Natale, Paolo

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Universidad Complutense de Madrid
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Ciencias Químicas
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Now showing 1 - 10 of 16
  • Publication
    The GDP-Bound State of Mitochondrial Mfn1 Induces Membrane Adhesion of Apposing Lipid Vesicles through a Cooperative Binding Mechanism
    (MDPI, 2020-07-21) Tolosa Díaz, Andrés; Almendro Vedia, Víctor G.; Natale, Paolo; López Montero, Iván
    Mitochondria are double-membrane organelles that continuously undergo fission and fusion. Outer mitochondrial membrane fusion is mediated by the membrane proteins mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2), carrying a GTP hydrolyzing domain (GTPase) and two coiled-coil repeats. The detailed mechanism on how the GTP hydrolysis allows Mfns to approach adjacent membranes into proximity and promote their fusion is currently under debate. Using model membranes built up as giant unilamellar vesicles (GUVs), we show here that Mfn1 promotes membrane adhesion of apposing lipid vesicles. The adhesion forces were sustained by the GDP-bound state of Mfn1 after GTP hydrolysis. In contrast, the incubation with the GDP:AlF− 4 , which mimics the GTP transition state, did not induce membrane adhesion. Due to the flexible nature of lipid membranes, the adhesion strength depended on the surface concentration of Mfn1 through a cooperative binding mechanism. We discuss a possible scenario for the outer mitochondrial membrane fusion based on the modulated action of Mfn1.
  • Publication
    Rhodamine-based sensor for real-time imaging of mitochondrial ATP in living fibroblasts
    (Elsevier, 2017) de la Fuente Herreruela, Diego; Gónzalez Charro, Vicente; Almendro Vedia, Víctor G.; Morán, María; Martín, Miguel Ángel; Lillo, M. Pilar; Natale, Paolo; López-Montero, Iván
    Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL+ into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL+ to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism.
  • Publication
    Enhanced Cytotoxic Activity of Mitochondrial Mechanical Effectors in Human Lung Carcinoma H520 Cells: Pharmaceutical Implications for Cancer Therapy
    (Frontiers Media, 2018-11) González Rubio, Sergio; Montero Pastor, Nuria; García, Carolina; Almendro Vedia, Víctor G.; Ferrer, Irene; Natale, Paolo; Paz-Ares, Luis; Lillo, M. Pilar; López-Montero, Iván
    Cancer cell mitochondria represent an attractive target for oncological treatment as they have unique hallmarks that differ from their healthy counterparts, as the presence of a stronger membrane potential that can be exploited to specifically accumulate cytotoxic cationic molecules. Here, we explore the selective cytotoxic effect of 10-N-nonyl acridine orange (NAO) on human lung carcinoma H520 cells and compare them with healthy human lung primary fibroblasts. NAO is a lipophilic and positively charged molecule that promotes mitochondrial membrane adhesion that eventually leads to apoptosis when incubated at high micromolar concentration. We found an enhanced cytotoxicity of NAO in H520 cancer cells. By means Fluorescence lifetime imaging microscopy (FLIM) we also confirmed the formation of H-dimeric aggregates originating from opposing adjacent membranes that interfere with the mitochondrial membrane structure. Based on our results, we suggest the mitochondrial membrane as a potential target in cancer therapy to mechanically control the cell proliferation of cancer cells.
  • Publication
    Thermomechanical Transitions of Egg-Ceramide Monolayers
    (American Chemical Society, 2015) Catapano, Elisa R.; Lillo, M. P.; García Rodríguez, C.; Natale, Paolo; Langevin, D.; Monroy Muñoz, Francisco; López-Montero, Iván
    Ceramides have unique biophysical properties. Their high melting temperature and their ability to form lateral domains have converted ceramides into the paradigm of rigid lipids. Here, using shear surface rheology of egg-ceramide Langmuir monolayers, a solid to fluid transition was evidenced as a vanishing shear rigidity at lower temperatures than the lipid melting temperature. Such a mechanical transition, which depends on the lipid lateral pressure, was found in a broad range temperature (40−50 °C). The solid to fluid transition was correlated to a LC to LC+LE phase transition, as confirmed by BAM experiments. Interestingly, together with the softening transition, a supercooling process compatible with a glassy behavior was found upon freezing. A new phase scenario is then depicted that broadens the mechanical behavior of natural ceramides. The phase diversity of ceramides might have important implications in their physiological roles.
