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  • 1. Amorim, Gisele C
    et al.
    Cisneros, David A.
    Unité de Génétique Moléculaire, Département de MicrobiologieInstitut Pasteur, Paris Cedex 15 France; CNRS, ERL 3526, Paris, France.
    Delepierre, Muriel
    Francetic, Olivera
    Izadi-Pruneyre, Nadia
    ¹H, ¹⁵N and ¹³C resonance assignments of PpdD, a type IV pilin from enterohemorrhagic Escherichia coli2014In: Biomolecular NMR Assignments, ISSN 1874-2718, E-ISSN 1874-270X, Vol. 8, no 1, p. 43-46Article in journal (Refereed)
    Abstract [en]

    Bacterial type 4 pili (T4P) are long flexible fibers involved in adhesion, DNA uptake, phage transduction, aggregation and a flagella-independent movement called "twitching motility". T4P comprise thousands of copies of the major pilin subunit, which is initially inserted in the plasma membrane, processed and assembled into dynamic helical filaments. T4P are crucial for host colonization and virulence of many Gram-negative bacteria. In enterohemorrhagic Escherichia coli the T4P, called hemorrhagic coli pili (HCP) promote cell adhesion, motility, biofilm formation and signaling. To understand the mechanism of HCP assembly and function, we analyzed the structure of the major subunit prepilin peptidase-dependent protein D (PpdD) (also called HcpA), a 15 kDa pilin with two potential disulfide bonds. Here we present the (1)H, (15)N and (13)C backbone and side chain resonance assignments of the C-terminal globular domain of PpdD as a first step to its structural determination.

  • 2. Campos, Manuel
    et al.
    Cisneros, David A.
    Institut Pasteur, Molecular Genetics Unit, Department of Microbiology, 75015 Paris, France.
    Nivaskumar, Mangayarkarasi
    Francetic, Olivera
    The type II secretion system: a dynamic fiber assembly nanomachine2013In: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 164, no 6, p. 545-555Article in journal (Refereed)
    Abstract [en]

    Type II secretion systems (T2SSs) share common origins and structure with archaeal flagella (archaella) and pili, bacterial competence systems and type IV pili. All of these systems use a conserved ATP-powered machinery to assemble helical fibers that are anchored in the plasma membrane. The T2SSs assemble pseudopili, periplasmic filaments that promote extracellular secretion of folded periplasmic proteins. Comparative analysis of T2SSs and related fiber assembly nanomachines might provide important clues on their functional specificities and dynamics. This review focuses on recent developments in the study of pseudopilus structure and biogenesis, and discusses mechanistic models of pseudopilus function in protein secretion.

  • 3. Campos, Manuel
    et al.
    Nilges, Michaël
    Cisneros, David A.
    Institut Pasteur, Unité de Génétique Moléculaire, Département de Microbiologie, F-75015 Paris, France.
    Francetic, Olivera
    Detailed structural and assembly model of the type II secretion pilus from sparse data2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 29, p. 13081-13086Article in journal (Refereed)
    Abstract [en]

    Many gram-negative bacteria secrete specific proteins via the type II secretion systems (T2SS). These complex machineries share with the related archaeal flagella and type IV pilus (T4P) biogenesis systems the ability to assemble thin, flexible filaments composed of small, initially inner membrane-localized proteins called "pilins." In the T2SS from Klebsiella oxytoca, periplasmic pseudopili that are essential for pullulanase (PulA) secretion extend beyond the bacterial surface and form pili when the major pilin PulG is overproduced. Here, we describe the detailed, experimentally validated structure of the PulG pilus generated from crystallographic and electron microscopy data by a molecular modeling approach. Two intermolecular salt bridges crucial for function were demonstrated using single and complementary charge inversions. Double-cysteine substitutions in the transmembrane segment of PulG led to position-specific cross-linking of protomers in assembled pili. These biochemical data provided information on residue distances in the filament that were used to derive a refined model of the T2SS pilus at pseudoatomic resolution. PulG is organized as a right-handed helix of subunits, consistent with protomer organization in gonococcal T4P. The conserved character of residues involved in key hydrophobic and electrostatic interactions within the major pseudopilin family supports the general relevance of this model for T2SS pseudopilus structure.

