Structural studies on the Radial Spoke proteins complex in Chlamydomonas reinhardtii
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1 Research Collection Doctoral Thesis Structural studies on the Radial Spoke proteins complex in Chlamydomonas reinhardtii Author(s): Poghosyan, Emiliya Publication Date: 2017 Permanent Link: Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library
2 DISS. ETH NO Structural studies on the Radial Spoke proteins complex in Chlamydomonas reinhardtii A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by EMILIYA POGHOSYAN Master of Science, Dresden University of Technology born on citizen of ARMENIA accepted on the recommendation of Prof. Gebhard F. X. Schertler Prof. Takashi Ishikawa Prof. Ritsu Kamiya Prof. Rudolf Glockshuber 2017
3 Summary The radial spokes (RS) are multi-protein complexes composed of at least 23 radial spoke proteins (RSP), decorating the microtubule doublets (MTD) of motile cilia and flagella 9 doublets + 2 singlets with 96nm periodic unit. The proximal stalk region of the complex is linked to the A-tubule of MTDs, inner arm dyneins and the dynein regulatory complex, while the distal head region of spokes is facing to central pair microtubules (CP). At least 12 RSPs have homologues in humans and among those, genes, encoding RSPH1, RSPH4a, RSPH3, RSPH9 and HSP40 (an RSP16 co-chaperone), carry primary ciliary dyskinesia (PCD) causing mutations. In Chlamydomonas, mutants with deficiencies in radial spoke proteins have disturbed motility or possess paralyzed phenotype. A functional role of the radial spokes is expected to be in mechano-chemical regulation of flagellar waveform by signal transduction between CP and inner dynein arms. However, the exact molecular mechanism underlying the mechano-chemical signal transduction between RS/CP and dyneins is not well understood and this is partially due to the missing information on the structural arrangement of RSPs. In this work, I have combined three different approaches to investigate the 3D molecular arrangement of individual radial spoke proteins within the complex. I examined the oligomerization states and interactions between spoke head and neck proteins in vitro. Through optimization of RS isolation and by applying single particle cryo-electron microscopy, I was able to obtain the 3D structure of the RS complex with the unprecedented resolution of 17 Å, which allowed me to conclude about symmetric positioning of the head sub-unit in the complex and which can serve as a template for future model building. With the help of cryo-electron tomography I was able to characterize three different RS mutants, Δ1, NDK5 and pf33, with absence or truncations of RSP3, RSP23 and RSP16, respectively. A comparative analysis with the wild type structure allowed deducing the role of those missing proteins in the structural integrity of the complex. i
4 Zusammenfassung Die radialen Speichen (RS) sind Multiproteinkomplexe, die aus mindestens 23 radialen Speichenproteinen (RSPs) bestehen, welche die Mikrotubuli- Dubletten (MTD) von beweglichen Zilien und Flagellen "9 Dubletten + 2 Einzeltubuli" mit 96nm Periodeneinheit dekorieren. Der proximale Stachelbereich des Komplexes ist mit dem A-Tubulus von MTDs, den inneren Dyneinarmen und dem Dynein-regulatorischen Komplex verbunden, während der distale Kopfbereich der Speichen dem zentralen Mikrotubuli-Paar (CP) gegenüberliegt. Mindestens 12 RSPs haben Homologe beim Menschen und unter diesen bewirken RSPH1-, RSPH4a-, RSPH3-, RSPH9- und HSP40-Co- Chaperon-Gene mutationsbedingte primäre ciliäre Dyskinesie (PCD). In Chlamydomonas haben Mutanten mit Defekten in radialen Speichenproteinen eine gestörte Motilität oder besitzen einen paralysierten Phänotyp. Wir gehen davon aus, dass die funktionelle Rolle der radialen Speichen in der Signaltransduktion zwischen CP und inneren Dyneinarmen liegt, welche die mechanisch-chemische Regulation der Wellenform der Flagellen steuert. Allerdings ist der genaue molekulare Mechanismus, welcher der mechanischchemischen Signaltransduktion zwischen RS/CP und Dyneinen zugrunde liegt, nicht gut verstanden, was teilweise auf das fehlende Wissen über die strukturelle Anordnung der RSPs zurückzuführen ist. In dieser Arbeit habe ich drei verschiedene Ansätze kombiniert, um die 3D- Molekülanordnung einzelner radialer Speichenproteine innerhalb des Komplexes zu charakterisieren. Ich untersuchte die Oligomerisierungszustände und Wechselwirkungen zwischen Speichenkopf und Halsproteinen in vitro. Aufgrund der erfolgreichen Optimierung der RS- Isolation und dank der Anwendung der Single-Particle-Kryo-Elektronen- Mikroskopie konnte ich die 3D-Struktur des RS-Komplexes mit einer noch nie da gewesenen Auflösung von 17 Å identifizieren. Dies erlaubte es mir, offene Fragen in Bezug auf die symmetrische Positionierung der Kopf-Untereinheit dieses Komplexes abschliessend zu beantworten. Die Ergebnisse können auch als Vorlage für zukünftige Modellierungen dienen. ii