  • Publication
    How rotating ATP synthases can modulate membrane structure
    (Elsevier, 2021-09-15) Almendro Vedia, Víctor G.; Natale, Paolo; Valdivieso González, David; Lillo, M. Pilar; Aragones, Juan L.; López-Montero, Iván
    F1Fo-ATP synthase (ATP synthase) is a central membrane protein that synthetizes most of the ATP in the cell through a rotational movement driven by a proton gradient across the hosting membrane. In mitochondria, ATP synthases can form dimers through specific interactions between some subunits of the protein. The dimeric form of ATP synthase provides the protein with a spontaneous curvature that sustain their arrangement at the rim of the high-curvature edges of mitochondrial membrane (cristae). Also, a direct interaction with cardiolipin, a lipid present in the inner mitochondrial membrane, induces the dimerization of ATP synthase molecules along cristae. The deletion of those biochemical interactions abolishes the protein dimerization producing an altered mitochondrial function and morphology. Mechanically, membrane bending is one of the key deformation modes by which mitochondrial membranes can be shaped. In particular, bending rigidity and spontaneous curvature are important physical factors for membrane remodelling. Here, we discuss a complementary mechanism whereby the rotatory movement of the ATP synthase might modify the mechanical properties of lipid bilayers and contribute to the formation and regulation of the membrane invaginations.
  • Publication
    pH-triggered endosomal escape of pore-forming Listeriolysin O toxin-coated gold nanoparticles
    (BMC, 2019) Plaza-GA, Ismael; Manzaneda González, Vanesa; Kisovec, Matic; Almendro Vedia, Víctor; Muñoz Úbeda, Mónica; Anderluh, Gregor; Guerrero Martínez, Andrés; Natale, Paolo; López-Montero, Iván
    Background: A major bottleneck in drug delivery is the breakdown and degradation of the delivery system through the endosomal/lysosomal network of the host cell, hampering the correct delivery of the drug of interest. In nature, the bacterial pathogen Listeria monocytogenes has developed a strategy to secrete Listeriolysin O (LLO) toxin as a tool to escape the eukaryotic lysosomal system upon infection, allowing it to grow and proliferate unharmed inside the host cell. Results: As a “proof of concept”, we present here the use of purifed His-LLO H311A mutant protein and its conjuga tion on the surface of gold nanoparticles to promote the lysosomal escape of 40 nm-sized nanoparticles in mouse embryonic fbroblasts. Surface immobilization of LLO was achieved after specifc functionalization of the nanoparti cles with nitrile acetic acid, enabling the specifc binding of histidine-tagged proteins. Conclusions: Endosomal acidifcation leads to release of the LLO protein from the nanoparticle surface and its self-assembly into a 300 Å pore that perforates the endosomal/lysosomal membrane, enabling the escape of nanoparticles.