  • 4.
    Cisneros, David A.
    et al.
    Unité de Génétique moléculaire, Départements de Microbiologie et Biologie Structurale et Chimie, Institut Pasteur, Paris, France.
    Bond, Peter J
    Pugsley, Anthony P
    Campos, Manuel
    Francetic, Olivera
    Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation2012In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 4, p. 1041-1053Article in journal (Refereed)
    Abstract [en]

    In Gram-negative bacteria, type II secretion systems (T2SS) assemble inner membrane proteins of the major pseudopilin PulG (GspG) family into periplasmic filaments, which could drive protein secretion in a piston-like manner. Three minor pseudopilins PulI, PulJ and PulK are essential for protein secretion in the Klebsiella oxytoca T2SS, but their molecular function is unknown. Here, we demonstrate that together these proteins prime pseudopilus assembly, without actively controlling its length or secretin channel opening. Using molecular dynamics, bacterial two-hybrid assays, cysteine crosslinking and functional analysis, we show that PulI and PulJ nucleate filament assembly by forming a staggered complex in the plasma membrane. Binding of PulK to this complex results in its partial extraction from the membrane and in a 1-nm shift between their transmembrane segments, equivalent to the major pseudopilin register in the assembled PulG filament. This promotes fully efficient pseudopilus assembly and protein secretion. Therefore, we propose that PulI, PulJ and PulK self-assembly is thermodynamically coupled to the initiation of pseudopilus assembly, possibly setting the assembly machinery in motion.

  • 5.
    Cisneros, David A.
    et al.
    BIOTEC, University of Technology Dresden, Tatzberg 47-51, 01307 Dresden, Germany..
    Friedrichs, Jens
    Taubenberger, Anna
    Franz, Clemens M
    Muller, Daniel J
    Creating ultrathin nanoscopic collagen matrices for biological and biotechnological applications2007In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 3, no 6, p. 956-963Article in journal (Refereed)
    Abstract [en]

    The biofunctionalization of materials creates interfaces on which proteins, cells, or tissues can fulfill native or desired tasks. Here we report how to control the assembly of type I collagen into well-defined nanoscopic matrices of different patterns. Collagen fibrils in these ultrathin (approximately 3 nm) matrices maintained their native structure as observed in vivo. This opens up the possibility to create programmable biofunctionalized matrices using collagen-binding proteins or proteins fused with collagen-binding domains. Applied to eukaryotic cells, these nanostructured matrices can direct cellular processes such as adhesion, orientation and migration.

  • 6.
    Cisneros, David A.
    et al.
    Biotechnology Center, University of Technology Dresden, 01307 Dresden, Germany.
    Hung, Carlos
    Franz, Clemens M
    Muller, Daniel J
    Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy2006In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 154, no 3, p. 232-245Article in journal (Refereed)
    Abstract [en]

    Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.

  • 7.
    Cisneros, David A.
    et al.
    Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), P.O. Box 70-159, Mexico City, D.F, 04510, Mexico.
    Montero-Morán, Gabriela M
    Lara-González, Samuel
    Calcagno, Mario L
    Inversion of the allosteric response of Escherichia coli glucosamine-6-P deaminase to N-acetylglucosamine 6-P, by single amino acid replacements2004In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 421, no 1, p. 77-84Article in journal (Refereed)
    Abstract [en]

    Amino acid replacements in the active site of glucosamine-6-P deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) involving the residues D141 and E148 produce atypical allosteric kinetics. These residues are located in the chain segment 139-156 which is part of the active site and which also forms several intersubunit contacts close to the allosteric site. In the D141N and E148Q mutant forms of this deaminase, there is an inversion of the effect of its physiological allosteric effector, N-acetylglucosamine 6-P, which becomes an inhibitor at substrate concentrations above a critical value. For both mutants, this particular point appears at low substrate concentration and the inhibition by the allosteric activator is the dominant effect in velocity versus substrate curves. These effects are analyzed as a particular case of the concerted allosteric model, assuming that the R state, the conformer displaying the higher affinity for the substrate, is the less catalytic state, thus producing an inverted allosteric response.

