id,collection,dc.contributor.author,dc.contributor.contact,dc.contributor.firstReferee,dc.contributor.furtherReferee,dc.contributor.gender,dc.date.accepted,dc.date.accessioned,dc.date.available,dc.date.issued,dc.description,dc.description.abstract[de],dc.description.abstract[en],dc.format.extent,dc.identifier.uri,dc.identifier.urn,dc.language,dc.rights.uri,dc.subject,dc.subject.ddc,dc.title,dc.title.translated[de],dc.type,dcterms.accessRights.dnb,dcterms.accessRights.openaire,dcterms.format[de],refubium.affiliation[de],refubium.mycore.derivateId,refubium.mycore.fudocsId "5d07be13-52b9-4a4f-8925-afe16820120b","fub188/14","Fröhlich, Chris","chris.froehlich@me.com","Prof. Dr. Udo Heinemann","Prof. Dr. Oliver Daumke","m","2013-07-29","2018-06-07T15:45:07Z","2013-11-07T14:59:47.136Z","2013","Contents ................................................................................................................................................... v List of Figures ........................................................................................................................................... ix List of Tables ............................................................................................................................................ xi 1\. Introduction ..................................................................................................................................... 1 1.1. Mitochondria ........................................................................................................................... 1 1.1.1. The mitochondrial compartment .................................................................................... 2 1.1.2. The mitochondrial network ............................................................................................. 5 1.1.3. Mitochondria associated diseases .................................................................................. 6 1.2. G proteins ................................................................................................................................ 8 1.3. The dynamin superfamily of G proteins ................................................................................ 10 1.3.1. Dynamin ........................................................................................................................ 12 1.3.1.1. Dynamins are key players in clathrin-mediated endocytosis ................................ 12 1.3.1.2. The G domain and the bundle signaling element ................................................. 14 1.3.1.3. G domain dimerization is crucial for dynamin function ........................................ 15 1.3.1.4. Dynamin's PH domain mediates lipid binding ....................................................... 17 1.3.1.5. The stalk is the central assembly hub for dynamin oligomerization ..................... 17 1.3.1.6. Regulatory functions of the stalk .......................................................................... 20 1.3.1.7. Functional models of dynamin's mechano-chemical action ................................. 20 1.3.2. Myxovirus resistance (Mx) proteins .............................................................................. 22 1.3.2.1. Mx proteins mediate antiviral host response ....................................................... 22 1.3.2.2. Different functions - similar structures: MxA and dynamin 1 ............................... 23 1.3.2.3. The MxA stalk mediates oligomerization and regulatory function ....................... 24 1.3.2.4. The MxA stalk mediates assembly in rings rather than helices ............................ 25 1.3.3. Bacterial dynamin-like proteins (BDLPs) ....................................................................... 26 1.3.4. Guanylate-binding proteins (GBPs) ............................................................................... 28 1.3.5. Eps15 homology-domain containing proteins (EHDs) ................................................... 29 1.3.6. Mitochondrial fusion dynamins..................................................................................... 31 1.3.6.1. Mitochondrial outer membrane fusion dynamins ................................................ 31 1.3.6.2. Mitochondrial inner membrane fusion dynamins ................................................ 32 1.3.7. The mitochondrial fission dynamin 1-like protein (DNM1L) ......................................... 32 1.3.7.1. DNM1L is a key player in mitochondrial fission .................................................... 32 1.3.7.2. Recruitment of DNM1L to mitochondria scission sites involves certain adaptor proteins and the endoplasmic reticulum (ER) ........................................................................... 33 1.3.7.3. Two-start versus one-start helix ............................................................................ 34 1.4. Scope of this work ................................................................................................................. 36 2\. Materials and Methods ................................................................................................................. 38 2.1. Materials ................................................................................................................................ 38 2.1.1. Chemicals ....................................................................................................................... 38 2.1.2. Antibodies...................................................................................................................... 38 2.1.3. Enzymes ......................................................................................................................... 38 2.1.4. Kits ................................................................................................................................. 39 2.1.5. Microorganisms and cell lines ....................................................................................... 39 2.1.6. Vectors ........................................................................................................................... 40 2.1.7. cDNA clone .................................................................................................................... 40 2.1.8. Primers .......................................................................................................................... 40 2.1.8.1. Cloning Primers ..................................................................................................... 40 2.1.8.2. Quick change mutagenesis primers ...................................................................... 40 2.1.9. Media and antibiotics .................................................................................................... 42 2.1.10. Buffers ........................................................................................................................... 42 2.2. Molecular biology methods................................................................................................... 43 2.2.1. Polymerase chain reaction ............................................................................................ 43 2.2.2. DNA digestion ................................................................................................................ 43 2.2.3. Agarose gel electrophoresis .......................................................................................... 43 2.2.4. DNA purification ............................................................................................................ 43 2.2.5. Ligation .......................................................................................................................... 44 2.2.6. Preparation of chemically competent E. coli ................................................................. 44 2.2.7. Transformation of chemically competent E. coli ........................................................... 44 2.2.8. Isolation of plasmid DNA ............................................................................................... 44 2.2.9. DNA sequencing ............................................................................................................ 44 2.2.10. Site specific mutagenesis............................................................................................... 44 2.2.11. Sequence alignments .................................................................................................... 45 2.2.12. Bacterial storage ............................................................................................................ 45 2.2.13. Construct design ............................................................................................................ 45 2.3. Biochemical methods ............................................................................................................ 45 2.3.1. SDS PAGE ....................................................................................................................... 45 2.3.2. Protein over-expression test in E. coli ........................................................................... 45 2.3.3. Protein solubility test .................................................................................................... 46 2.3.4. Large scale protein over-expression in E. coli ............................................................... 46 2.3.5. Protein purification / AC and SEC .................................................................................. 46 2.3.6. Protein concentration ................................................................................................... 47 2.3.7. Determination of protein concentration ....................................................................... 47 2.3.8. Protein storage .............................................................................................................. 47 2.3.9. Western Blot .................................................................................................................. 47 2.3.10. Nucleotide detection using reversed-phase HPLC ........................................................ 47 2.3.11. Isothermal titration calorimetry (ITC) ........................................................................... 48 2.3.12. Nucleotide hydrolysis assays ......................................................................................... 48 2.3.13. Analytical ultracentrifugation ........................................................................................ 49 2.3.14. Analytical gelfiltration and right angle light scattering (RALS) ...................................... 49 2.3.15. Oligomerization and liposome co-sedimentation assays .............................................. 50 2.3.16. Floatation assays ........................................................................................................... 50 2.3.17. Electron microscopy ...................................................................................................... 50 2.4. Crystallographic and computational methods ...................................................................... 51 2.4.1. Protein crystallization .................................................................................................... 51 2.4.2. Cryo-protection of crystals ............................................................................................ 51 2.4.3. Data collection ............................................................................................................... 51 2.4.4. Protein structure solution ............................................................................................. 52 2.4.5. Atomic model building and refinement ........................................................................ 55 2.4.6. Structure analysis and figure preparation ..................................................................... 55 2.4.7. Protein structure validation and deposition ................................................................. 