5 Mit Hilfe der Kryo-Elektronen-Tomographie konnte ich drei verschiedene RS- Mutanten, Δ1, NDK5 und pf33, mit fehlenden oder trunkierten/verkürzten RSP3, RSP23 bzw. RSP16 Proteinen charakterisieren. Eine vergleichende Analyse mit der Wildtypstruktur erlaubte, es, Schlüsse zu ziehen über die Rolle jener fehlenden Proteine für die strukturelle Integrität des Komplexes. iii
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Research Collection Doctoral Thesis Engineering of synthetic circuits for biomedical applications Author(s): Ye, Haifeng Publication Date: 2012 Permanent Link: https://doi.org/10.3929/ethz-a-007563440
Robust Indoor Positioning through Adaptive Collaborative Labeling of Location Fingerprints
Research Collection Doctoral Thesis Robust Indoor Positioning through Adaptive Collaborative Labeling of Location Fingerprints Author(s): Bolliger, Philipp L. Publication Date: 2011 Permanent Link: https://doi.org/10.3929/ethz-a-007052434
Metabolic engineering of Vitamin B1 and Vitamin B6 in staple crops
Research Collection Doctoral Thesis Metabolic engineering of Vitamin B1 and Vitamin B6 in staple crops Author(s): Li, Kuan-Te Publication Date: 2014 Permanent Link: https://doi.org/10.3929/ethz-a-010192503
Polarization effects of the spectral thermal emission from ferromagnets
Research Collection Doctoral Thesis Polarization effects of the spectral thermal emission from ferromagnets Author(s): Brönnimann, Rolf Publication Date: 1986 Permanent Link: https://doi.org/10.3929/ethz-a-000361763
Research Collection. Individual and social determinants of healthy eating: the role of people's nutrition knowledge and their regular eating networks
Research Collection Doctoral Thesis Individual and social determinants of healthy eating: the role of people's nutrition knowledge and their regular eating networks Author(s): Mötteli, Sonja Publication
Optimization of levodopa therapy in in Parkinson's disease
Research Collection Doctoral Thesis Optimization of levodopa therapy in in Parkinson's disease Author(s): Hacisalihzade, Selim Sedat Publication Date: 1986 Permanent Link: https://doi.org/10.3929/ethz-a-000409859
Research Collection. Exploration of augmented reality technology for surgical training simulators. Doctoral Thesis. ETH Library
Research Collection Doctoral Thesis Exploration of augmented reality technology for surgical training simulators Author(s): Bianchi, Gérald Publication Date: 2007 Permanent Link: https://doi.org/10.3929/ethz-a-005307590
Improving inter-organizational knowledge transfer to gain development project success
Research Collection Doctoral Thesis Improving inter-organizational knowledge transfer to gain development project success Author(s): Brojerdi-Heyn, Gundula Janin Christina Publication Date: 2011 Permanent
Research Collection. Loga-Rechenwalzen Modellverzeichnis und Preisliste. Report. ETH Library. Author(s): Bruderer, Herbert. Publication Date: 2014
Research Collection Report Loga-Rechenwalzen Modellverzeichnis und Preisliste Author(s): Bruderer, Herbert Publication Date: 2014 Permanent Link: https://doi.org/10.3929/ethz-a-010095617 Rights / License:
Role of Foxa2 Acetylation in Diabetes and Obesity in Vivo
Research Collection Doctoral Thesis Role of Foxa2 Acetylation in Diabetes and Obesity in Vivo Author(s): Kondratiuk, Nadiia Publication Date: 2015 Permanent Link: https://doi.org/10.3929/ethz-a-010464159
Structural Studies of Crosslinked Nucleosome Core Particles
Research Collection Doctoral Thesis Structural Studies of Crosslinked Nucleosome Core Particles Author(s): Barth, Philip Publication Date: 2016 Permanent Link: https://doi.org/10.3929/ethz-a-010807741
Research Collection. To sense or not to sense viral RNA - essentials of coronavirus innate immune evasion. Doctoral Thesis.
Research Collection Doctoral Thesis To sense or not to sense viral RNA - essentials of coronavirus innate immune evasion Author(s): Kindler, Eveline Patricia Publication Date: 2015 Permanent Link: https://doi.org/10.3929/ethz-a-010560814
A communication Architecture for Telepresence in Virtual Environments
Research Collection Doctoral Thesis A communication Architecture for Telepresence in Virtual Environments Author(s): Lamboray, Edouard C. Publication Date: 2004 Permanent Link: https://doi.org/10.3929/ethz-a-004845157
Geometric Change Detection and Image Registration in Large Scale Urban Environments
Research Collection Doctoral Thesis Geometric Change Detection and Image Registration in Large Scale Urban Environments Author(s): Taneja, Aparna Publication Date: 2014 Permanent Link: https://doi.org/10.3929/ethz-a-010394826
Research Collection. Live 3D Reconstruction on Mobile Phones. Doctoral Thesis. ETH Library. Author(s): Tanskanen, Petri. Publication Date: 2016
Research Collection Doctoral Thesis Live 3D Reconstruction on Mobile Phones Author(s): Tanskanen, Petri Publication Date: 2016 Permanent Link: https://doi.org/10.3929/ethz-a-010636796 Rights / License: