Crosslinking studies on the conventional kinesin of Neurospora crassa [Elektronische Ressource] / vorgelegt von Katrin Hahlen

Crosslinking studies on the conventional
kinesin of Neurospora crassa










Dissertation
der Fakultät für Biologie der
Ludwig-Maximilians Universität
München








vorgelegt von
Katrin Hahlen
aus Köln
2004




Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig und ohne unerlaubte Hilfsmittel angefertigt.




Katrin Hahlen


















Erstgutachter: PD Dr. Günther Woehlke
Zweitgutachter: Prof. Dr. Charles N. David
eingereicht: 30.4.2004
Tag der mündlichen Prüfung: 22.7.2004

Meeting abstracts:

Hahlen K, Woehlke G and Schliwa M (2002)
"Testing kinesin’s flexibility by crosslinking: Which domains need to be mobile to allow
mobility?"
2nd Munich Symposium on Cell Dynamics

Hahlen K, Woehlke G and Schliwa M (2003)
"Testing kinesin’s flexibility by crosslinking: Which domains need to be mobile to allow
mobility?"
47th Annual Meeting of the Biophysical Society

Hahlen K, Schäfer F, Schliwa M and Woehlke G (2003)
"The role of the neck in fast kinesins"
Jahrestagung der Gesellschaften für Zellbiologie und Entwicklungsbiologie

Hahlen K, Schliwa M and Woehlke G (2003)
"Crosslinking kinesin’s neck-linker to the motor core"
Workshop Molecular Motors and New Microscopic Techniques










The work presented here was carried out in the laboratory of Prof. Dr. Manfred Schliwa
(Adolf-Butenandt-Institut of the Ludwig-Maximilians Universität, München) from January
2000 to December 2003. The work was supported by the Deutsche Forschungsgemeinschaft.

Contents I
Contents

Abbreviations........................................................................................................................... V
1 Introduction..................................................................................................................... 1
1.1 Molecular Motors.........................................................................................................1
1.1.1 Kinesin Superfamily ...................................................................................................... 2
1.1.2 Similarities between Kinesins, Myosins and G-Proteins............................................... 3
1.2 Conventional Kinesins.................................................................................................4
1.2.1 Functional Anatomy 4
1.2.2 Model of Motion............................................................................................................ 6
1.3 Neurospora crassa Kinesins ........................................................................................ 7
1.4 Goals of the Present Work ........................................................................................... 8
2 Materials and Methods................................................................................................. 11
2.1 Materials..................................................................................................................... 11
2.1.1 Reagents and other Materials....................................................................................... 11
2.1.2 Vectors.........................................................................................................................11
2.1.3 Bacterial Strains........................................................................................................... 11
2.1.4 Media and Cultivation of E. coli.................................................................................. 11
2.2 Molecular Biology Methods ...................................................................................... 12
2.2.1 Agarose Gel Electrophoresis 12
2.2.2 DNA Extraction from Agarose Gels............................................................................ 12
2.2.3 Determination of DNA Concentration......................................................................... 12
2.2.4 Preparation of Plasmid DNA....................................................................................... 12
2.2.5 DNA cleavage with Restriction Endonucleases .......................................................... 12
2.2.6 Ligation of DNA into Plasmid Vectors ....................................................................... 13
2.2.7 Preparation and Transformation of Competent Cells .................................................. 13
2.2.7.1 Preparation of Electrocompetent Cells................................................................................ 13
2.2.7.2 Electroporation.................................................................................................................... 13
2.2.7.3 Preparation of SEM Competent Cells ................................................................................. 13
2.2.7.4 Heat Shock Transformation................................................................................................. 14
2.2.8 Identification of Transformed Clones in E. coli .......................................................... 14
2.2.9 Polymerase Chain Reaction (PCR).............................................................................. 14
2.2.10 Introduction of Point Mutations................................................................................... 15
2.2.11 Oligonucleotides.......................................................................................................... 15
2.2.11.1 Primers for the Removal or Insertion of Restriction Sites .............................................. 15
2.2.11.2 Prim Removal of Cysteine residues 38, 59 and 307 ....................................... 15 Contents II
2.2.11.3 Primers for the Insertion of Cysteine residues ................................................................ 16
2.2.11.4 Prim Construction of Monomeric Constructs ................................................. 17
2.2.11.5 Primer for the Monomeric Human Kinesin Construct .................................................... 17
2.2.11.6 Sequencing Primers ........................................................................................................ 17
2.2.12 Generation of Constructs ............................................................................................. 17
2.2.13 Summary of all Measured Constructs.......................................................................... 21
2.3 Biochemical Methods................................................................................................22
2.3.1 SDS-Polyacrylamide Gel Electrophoresis (PAGE)..................................................... 22
2.3.2 Coomassie Staining ..................................................................................................... 23
2.3.3 Colloidal Coomassie Staining...................................................................................... 23
2.3.4 Expression of Kinesin Constructs................................................................................ 23
2.3.5 Protein Purification...................................................................................................... 23
2.3.5.1 Purification of Bacterially Expressed NcKin ...................................................................... 23
2.3.5.2 PuriBacteriaed HsKin....................................................................... 24
2.3.5.3 Purification of Tubulin........................................................................................................ 25
2.3.6 Determination of Protein Concentration 27
2.3.7 Polymerisation of Microtubules .................................................................................. 27
2.3.8 Determination of Microtubule Concentration.............................................................. 27
2.3.9 Microscopy .................................................................................................................. 28
2.3.9.1 Video Enhanced Light Microscopy..................................................................................... 28
2.3.9.2 Motility Assay..................................................................................................................... 28
2.3.10 ATPase Assays ............................................................................................................ 29
2.3.10.1 Basal ATPase Assay ....................................................................................................... 29
2.3.10.2 Microtubule-activated ATPase Assay............................................................................. 30
2.3.10.3 Calculations for the ATPase Assay................................................................................. 31
2.3.11 mantADP Experiments ................................................................................................ 32
2.3.11.1 Loading of Kinesin Motor Domains with mantADP ...................................................... 32
2.3.11.2 Stoichiometry of Kinesin mantADP Comp