  • 8.
    Cisneros, David A.
    et al.
    Biotechnologisches Zentrum, Technische Universität Dresden, Tatzberg 49, 01307 Dresden, Germany.
    Muller, Daniel J
    Daud, Sofian M
    Lakey, Jeremy H
    An approach to prepare membrane proteins for single-molecule imaging2006In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 45, no 20, p. 3252-3256Article in journal (Refereed)
  • 9.
    Cisneros, David A.
    et al.
    Biotechnology Center, University of Technology, Dresden, Germany.
    Oberbarnscheidt, Leoni
    Pannier, Angela
    Klare, Johann P.
    Helenius, Jonne
    Engelhard, Martin
    Oesterhelt, Filipp
    Muller, Daniel J.
    Transducer binding establishes localized interactions to tune sensory rhodopsin II2008In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 16, no 8, p. 1206-1213Article in journal (Refereed)
    Abstract [en]

    In haloarchaea, sensory rhodopsin II (SRII) mediates a photophobic response to avoid photo-oxidative damage in bright light. Upon light activation the receptor undergoes a conformational change that activates a tightly bound transducer molecule (HtrII), which in turn by a chain of homologous reactions transmits the signal to the chemotactic eubacterial two-component system. Here, using single-molecule force spectroscopy, we localize and quantify changes to the intramolecular interactions within SRII of Natronomonas pharaonis (NpSRII) upon NpHtrII binding. Transducer binding affected the interactions at transmembrane alpha helices F and G of NpSRII to which the transducer was in contact. Remarkably, the interactions were distributed asymmetrically and significantly stabilized alpha helix G entirely but alpha helix F only at its extracellular tip. These findings provide unique insights into molecular mechanisms that "prime" the complex for signaling, and guide the receptor toward transmitting light-activated structural changes to its cognate transducer.

  • 10.
    Cisneros, David A.
    et al.
    BIOTEC, University of Technology, 01307 Dresden, Germany..
    Oesterhelt, Dieter
    Müller, Daniel J
    Probing origins of molecular interactions stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin2005In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 13, no 2, p. 235-242Article in journal (Refereed)
    Abstract [en]

    Single-molecule atomic force microscopy and spectroscopy were applied to detect molecular interactions stabilizing the structure of halorhodopsin (HR), a light-driven chloride pump from Halobacterium salinarum. Because of the high structural and sequence similarities between HR and bacteriorhodopsin, we compared their unfolding pathways and polypeptide regions that established structurally stable segments against unfolding. Unfolding pathways and structural segments stabilizing the proteins both exhibited a remarkably high similarity. This suggests that different amino acid compositions can establish structurally indistinguishable energetic barriers. These stabilizing domains rather result from comprehensive interactions of all amino acids within a structural region than from specific interactions. However, one additional unfolding barrier located within a short segment of helix E was detected for HR. This barrier correlated with a Pi-bulk interaction, which locally disrupts helix E and divides a structural stabilizing segment.

  • 11.
    Cisneros, David A.
    et al.
    Molecular Genetics Unit, Department of Microbiology, Institut Pasteur, 75015, Paris, France; CNRS ERL3526, 75015 Paris, France..
    Pehau-Arnaudet, Gerard
    Francetic, Olivera
    Heterologous assembly of type IV pili by a type II secretion system reveals the role of minor pilins in assembly initiation2012In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 86, no 4, p. 805-818Article in journal (Refereed)
    Abstract [en]

