Automatización de la adquisición y procesamiento de datos en microscopía electrónica tridimensional

257 P.La presente tesis ha sido realizada en la unidad de biología estructural del Centro de Investigación Cooperativa en biociencias (CIC bioGUNE) en colaboración con el Grupo de Control Automático (GCA) del departamento de Ingeniería de Sistemas y Automática de la Universidad de País Vasco (UPV/EHU). Esta tesis se centra en el desarrollo de un nuevo esquema de control inteligente utilizando diferentes algoritmos de inteligencia artificial para automatizar la adquisición de datos en un TEM dedicado a experimentos de cryo-EM. Este trabajo ha sido realizado utilizando el equipamiento de la plataforma de microscopía electrónica del CIC bioGUNE, especializada en el análisis de muestras a temperaturas criogénicas con el microscopio electrónico de transmisión modelo JEM-2200FS/CR de la compañía Jeol. El esquema de control inteligente permite el control remoto del microscopio y la monitorización en tiempo real del proceso de adquisición y análisis de las imágenes en una sesión cryo-TEM en modo automático. Uno de los objetivos de este trabajo es que el sistema de control inteligente lleve a cabo la misma tarea y de forma similar a cómo lo realizaría un microscopista experto en sesiones cryo-TEM, evaluando la calidad de las imágenes en tiempo real

Photoacoustic efect applied on model membranes and living cells: direct observation with multiphoton excitation microscopy and long-term viability analysis

The photoacoustic efect is generated when a variable light interacts with a strongly light-absorbing material. In water, it may produce hot bubbles and shock waves that could afect the integrity of nearby cellular membranes, opening transient pores (photoporation). In this study, we have evaluated the efect of pulsed laser-irradiated carbon nanoparticles (cNP) on model membranes and on Chinese hamster ovary (CHO) cells. Fluorescence lifetime measurements of calcein-loaded liposomes support the notion that the photoacoustic efect causes transient openings in membranes, allowing difusion fuxes driven by gradient concentrations. With CHO cells, we have shown that this efect can induce either intracellular delivery of calcein, or release of cellular compounds. The latter process has been recorded live with multiphoton excitation microscopy during pulsed infrared laser irradiation. Calcein loading and cell viability were assayed by fow cytometry, measuring necrotic cells as well as those in early apoptosis. To further assess long-term cell recovery after the rather harsh treatment, cells were reseeded and their behaviour recorded for 48h. These extended studies on cell viability show that pulsed laser cNP photoporation may be considered an adequate intracellular delivery technique only if employed with soft irradiation conditions (below 50mJ/cm2).Spanish Ministry of Economy FEDER MINECO PGC2018-099857-B-I00Basque Government IT1264-19 IT1270-19University of Basque Countr

CryoEM structural exploration of catalytically active enzyme pyruvate carboxylase

Pyruvate carboxylase (PC) is a tetrameric enzyme that contains two active sites per subunit that catalyze two consecutive reactions. A mobile domain with an attached prosthetic biotin links both reactions, an initial biotin carboxylation and the subsequent carboxyl transfer to pyruvate substrate to produce oxaloacetate. Reaction sites are at long distance, and there are several co-factors that play as allosteric regulators. Here, using cryoEM we explore the structure of active PC tetramers focusing on active sites and on the conformational space of the oligomers. The results capture the mobile domain at both active sites and expose catalytic steps of both reactions at high resolution, allowing the identification of substrates and products. The analysis of catalytically active PC tetramers reveals the role of certain motions during enzyme functioning, and the structural changes in the presence of additional cofactors expose the mechanism for allosteric regulation.This study was supported by grants from the HFSP (RGP0062), and from the Spanish Ministerio de Ciencia e Innovacion (PGC2018-098996-B-100) to M.V., and grants from the NIGMS (R35GM118093) and the NIAID (R01AI116669) to L.T

Elucidating the role of shape anisotropy infaceted magnetic nanoparticles using biogenicmagnetosomes as a model

Shape anisotropy is of primary importance to understand the magnetic behavior of nanoparticles, but a rigorous analysis in polyhedral morphologies is missing. In this work, a model based on finite element techniques has been developed to calculate the shape anisotropy energy landscape for cubic, octahedral, and truncated-octahedral morphologies. In all cases, a cubic shape anisotropy is found that evolves to quasi-uniaxial anisotropy when the nanoparticle is elongated >= 2%. This model is tested on magnetosomes, similar to 45 nm truncated octahedral magnetite nanoparticles forming a chain inside Magnetospirillum gryphiswaldense MSR-1 bacteria. This chain presents a slightly bent helical configuration due to a 20 degrees tilting of the magnetic moment of each magnetosome out of chain axis. Electron cryotomography images reveal that these magnetosomes are not ideal truncated-octahedrons but present approximate to 7.5% extrusion of one of the {001} square faces and approximate to 10% extrusion of an adjacent {111} hexagonal face. Our model shows that this deformation gives rise to a quasi-uniaxial shape anisotropy, a result of the combination of a uniaxial (Ksh-u = 7 kJm(-3)) and a cubic (Ksh-c = 1.5 kJ m(-3)) contribution, which is responsible for the 20 degrees tilting of the magnetic moment. Finally, our results have allowed us to accurately reproduce, within the framework of the Landau-Lifshitz-Gilbert model, the experimental AC loops measured for these magnetotactic bacteria.Spanish Government is acknowledged for funding under the project number MAT2017-83631-C3. Basque Government is acknowledged for funding under the project number IT124519. HRTEM images were obtained in the Laboratorio de Microscopias Avanzadas at Instituto de Nanociencia de Aragon -Universidad de Zaragoza (LMA-INA). Authors acknowledge the LMA-INA for offering access to their instruments and expertise. Authors thank Prof. J. A. Garcia and I. Rodrigo for providing AC hysteresis loops