  • Publication
    ATP Synthesis and Biosensing Coupled to the Electroenzymatic Activity of a Hydrogenase on an Electrode/Biomimetic Membrane Interface
    (MDPI, 2017-11-29) Pita, Marcos; Gutierrez-Sanchez, Cristina; Natale, Paolo; García-Molina, Gabriel; Marquez, Ileana F.; Marques, Marta C.; Zacarias, Sonia; Pereira, Ines A. C.; López Montero, Iván; Velez, Marisela; Lacey, Antonio L. De
  • Publication
    Rheology of Pseudomonas fluorescens biofilms: From experiments to predictive DPD mesoscopic modeling
    (American Institute of Physics, 2023-02-21) Martín Roca, José; Bianco, Valentino; Alarcón, Francisco; Monnappa, Ajay K.; Natale, Paolo; Monroy, Francisco; Orgaz, Belén; López Montero, Iván; Valeriani, Chantal
    Bacterial biofilms mechanically behave as viscoelastic media consisting of micron-sized bacteria cross-linked to a self-produced network of extracellular polymeric substances (EPSs) embedded in water. Structural principles for numerical modeling aim at describing mesoscopic viscoelasticity without losing details on the underlying interactions existing in wide regimes of deformation under hydrodynamic stress. Here, we approach the computational challenge to model bacterial biofilms for predictive mechanics in silico under variable stress conditions. Up-to-date models are not entirely satisfactory due to the plethora of parameters required to make them functioning under the effects of stress. As guided by the structural depiction gained in a previous work with Pseudomonas fluorescens [Jara et al., Front. Microbiol. 11, 588884 (2021)], we propose a mechanical modeling by means of Dissipative Particle Dynamics (DPD), which captures the essentials of topological and compositional interactions between bacterial particles and cross-linked EPS-embedding under imposed shear. The P. fluorescens biofilms have been modeled under mechanical stress mimicking shear stresses as undergone in vitro. The predictive capacity for mechanical features in DPD-simulated biofilms has been investigated by varying the externally imposed field of shear strain at variable amplitude and frequency. The parametric map of essential biofilm ingredients has been explored by making the rheological responses to emerge among conservative mesoscopic interactions and frictional dissipation in the underlying microscale. The proposed coarse grained DPD simulation qualitatively catches the rheology of the P. fluorescens biofilm over several decades of dynamic scaling. Published under an exclusive license by AIP Publishing.
  • Publication
    Nonequilibrium fluctuations of lipid membranes by the rotating motor protein F1F0-ATP synthase
    (National Academy of Sciences, 2017) Almendro-Vedia, Víctor G.; Natale, Paolo; Mell, Michael; Bonneau, Stephanie; Monroy Muñoz, Francisco; Joubert, Frederic; López-Montero, Iván
    ATP synthase is a rotating membrane protein that synthesizes ATP through proton-pumping activity across the membrane. To unveil the mechanical impact of this molecular active pump on the bending properties of its lipid environment, we have functionally reconstituted the ATP synthase in giant unilamellar vesicles and tracked the membrane fluctuations by means of flickering spectroscopy. We find that ATP synthase rotates at a frequency of about 20 Hz, promoting large nonequilibrium deformations at discrete hot spots in lipid vesicles and thus inducing an overall membrane softening. The enhanced nonequilibrium fluctuations are compatible with an accumulation of active proteins at highly curved membrane sites through a curvature−protein coupling mechanism that supports the emergence of collective effects of rotating ATP synthases in lipid membranes.
  • Publication
    The enzymatic sphingomyelin to ceramide conversion increases the shear membrane viscosity at the air-water interface
    (Elsevier, 2017) Catapano, Elisa R.; Natale, Paolo; Monroy Muñoz, Francisco; López-Montero, Iván
    Whereas most of lipids have viscous properties and they do not have significant elastic features, ceramides behave as very rigid solid assemblies, displaying viscoelastic behaviour at physiological temperatures. The present review addresses the surface rheology of lipid binary mixtures made of sphingomyelin and ceramide. However, ceramide is formed by the enzymatic cleavage of sphingomyelin in cell plasma membranes. The consequences of the enzymatically-driven ceramide formation involve mechanical alterations of the embedding membrane. Here, an increase on surface shear viscosity was evidenced upon enzymatic incubation of sphingomyelin monolayers. The overall rheological data are discussed in terms of the current knowledge of the thermotropic behaviour of ceramide-containing model membranes.