    In Gram-negative bacteria, type IV pilus assembly (T4PS) and type II secretion (T2SS) systems polymerize inner membrane proteins called major pilins or pseudopilins respectively, into thin filaments. Four minor pilins are required in both systems for efficient fibre assembly. Escherichia coli K-12 has a set of T4PS assembly genes that are silent under standard growth conditions. We studied the heterologous assembly of the E. coli type IV pilin PpdD by the Klebsiella oxytoca T2SS called the Pul system. PpdD pilus assembly in this context depended on the expression of the K. oxytoca minor pseudopilin genes pulHIJK or of the E. coli minor pilin genes ppdAB-ygdB-ppdC. The E. coli minor pilins restored assembly of the major pseudopilin PulG in a pulHIJK mutant, but not the secretion of the T2SS substrate pullulanase. Thus, minor pilins and minor pseudopilins are functionally interchangeable in initiating major pilin assembly, further extending the fundamental similarities between the two systems. The data suggest that, in both systems, minor pilins activate the assembly machinery through a common self-assembly mechanism. When produced together, PulG and PpdD assembled into distinct homopolymers, establishing major pilins as key determinants of pilus elongation and structure.

  • 12. Davidson, Iain F.
    et al.
    Goetz, Daniela
    Zaczek, Maciej P.
    Molodtsov, Maxim I.
    in't Veld, Pim J. Huis
    Weissmann, Florian
    Litos, Gabriele
    Cisneros, David A.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Research Institute of Molecular Pathology (IMP), Vienna, Austria.
    Ocampo-Hafalla, Maria
    Ladurner, Rene
    Uhlmann, Frank
    Vaziri, Alipasha
    Peters, Jan-Michael
    Rapid movement and transcriptional re-localization of human cohesin on DNA2016In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 35, no 24, p. 2671-2685Article in journal (Refereed)
    Abstract [en]

    The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA. It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.

  • 13. Janovjak, Harald
    et al.
    Kedrov, Alexej
    Cisneros, David A.
    Center of Biotechnology, University of Technology and Max-Planck-Institute of Molecular Cell Biology and Genetics, Tatzberg 49, D-01307 Dresden, Germany.
    Sapra, K Tanuj
    Struckmeier, Jens
    Muller, Daniel J
    Imaging and detecting molecular interactions of single transmembrane proteins2006In: Neurobiology of Aging, ISSN 0197-4580, E-ISSN 1558-1497, Vol. 27, no 4, p. 546-561Article in journal (Refereed)
    Abstract [en]

    Single-molecule atomic force microscopy (AFM) provides novel ways to characterize structure-function relationships of native membrane proteins. High-resolution AFM-topographs allow observing substructures of single membrane proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. Complementary to AFM imaging, single-molecule force spectroscopy experiments allow detecting molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to detect the interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the position of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes. We review current and future potential of these approaches to reveal insights into membrane protein structure, function, and unfolding as we recognize that they could help answering key questions in the molecular basis of certain neuro-pathological dysfunctions.

  • 14. Ladurner, Rene
    et al.
    Kreidl, Emanuel
    Ivanov, Miroslav P
    Ekker, Heinz
    Idarraga-Amado, Maria Helena
    Busslinger, Georg A
    Wutz, Gordana
    Cisneros, David A.
    Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.
    Peters, Jan-Michael
    Sororin actively maintains sister chromatid cohesion2016In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 35, no 6, p. 635-653Article in journal (Refereed)
    Abstract [en]

    Cohesion between sister chromatids is established during DNA replication but needs to be maintained to enable proper chromosome-spindle attachments in mitosis or meiosis. Cohesion is mediated by cohesin, but also depends on cohesin acetylation and sororin. Sororin contributes to cohesion by stabilizing cohesin on DNA. Sororin achieves this by inhibiting WAPL, which otherwise releases cohesin from DNA and destroys cohesion. Here we describe mouse models which enable the controlled depletion of sororin by gene deletion or auxin-induced degradation. We show that sororin is essential for embryonic development, cohesion maintenance, and proper chromosome segregation. We further show that the acetyltransferases ESCO1 and ESCO2 are essential for stabilizing cohesin on chromatin, that their only function in this process is to acetylate cohesin's SMC3 subunit, and that DNA replication is also required for stable cohesin-chromatin interactions. Unexpectedly, we find that sororin interacts dynamically with the cohesin complexes it stabilizes. This implies that sororin recruitment to cohesin does not depend on the DNA replication machinery or process itself, but on a property that cohesin acquires during cohesion establishment.

