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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.

DNA lesions threaten cell life and must be repaired to maintain genome integrity. During transcription, RNA polymerases actively scan DNA to find lesions and trigger their repair. In growing eukaryotic cells, about 60% of the total transcriptional activity involves the synthesis of ribosomal RNA (rRNA) by RNA polymerase I (Pol I), a 14-subunit macromolecular machine with unique regulatory features. Accordingly, transcribing Pol I monitors ribosomal DNA (rDNA) integrity and influences cell survival, but how this enzyme handles DNA lesions remains largely unknown. We used cryo-EM and in vitro transcription tests to investigate Pol I transcriptional stalling by one of the most common UV light-induced lesions, i.e. cyclobutane pyrimidine dimers (CPD) [3]. A two-step mechanism operates in Pol I to firmly stall the enzyme at CPD lesions, whereas RNA polymerase II (Pol II), which scans for damage within protein-coding genes, is able to bypass such lesions. First, bridge helix residue Arg1015, which is unique in Pol I, contacts the lesion and significantly reduces the bypass rate, as we confirm by mutational analysis. Second, the intrinsic RNA cleavage activity of Pol I, which in Pol II requires binding of transcription factor IIS (TFIIS), removes RNA nucleotides opposite the lesion. Our results suggest that this dual mechanism strongly blocks rRNA synthesis to hamper incorporation of mutations into ribosomes. These findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA.

The original version of this article omitted from the author list the 5th author Alexandra Dodu...

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.

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.

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the ribosomal RNA (rRNA) precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes UV-induced lesions. We report the electron cryomicroscopy structure of Pol I in an elongation complex containing a cyclobutane pyrimidine dimer (CPD) at a resolution of 3.6 Å. The structure shows that the lesion induces an early translocation intermediate exhibiting unique features. The bridge helix residue Arg1015 plays a major role in CPD-induced Pol I stalling, as confirmed by mutational analysis. These results, together with biochemical data presented here, reveal the molecular mechanism of Pol I stalling by CPD lesions, which is distinct from Pol II arrest by CPD lesions. Our findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA.

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.

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 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).

The human cathelicidin LL-37 serves a critical role in the innate immune system defending bacterial infections. LL-37 can interact with molecules of the cell wall and perforate cytoplasmic membranes resulting in bacterial cell death. To test the interactions of LL-37 and bacterial cell wall components we crystallized LL-37 in the presence of detergents and obtained the structure of a narrow tetrameric channel with a strongly charged core. The formation of a tetramer was further studied by cross-linking in the presence of detergents and lipids. Using planar lipid membranes a small but defined conductivity of this channel could be demonstrated. Molecular dynamic simulations underline the stability of this channel in membranes and demonstrate pathways for the passage of water molecules. Time lapse studies of E. coli cells treated with LL-37 show membrane discontinuities in the outer membrane followed by cell wall damage and cell death. Collectively, our results open a venue to the understanding of a novel AMP killing mechanism and allows the rational design of LL-37 derivatives with enhanced bactericidal activity.

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).

Magnetospirillum gryphiswaldense is a microorganism with the ability to biomineralize magnetite nanoparticles, called magnetosomes, and arrange them into a chain that behaves like a magnetic compass. Rather than straight lines, magnetosome chains are slightly bent, as evidenced by electron cryotomography. Our experimental and theoretical results suggest that due to the competition between the magnetocrystalline and shape anisotropies, the effective magnetic moment of individual magnetosomes is tilted out of the [111] crystallographic easy axis of magnetite. This tilt does not affect the direction of the chain net magnetic moment, which remains along the [111] axis, but explains the arrangement of magnetosomes in helical-like shaped chains. Indeed, we demonstrate that the chain shape can be reproduced by considering an interplay between the magnetic dipolar interactions between magnetosomes, ruled by the orientation of the magnetosome magnetic moment, and a lipid/protein-based mechanism, modeled as an elastic recovery force exerted on the magnetosomes.

The R2TP complex, comprising the Rvb1p-Rvb2p AAA-ATPases, Tah1p, and Pih1p in yeast, is a specialized Hsp90 co-chaperone required for the assembly and maturation of multi-subunit complexes. These include the small nucleolar ribonucleoproteins, RNA polymerase II, and complexes containing phosphatidylinositol-3-kinase-like kinases. The structure and stoichiometry of yeast R2TP and how it couples to Hsp90 are currently unknown. Here, we determine the 3D organization of yeast R2TP using sedimentation velocity analysis and cryo-electron microscopy. The 359-kDa complex comprises one Rvb1p/Rvb2p hetero-hexamer with domains II (DIIs) forming an open basket that accommodates a single copy of Tah1p-Pih1p. Tah1p-Pih1p binding to multiple DII domains regulates Rvb1p/Rvb2p ATPase activity. Using domain dissection and cross-linking mass spectrometry, we identified a unique region of Pih1p that is essential for interaction with Rvb1p/Rvb2p. These data provide a structural basis for understanding how R2TP couples an Hsp90 dimer to a diverse set of client proteins and complexes.

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.

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.

The intrinsically disordered protein p15(PAF) regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15(PAF)-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15(PAF) tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15(PAF) also contacts the inside of, and passes through, the PCNA ring. The disordered p15(PAF) termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15(PAF) binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15(PAF) acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

In a previous article, we demonstrated that histones (H1 or histone octamers) interact with negatively charged bilayers and induce extensive aggregation of vesicles containing phosphatidylinositol-4-phosphate (PIP) and, to a lesser extent, vesicles containing phosphatidylinositol (PI). Here, we found that vesicles containing PIP, but not those containing PI, can undergo fusion induced by histones. Fusion was demonstrated through the observation of intervesicular mixing of total lipids and inner monolayer lipids, and by ultrastructural and confocal microscopy studies. Moreover, in both PI- and PIP-containing vesicles, histones caused permeabilization and release of vesicular aqueous contents, but the leakage mechanism was different (all-or-none for PI and graded release for PIP vesicles). These results indicate that histones could play a role in the remodeling of the nuclear envelope that takes place during the mitotic cycle.

Hepatitis C virus infects almost 170 million people per year but its assembly pathway, architecture and the structures of its envelope proteins are poorly understood. Using electron tomography of plastic-embedded sections of insect cells, we have visualized the morphogenesis of recombinant Hepatitis C virus-like particles. Our data provide a three-dimensional sketch of viral assembly at the endoplasmic reticulum showing different budding stages and contiguity of buds. This latter phenomenon could play an important role during the assembly of . wt-HCV and explain the size-heterogeneity of its particles.