55 2.4.8. Electron microscopy model fit....................................................................................... 55 2.5. Cell biological methods ......................................................................................................... 56 2.5.1. Cell culture and transfection ......................................................................................... 56 2.5.2. Live cell microscopy ....................................................................................................... 56 2.5.3. Mitochondrial connectivity FRAP assay ........................................................................ 56 3\. Results ........................................................................................................................................... 57 3.1. Protein production and biochemistry ................................................................................... 57 3.1.1. Protein over-expression ................................................................................................ 57 3.1.2. Protein solubility and purification ................................................................................. 58 3.1.3. Nucleotide binding and affinity ..................................................................................... 59 3.2. Structural analysis of DNM1L ................................................................................................ 61 3.2.1. Crystallization and structure determination ................................................................. 61 3.2.2. Structure of DNM1L....................................................................................................... 65 3.2.3. The stalk interfaces ....................................................................................................... 68 3.2.4. Localization of the B insert and the GPRP motif ........................................................... 72 3.3. Structure- based mutational analysis ..................................................................................... 73 3.3.1. Stalk interface 2 mediates dimerization ........................................................................ 73 3.3.2. Functional importance of the B insert and the GPRP motif .......................................... 83 3.3.3. Stalk interface 4 is important for liposome tubulation in vitro and mitochondrial remodeling in vivo ......................................................................................................................... 88 3.4. A helical model for DNM1L assembly.................................................................................... 92 4\. Discussion ...................................................................................................................................... 94 4.1. Similarities and differences in the assembly of dynamin superfamily proteins ................... 94 4.2. Mechano-chemical coupling in DNM1L ................................................................................ 97 4.3. DNM1L oligomers might be adapted to the size of mitochondria ........................................ 99 4.4. The molecular basis for DNM1L caused diseases................................................................ 101 4.5. Open questions and outlook ............................................................................................... 105 Appendix A - Instruments .................................................................................................................... 107 Appendix B - Chemicals ....................................................................................................................... 108 Appendix C - Clone list ......................................................................................................................... 110 Appendix D - Alignment ...................................................................................................................... 111 Appendix E - Abbreviation ................................................................................................................... 114 Bibliography ......................................................................................................................................... 116 Abstract ............................................................................................................................................... 125 Zusammenfassung ............................................................................................................................... 127 Publications ......................................................................................................................................... 129 Acknowledgement............................................................................................................................... 130 Erklärung ............................................................................................................................................. 131","Mitochondria form a dynamic cytoskeleton-associated tubulovesicular network throughout the whole cell which continuously undergoes fusion- and division processes; collectively termed mitochondrial dynamics. Mitochondrial dynamics is an important cellular tool for proper organelle transport to places of high energy demand inside the cell. Furthermore, mitochondrial dynamics functions as quality control mechanism. During lifetime the mitochondrial genome gets more and more heteroplasmic and mitochondrial fusion is an important process to keep the mtDNA content of a cell homogenous. Thus, fusion enables mitochondria with mutations in different genes to cross-complement each other. In contrast, fission can separate mitochondria from the network which are damaged by, for example, reactive oxygen species for mitophagy and recycling. Since mitochondria cannot be created de novo, fission is also required to equally distribute mitochondria during cytogenesis. Several studies showed that disturbed