  • 15. Muhar, Matthias
    et al.
    Ebert, Anja
    Neumann, Tobias
    Umkehrer, Christian
    Jude, Julian
    Wieshofer, Corinna
    Rescheneder, Philipp
    Lipp, Jesse J.
    Herzog, Veronika A.
    Reichholf, Brian
    Cisneros, David A.
    Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), 1030 Vienna, Austria..
    Hoffmann, Thomas
    Schlapansky, Moritz F.
    Bhat, Pooja
    von Haeseler, Arndt
    Köcher, Thomas
    Obenauf, Anna C.
    Popow, Johannes
    Ameres, Stefan L.
    Zuber, Johannes
    SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis2018In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 360, no 6390, p. 800-805Article in journal (Refereed)
    Abstract [en]

    Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

  • 16. Poole, Kate
    et al.
    Khairy, Khaled
    Friedrichs, Jens
    Franz, Clemens
    Cisneros, David A.
    BioTechnological Center, University of Technology Dresden, 01307 Dresden, Germany.
    Howard, Jonathon
    Mueller, Daniel
    Molecular-scale topographic cues induce the orientation and directional movement of fibroblasts on two-dimensional collagen surfaces2005In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 349, no 2, p. 380-386Article in journal (Refereed)
    Abstract [en]

    Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a functional scaffold for cells. Cells can move directionally along the axis of fibrous structures, in a process important in wound healing and cell migration. The precise nature of the structural cues within the collagen fibrils that can direct cell movement are not known. We have investigated the structural features of collagen that are required for directional motility of mouse dermal fibroblasts, by analysing cell movement on two-dimensional collagen surfaces. The surfaces were prepared with aligned fibrils of collagen type I, oriented in a predefined direction. These collagen-coated surfaces were generated with or without the characteristic 67 nm D-periodic banding. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation and motional directionality with the orientation of D-periodic collagen microfibrils. Neither directed motility, nor cell body alignment, was observed on aligned collagen lacking D-periodicity, or on D-periodic collagen in the presence of peptide containing an RGD motif. The directional motility of fibroblast cells on aligned collagen type I fibrils cannot be attributed to contact guidance, but requires additional structural information. This allows us to postulate a physiological function for the 67 nm periodicity.

  • 17. Preiner, Johannes
    et al.
    Janovjak, Harald
    Rankl, Christian
    Knaus, Helene
    Cisneros, David A.
    BioTec, University of Technology Dresden, Dresden, Germany.
    Kedrov, Alexej
    Kienberger, Ferry
    Muller, Daniel J
    Hinterdorfer, Peter
    Free energy of membrane protein unfolding derived from single-molecule force measurements2007In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 93, no 3, p. 930-937Article in journal (Refereed)
    Abstract [en]

    Mechanical single-molecule techniques offer exciting possibilities to investigate protein folding and stability in native environments at submolecular resolution. By applying a free-energy reconstruction procedure developed by Hummer and Szabo, which is based on a statistical theorem introduced by Jarzynski, we determined the unfolding free energy of the membrane proteins bacteriorhodopsin (BR), halorhodopsin, and the sodium-proton antiporter NhaA. The calculated energies ranged from 290.5 kcal/mol for BR to 485.5 kcal/mol for NhaA. For the remarkably stable BR, the equilibrium unfolding free energy was independent of pulling rate and temperature ranging between 18 and 42 degrees C. Our experiments also revealed heterogeneous energetic properties in individual transmembrane helices. In halorhodopsin, the stabilization of a short helical segment yielded a characteristic signature in the energy profile. In NhaA, a pronounced peak was observed at a functionally important site in the protein. Since a large variety of single- and multispan membrane proteins can be tackled in mechanical unfolding experiments, our approach provides a basis for systematically elucidating energetic properties of membrane proteins with the resolution of individual secondary-structure elements.