Structure of p15PAF–PCNA complex and implications for clamp sliding during DNA replication and repair

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The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.The project was supported by grant BFU2013-48374-P of the Spanish MINECO and by the Ramón Areces Foundation. O.G. held a research contract under the Ramón y Cajal program of the Spanish MINECO (RYC-2011-07967). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from the Spanish MINECO.Peer reviewe

Architecture of the ESCPE-1 membrane coat

Recycling of membrane proteins enables the reuse of receptors, ion channels and transporters. A key component of the recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), which rescues transmembrane proteins from the endolysosomal pathway for transport to the trans-Golgi network and the plasma membrane. This rescue entails the formation of recycling tubules through ESCPE-1 recruitment, cargo capture, coat assembly and membrane sculpting by mechanisms that remain largely unknown. Herein, we show that ESCPE-1 has a single-layer coat organization and suggest how synergistic interactions between ESCPE-1 protomers, phosphoinositides and cargo molecules result in a global arrangement of amphipathic helices to drive tubule formation. Our results thus define a key process of tubule-based endosomal sorting.This work was funded by MCIN/AEI/10.13039/501100011033 (PID2020- 119132GB-I00, CEX2021-001136-S) (to A.H.), the Intramural Program of NICHD, NIH (ZIA HD001607 to J.S.B.), the Swiss National Science Foundation grant 205321 179041 (to D.C.-D.), the Human Frontiers Science Program grant RGP0017/2020 (to D.C.-D.) and the PID2021- 127309NB-I00 funded by AEI/10.13039/501100011033/ FEDER, UE (to D.C.-D.). This study made use of the Diamond Light Source proposal MX20113, ALBA synchrotron beamline BL13-XALOC, the cryo-EM facilities at the UK Electron Bio-Imaging Centre, proposals EM17171- 6 and EM17171, and the Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB). We thank C. Savva (LISCB, University of Leicester) for his help in cryo-EM data collection. With funding from the Spanish government through the Severo Ochoa Centre of Excellence’ accreditation (CEX2021-001136-S

Automatización de la adquisición y procesamiento de datos en microscopía electrónica tridimensional

257 P.La presente tesis ha sido realizada en la unidad de biología estructural del Centro de Investigación Cooperativa en biociencias (CIC bioGUNE) en colaboración con el Grupo de Control Automático (GCA) del departamento de Ingeniería de Sistemas y Automática de la Universidad de País Vasco (UPV/EHU). Esta tesis se centra en el desarrollo de un nuevo esquema de control inteligente utilizando diferentes algoritmos de inteligencia artificial para automatizar la adquisición de datos en un TEM dedicado a experimentos de cryo-EM. Este trabajo ha sido realizado utilizando el equipamiento de la plataforma de microscopía electrónica del CIC bioGUNE, especializada en el análisis de muestras a temperaturas criogénicas con el microscopio electrónico de transmisión modelo JEM-2200FS/CR de la compañía Jeol. El esquema de control inteligente permite el control remoto del microscopio y la monitorización en tiempo real del proceso de adquisición y análisis de las imágenes en una sesión cryo-TEM en modo automático. Uno de los objetivos de este trabajo es que el sistema de control inteligente lleve a cabo la misma tarea y de forma similar a cómo lo realizaría un microscopista experto en sesiones cryo-TEM, evaluando la calidad de las imágenes en tiempo real

Photoacoustic effect applied on model membranes and living cells: direct observation with multiphoton excitation microscopy and long-term viability analysis

The photoacoustic effect is generated when a variable light interacts with a strongly light-absorbing material. In water, it may produce hot bubbles and shock waves that could affect the integrity of nearby cellular membranes, opening transient pores (photoporation). In this study, we have evaluated the effect of pulsed laser-irradiated carbon nanoparticles (cNP) on model membranes and on Chinese hamster ovary (CHO) cells. Fluorescence lifetime measurements of calcein-loaded liposomes support the notion that the photoacoustic effect causes transient openings in membranes, allowing diffusion fluxes driven by gradient concentrations. With CHO cells, we have shown that this effect can induce either intracellular delivery of calcein, or release of cellular compounds. The latter process has been recorded live with multiphoton excitation microscopy during pulsed infrared laser irradiation. Calcein loading and cell viability were assayed by flow cytometry, measuring necrotic cells as well as those in early apoptosis. To further assess long-term cell recovery after the rather harsh treatment, cells were reseeded and their behaviour recorded for 48 h. These extended studies on cell viability show that pulsed laser cNP photoporation may be considered an adequate intracellular delivery technique only if employed with soft irradiation conditions (below 50 mJ/cm2).This work was supported in part by the Spanish Ministry of Economy (grant No. FEDER MINECO PGC2018-099857-B-I00) and the Basque Government (grants No. IT1264-19 and IT1270-19). BGM was a recipient of a predoctoral grant from the University of the Basque Country.Peer reviewe