de Proteínas , ViscosidadRESUMEN
Multicellular spheroid is a three-dimensional (3D) cell culture model that mimics cancer tumor environment. Its widespread use for anticancer therapy evaluation is nowadays limited by accessibility of 3D compatible assays. Here, a microfluidic system for spheroid formation, culture and analysis is presented. The system is compatible with standard microplate readers. The microfluidic chip enables long-term 3D cell culture and in situ monitoring of cellular viability. Moreover, design of the assay enables observation of delayed type of toxicity or application of repeated doses of a drug.