  • 18. Pérez-Armendariz, E. Martha
    et al.
    Lamoyi, Edmundo
    Mason, J. Ian
    Cisneros-Armas, David
    Departamento de Biología Celular, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, D. F., 04510. .
    Luu-The, Van
    Bravo Moreno, J. Francisco
    Developmental regulation of connexin 43 expression in fetal mouse testicular cells2001In: Anatomical Record, ISSN 0003-276X, E-ISSN 1097-0185, Vol. 264, no 3, p. 237-46Article in journal (Refereed)
    Abstract [en]

    Multiple connexins have been identified in testicular cells. Several lines of evidences indicate that, among them, connexin 43 (Cx43) may be unique for control of gonad development and spermatogenesis. To date, however, it is not known whether Cx43 is expressed in the fetal testis and what possible types of cellular interactions mediated by this connexin are critical to male fertility. In the present work, expression of Cx43 was investigated at various developmental ages in cryosections from mouse testis by using specific antibodies against Cx43. In serial or double‐labeled sections, Cx43 localization was compared with immunocytochemical distribution of steroidogenic enzyme, 3β‐hydroxysteroid dehydrogenase (3βHSD), Mullerian inhibitory hormone (MIH), and germinal nuclear cell antigen (GCNA1), which are specific markers, respectively, of interstitial Leydig, Sertoli, and germinal cells. Sections were analyzed by fluorescence microcopy. We found that Cx43 immunofluorescence (IF) was uniformly distributed in the undifferentiated gonad at 11.5 days post coitus (dpc) and in cells of the mesonephric tubules. In the undifferentiated gonad, Cx43 was localized between primordial germ cells and somatic cells. At 12.5 dpc, when the gonad has undergone sexual differentiation, in the interstitium Cx43 was localized in Leydig cells and in the seminiferous cord it was localized between adjacent Sertoli cells. In Leydig and Sertoli cells, Cx43 labeling increased at 14.5, 16.5, and 18.5 dpc. From day 12.5 up to 18.5 dpc, Cx43 was also localized in cell borders between germinal and Sertoli cells. In conclusion, this study demonstrates that from the earliest stages of gonadal development, Cx43 is expressed in the principal cell types that participate in the control of male fertility. It also shows that Cx43 expression in Leydig and Sertoli cells increase during fetal life. Finally, it provides evidence that, throughout embryonic life, Cx43 forms gap junctions between Sertoli and germinal cells.

  • 19. Shah, D S
    et al.
    Thomas, M B
    Phillips, S
    Cisneros, David A.
    Biotechnology Center, Dresden University of Technology, Tatzberg 49, 01307 Dresden, Germany‡Biotechnology Center, Dresden University of Technology, Tatzberg 49, 01307 Dresden, Germany‡Biotechnology Center, Dresden University of Technology, Tatzberg 49, 01307 Dresden, Germany.
    Le Brun, A P
    Holt, S A
    Lakey, J H
    Self-assembling layers created by membrane proteins on gold2007In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 35, no 3, p. 522-526Article in journal (Refereed)
    Abstract [en]

    Membrane systems are based on several types of organization. First, amphiphilic lipids are able to create monolayer and bilayer structures which may be flat, vesicular or micellar. Into these structures membrane proteins can be inserted which use the membrane to provide signals for lateral and orientational organization. Furthermore, the proteins are the product of highly specific self-assembly otherwise known as folding, which mostly places individual atoms at precise places in three dimensions. These structures all have dimensions in the nanoscale, except for the size of membrane planes which may extend for millimetres in large liposomes or centimetres on planar surfaces such as monolayers at the air/water interface. Membrane systems can be assembled on to surfaces to create supported bilayers and these have uses in biosensors and in electrical measurements using modified ion channels. The supported systems also allow for measurements using spectroscopy, surface plasmon resonance and atomic force microscopy. By combining the roles of lipids and proteins, highly ordered and specific structures can be self-assembled in aqueous solution at the nanoscale.