mitochondrial dynamics is implicated in several severe neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease or Huntington's disease. The key player in the process of mitochondrial division is the large G protein dynamin 1-like protein (DNM1L). In this work DNM1L was structurally and functionally characterized to better understand the process of mitochondrial division and to explain the causes of DNM1L associated diseases on a molecular level. Despite limited sequence identity, the domain architecture of DNM1L is similar to that observed for dynamin and MxA. DNM1L is composed of a dynamin-specific N-terminal G domain and an elongated four-helical stalk. Both domains are connected via a three-helical bundle; the bundle signaling element (BSE). The stalk is intersected by another domain, termed B insert which is located at equivalent positions like the lipid binding PH domain in dynamin or the substrate binding loop in MxA. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodeling. Based on these results and electron microscopy reconstructions, we propose an oligomerization model for DNM1L which differs from that of dynamin and might be adapted to the remodeling of mitochondria.","Mitochondrien formen ein tubolovesikuläres Netzwerk, welches die gesamte Zelle durchspannt und eng an das Zytoskelett assoziiert ist. Außerdem ist das mitochondriale Netzwerk sehr dynamisch und kontinuierlichen Teilungs- und Fusionsprozessen unterworfen. Dies ermöglicht der Zelle den gezielten Transport von einzelnen Teilen des mitochondrialen Netzwerks zu Orten hohen Energie- (ATP)-verbrauchs innerhalb der Zelle. Außerdem funktionieren Fusions- und Teilungsprozesse als Qualitätssicherungsmaßnahme. Mitochondriale Fusion er-möglicht es der Zelle zum Beispiel, dass mit fortschreitendem Alter zunehmend heteroplasmische mitochondriale Genom homogen zu halten. Mitochondrien mit verschiedenen Mutationen innerhalb ihres Genoms können gezielt verschmelzen und damit ihr Genom gegenseitig komplementieren. Im Gegensatz dazu ermöglicht mitochondriale Teilung Mitochondrien, die z.B. durch reaktive Sauerstoffspezies stark beschädigt worden sind, vom Netzwerk abzu- koppeln und während des mitophagialen Stoffwechselweges gänzlich abzubauen bzw. deren molekulare Bausteine wieder aufzubereiten. Da Mitochondrien zellulär nicht de novo synthetisiert werden können, benötigt die Zelle außerdem einen Mechanismus, um während der Zellteilung das mitochondriale Netzwerk gleichmäßig auf beide Tochterzellen aufzuteilen. Fehlfunktionen, die zu gestörten mitochondrialen Fusions- oder Teilungsprozessen führen, wurden in mehreren unabhängigen Studien in Zusammenhang mit ernsthaften neurodegenerativen Erkrankungen wie Alzheimer, Parkinson oder Huntington gebracht. Der Hauptakteur mitochondrialer Teilung ist das große G protein ""Dynamin 1-like Protein"" (DNM1L). In dieser Arbeit wurde DNM1L strukturell und biochemisch charakterisiert mit dem Ziel, den molekularen Mechanismus besser zu verstehen, der hinter dem Prozess der mitochondrialen Teilung steckt und um mögliche Ursachen der oben genannten Krankheiten auf molekularer Ebene betrachten zu können. Obwohl die Sequenzhomologie nur sehr gering ist, zeigt die Struktur von DNM1L große Ähnlichkeit mit den Strukturen klassischen Dynamins und Myxovirusresistenz-proteins A (Mx A). DNM1L besteht aus der für Dynaminproteine typischen N-terminalen G domäne und einer elongierten, stielartigen Domäne (engl. stalk), welche aus vier α-Helices besteht. Beide Domänen sind durch ein Dreihelixbündel 128 miteinander verbunden (Bündelsignalelement; engl. bundle signaling element: BSE). Die stielartige Domäne wird durch eine weitere Domäne unterbrochen, welche B Domäne (engl. B insert) genannt wird und an äquivalenter Position zu den Substratbindedomänen von Dynamin und MxA lokalisiert ist. In dieser Arbeit konnte gezeigt werden, dass DNM1L analog zu Dynamin und MxA über eine konservierte Interaktionsschnittstelle in der stielartigen Domäne dimerisiert. Mutationsanalysen zeigten, dass die Dimerisierung über diese Schnittstelle von zentraler Bedeutung für die Membranbindung und mitochondriale Remodelierung ist. Außerdem deuten die Ergebnisse dieser Arbeit darauf hin, dass die B Domäne die Bindung von DNM1L an die mitochondriale Außenmembran vermittelt. Außerdem ist ein weiterer ungeordneter Bereich am distalen Ende der stielartigen Domäne wichtig für die geordnete Oligomerisierung von DNM1L und für dessen Membranbindung. Im Kristall lagerten sich die DNM1L Dimere über eine weitere Schnittstelle in der stielartigen Domäne zusammen, welche bisher für kein Protein der Dynaminsuper-familie beschrieben worden ist. Mutationen, die diese Schnittstelle auflösen, führten zu einer gestörten Tubulierung von Liposomen in vitro und zum Ausbleiben der Lokalisierung von DNM1L an die mitochondraile Außenmebran in vivo. Damit verbunden war ein fehlerhafter mitochondrialer Teilungsprozess, der zu vergrößer-ten, teilweise mehrere μm langen Mitochondrien führte. Basierend auf den Ergebnissen dieser Arbeit und cryoelektronenmikroskopischen Studien, schlagen wir ein Oligomerisierungsmodell vor, welches sich von Dynamin und MxA unterscheidet und möglicherweise an die besonderen Anforderungen für den Teilungsprozess der mitochondrialen Doppelmembran und die Größe von Mito-chondrien angepasst ist.","XI, 131 S.","https://refubium.fu-berlin.de/handle/fub188/1546||http://dx.doi.org/10.17169/refubium-5748","urn:nbn:de:kobv:188-fudissthesis000000095444-6","eng","http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen","DRP1||DNM1L||DLP1||dynamin||dynamin 1-like||mitochondrial remodeling||stalk||two-start helix||mitochondrial fission","500 Naturwissenschaften und Mathematik::540 Chemie","Structural insights into oligomerization and mitochondrial remodeling of dynamin 1-like protein","Einblicke in die Struktur, Oligomerisierung und mitochondrialer Remodellierung von Dynamin 1-like protein","Dissertation","free","open access","Text","Biologie, Chemie, Pharmazie","FUDISS_derivate_000000014322","FUDISS_thesis_000000095444"