The 3D architecture of interphase microtubule cytoskeleton and functions of microtubule plus end tracking proteins in fission yeast [Elektronische Ressource] / presented by Johanna Höög

Johanna Hoog

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences









presented by

thBSc Cell and Molecular Biology Johanna Höög, 17 of July 2003, Oxford Brookes
University

born in: Trollhättan, Sweden

Oral-examination:: ................................................
2


The 3D Architecture of Interphase Microtubule
Cytoskeleton and Functions of Microtubule
Plus End Tracking Proteins in Fission Yeast



















Referees: Dr. Damian Brunner
PD Dr. Harald Herrmann-Lerdon
3

THE 3D ARCHITECTURE OF INTERPHASE MICROTUBULE CYTOSKELETON AND
FUNCTIONS OF MICROTUBULE PLUS END TRACKING PROTEINS IN FISSION YEAST ......3
CHAPTER 1 ..................................................................................................................................................9
INTRODUCTION........................................................................................................................................... 9
Morphogenesis and Cell Polarity in Eukaryotes ........................................................ 11
1. Cellular Polarization and Cell Shape ............................................................................................................. 11
2. General Principles of Polarization.................................................................................................................. 11
The Cytoskeleton................................................................................................................................. 14
1. Actin................................................................................................................................................................ 15
2. Intermediate Filaments ................................................................................................................................... 16
3. Microtubules ................................................................................................................................................... 17
A. The Structure and Polarity of Eukaryotic Tubulin and MTs................................................................... 17
B. The MT Dynamic Instability Model ........................................................................................................ 18
C. MT Nucleation by Centrosomes and other MTOCs................................................................................ 19
D. Structure of Centrosomes and SPBs ........................................................................................................ 20
E. Local Regulation of MT Dynamics in the Cell........................................................................................ 22
Polarity and Morphogenesis in the Fission yeast ............................................................................... 23
1. The Fission Yeast Growth Cycle.................................................................................................................... 23
2. The Growth Zones .......................................................................................................................................... 24
3. The Establishment of Growth Zones.............................................................................................................. 25
The Fission Yeast Microtubule Cytoskeleton...................................................................................... 26
1. The Interphase MT Cytoskeleton is involved in Cellular Morphogenesis.................................................... 26
2. Interphase MTs Extend From the Nucleus Toward Both Cell Ends ............................................................. 27
3. Fission Yeast MT Organizing Centers........................................................................................................... 28
4. MT Organization During Closed Mitosis ...................................................................................................... 29
MT’s Role in Organelle Positioning ................................................................................................... 30
1. Positioning of the Nucleus and the Site of Cytokinesis by MTs ................................................................... 30
2. Golgi and Mitochondria Morphology is MT Dependent............................................................................... 30
3. Most Intracellular Vesicle Trafficking is Performed by Actin...................................................................... 31
MT-Associated Proteins in the Fission Yeast...................................................................................... 33
1. MT Plus-End Tracking MAPs Regulate MT Dynamics................................................................................ 33
A. The EB1 Protein Family and Mal3 .......................................................................................................... 34
B. The Clip-170 Family and Tip1................................................................................................................. 35
C. +TIPs as Centrosomal Proteins ................................................................................................................ 36
2. Molecular Motors ........................................................................................................................................... 36
3. MT Bundlers................................................................................................................................................... 37
Tomography........................................................................................................................................ 39
1. 3D Reconstruction of Cells Using Electron Tomography ............................................................................. 39
2. MT End Structure can show MT Polarity and Dynamics.............................................................................. 40
CHAPTER 2 ................................................................................................................................................43
MATERIALS AND METHODS ...................................................................................................................... 43
Summary .................................................................................. 43
Methods....................................................................................................................... 44
Sample preparation.................................................................. 44
1. Cell culture...................................................................................................................................................... 44
2. High Pressure Freezing (HPF) ....................................................................................................................... 44
3. Freeze substitution.......................................................................................................................................... 45
4. Choice of FS resin .......................................................................................................................................... 46
5. Serial sectioning.............................................................................................................................................. 47
6. Contrasting and application of fiducial markers for tilt alignment................................................................ 48
Tomogram acquisition ........................................................................................................................ 48
1. Large scale adaptations................................................................................................................................... 48
2. Image Acquisition........................................................................................................................................... 51
Tomogram calculation and 3D model reconstruction ........................................................................ 52
1. eTomo and tomogram calculation.................................................................................................................. 52
2. Joining of serial tomograms ........................................................................................................................... 53
4
3. Taking a snapshot from the tomogram........................................................................................................... 54
4. Construction of a 3D model ........................................................................................................................... 56
A. Tracking MTs and other filaments using 3dmod..................................................................................... 56
B. Modeling nucleus, PM and other membrane-bound organelles in 3dmod............................................. 59
5. Analysis of 3D data ................................................................................................................