  • 20. Taubenberger, Anna
    et al.
    Cisneros, David A.
    BioTechnological Center, University of Technology Dresden, 01307 Dresden, Germany.
    Friedrichs, Jens
    Puech, Pierre-Henri
    Muller, Daniel J
    Franz, Clemens M.
    Revealing early steps of alpha2beta1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy2007In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 18, no 5, p. 1634-1644Article in journal (Refereed)
    Abstract [en]

    We have characterized early steps of alpha(2)beta(1) integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of alpha(2)beta(1) as a collagen type I receptor, alpha(2)beta(1)-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas alpha(2)beta(1)-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin-collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin-collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of alpha(2)beta(1)-mediated adhesion as weak initial, single-integrin-mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility.

  • 21. Tedeschi, Antonio
    et al.
    Wutz, Gordana
    Huet, Sébastien
    Jaritz, Markus
    Wuensche, Annelie
    Schirghuber, Erika
    Davidson, Iain Finley
    Tang, Wen
    Cisneros, David A.
    Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria..
    Bhaskara, Venugopal
    Nishiyama, Tomoko
    Vaziri, Alipasha
    Wutz, Anton
    Ellenberg, Jan
    Peters, Jan-Michael
    Wapl is an essential regulator of chromatin structure and chromosome segregation2013In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 501, no 7468, p. 564-568Article in journal (Refereed)
    Abstract [en]

    Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.

  • 22. Tellez, Luis A.
    et al.
    Blancas-Mejia, Luis M.
    Carrillo-Nava, Ernesto
    Mendoza-Hernández, Guillermo
    Cisneros, David A.
    Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apdo. Postal 70-159, 04510 México.
    Fernández-Velasco, D. Alejandro
    Thermal unfolding of triosephosphate isomerase from Entamoeba histolytica: dimer dissociation leads to extensive unfolding2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 44, p. 11665-11673Article in journal (Refereed)
    Abstract [en]

    In mesophiles, triosephosphate isomerase (TIM) is an obligated homodimer. We have previously shown that monomeric folding intermediates are common in the chemical unfolding of TIM, where dissociation provides 75% of the overall conformational stability of the dimer. However, analysis of the crystallographic structure shows that, during unfolding, intermonomeric contacts contribute to only 5% of the overall increase in accessible surface area. In this work several methodologies were used to characterize the thermal dissociation and unfolding of the TIM from Entamoeba histolytica (EhTIM) and a monomeric variant obtained by chemical derivatization (mEhTIM). During EhTIM unfolding, sequential transitions corresponding to dimer dissociation into a compact monomeric intermediate followed by unfolding and further aggregation of the intermediate occurred. In the case of mEhTIM, a single transition, analogous to the second transition of EhTIM, was observed. Calorimetric, spectroscopic, hydrodynamic, and functional evidence shows that dimer dissociation is not restricted to localized interface reorganization. Dissociation represents 55% (DeltaH(Diss) = 146.8 kcal mol(-1)) of the total enthalpy change (DeltaH(Tot) = 266 kcal mol(-1)), indicating that this process is linked to substantial unfolding. We propose that, rather than a rigid body process, subunit assembly is best represented by a fly-casting mechanism. In TIM, catalysis is restricted to the dimer; therefore, the interface can be viewed as the final nucleation motif that directs assembly, folding, and function.

  • 23. Wutz, Gordana
    et al.
    Várnai, Csilla
    Nagasaka, Kota
    Cisneros, David A.
    Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.
    Stocsits, Roman R
    Tang, Wen
    Schoenfelder, Stefan
    Jessberger, Gregor
    Muhar, Matthias
    Hossain, M Julius
    Walther, Nike
    Koch, Birgit
    Kueblbeck, Moritz
    Ellenberg, Jan
    Zuber, Johannes
    Fraser, Peter
    Peters, Jan-Michael
    Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins2017In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 36, no 24, p. 3573-3599Article in journal (Refereed)
    Abstract [en]

    Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.

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