Measuring mitotic spindle dynamics in budding yeast Kemp Plumb B.Sc. Department of Physics McGill University Montreal,Qu´ ebec´ July, 2009 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science c Kemp Plumb, 2009 Library and Archives Bibliothèque et Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l’édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-66191-8 Our file Notre référence ISBN: 978-0-494-66191-8 NOTICE: AVIS: The author has granted a non- L’auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l’Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distribute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor, Dr. Maria Kilfoil, whose patience and guidance were invaluable at every step of this thesis project. I am grateful to Dr. Kilfoil for introducing me to the field of Biophysics and for giving me the opportunity to work on this exciting project. She has been a continuous source of support and guidance throughout my time here at McGill. This work would not have been possible without the help and support of Dr. Jackie Vogel, in the department of Biology. I would like to thank Dr. Vogel for sharing her biological expertise and for allowing me to work in her lab. I acknowledge the work Dr. Vogel has done preparing budding yeast strains for fluorescence microscopy and performing genetic analysis of those yeast strains. Dr. Vogel has helped me to appreciate and better understand the field of cell biology, an area of science that I had not been exposed to prior to my graduate work. I am extremely grateful to Dr. Vincent Pelletier and would like to acknowledge his work writing the majority of the feature finding and volume reconstruction code. I would also like to thank Dr. Pelletier for introducing me to the optical equipment in the Kilfoil lab and for continually providing helpful advice whenever a problem arose. I extend my gratitude to past and present members of the Kilfoil group: Dr. Yongx- iang Gao, Elvis Prandzic, Stefan Nicolau, and Dr. Stephanie Deboeuf for providing an intellectually stimulating and pleasant working atmosphere. In particular, I would like to thank Stefan Nicolau for implementing extensive error checking in the analysis ii code, which made my life much easier. I am additionally grateful to Dr. Deboeuf for translating the abstract to French. I would also like to thank Dr. Susi Kaitna and Elena Nazarova for their help with biological aspects of this thesis. Dr. Kaitna has put much work into developing motor- mutant budding yeast strains. Both Dr. Kaitna and Elena were always available to help when I had trouble culturing and preparing yeast cells for microscopy, and to elucidate any aspects of the biology that were unclear to me. Finally, thank you to Courtney Ross for editing this thesis, for her endless support, and for just generally putting up with me throughout the writing of this thesis. I could not have done this without her. iii ABSTRACT In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells. iv ABREG´ E´ La regulation´ du cycle cellulaire et la proliferation´ de gen´ erations´ viables requierent` a` la fois la reproductibilite´ et la coordination des differents´ processus complexes en jeu dans un environnement toutefois domine´ par l’agitation thermique. Un exemple essentiel est l’assemblage et la migration du fuseau mitotique qui doivent avoir lieu correctement avant mme la segr´ egation´ des chromosomes. Le fuseau mitotique est une structure transitoire composee´ de deux polesˆ separ´ es´ par des filaments de proteines´ appeles´ les microtubules, auxquelles est rattache´ le materiel´ gen´ etique´ de la cellule. Le fuseau mitotique est notamment implique´ dans l’alignement des chromosomes, puis leur segr´ egation´ vers des polesˆ opposes´ de la cellule ; d’ou` la necessit´ e´ d’un positionnement spatial precis´ et temporellement coordonne´ du fuseau mitotique pour assurer la division correcte du materiel´ gen´ etique´ entre les cellules mere` et fille. Dans ce memoire,´ je decrirai´ les techniques de microscopie confocale par fluores- cence ainsi que les algorithmes automatises´ d’analyse d’image, que j’ai mis en oeuvre pour observer et analyser la dynamique spatiale en temps reel´ du fuseau mitotique chez la levure bourgeonnante. Les programmes developp´ es´ permettent de localiser dans l’espace tridimensionnel des structures subcellulaires et de detecter´ leurs deplacements´ au cours du temps. Le marquage par proteines´ fluorescentes des polesˆ du fuseau mitotique et de la membrane cellulaire a permis de quantifier la dynamique du fuseau mitotique dans un systeme` de coordonnees´ pertinent pour la division cellulaire. Je decrirai´ des simulations numeriques´ de signaux fluorescents de ces structures subcellulaires qui m’ont permis de caracteriser´ la fiabilite´ et de quantifier la precision´ v des programmes d’analyse. Je terminerai ce memoire´ par la description d’un dispositif microfluidique permettant a` la fois la culture de cellules et la caracterisation´ de leur dynamique a` l’echelle´ individuelle, et ce sur de longues echelles´ de temps. Les outils developp´ es´ au cours de cette these` et present´ es´ dans ce memoire´ offrent la possibilite´ d’analyses quantitatives nouvelles des processus hors equilibre´ en jeu chez les cellules vivantes en gen´ eral.´ vi TABLE OF CONTENTS ACKNOWLEDGEMENTS . ii ABSTRACT . iv ABREG´ E.......................................´ v LIST OF TABLES . ix LIST OF FIGURES . x 1 The cell cycle and the mitotic spindle . 1 1.1 The cell cycle control system . 1 1.2 Mitosis . 3 1.2.1 Budding yeast: a model system for study of mitosis . 4 1.3 Structure of the mitotic spindle in budding yeast . 5 1.3.1 Microtubules . 6 1.3.2 Spindle poles . 8 1.3.3 Chromatin . 8 1.3.4 Kinetochores . 9 1.3.5 Mitotic motors . 10 1.4 Spindle assembly and positioning . 12 1.4.1 Measurements of spindle dynamics . 13 1.4.2 Modelling spindle assembly . 16 2 Microscopy . 19 2.1 Overview of optical microscopy . 19 2.1.1 Fluorescence microscopy . 20 2.1.2 The confocal microscope . 24 2.2 Optical resolution limits for an objective lens . 28 2.3 Image formation in a confocal microscope . 31 2.3.1 Fluorescence microscope as a linear translation invariant system 31 2.3.2 The point spread function in a confocal fluorescence microscope 34 vii 2.3.3 CCD array for image detection . 37 2.3.4 Noise sources . 38 3 Feature finding and tracking . 41 3.1 Overview of feature localization and tracking in biology . 41 3.2 Automated feature finding in a cell population . 50 3.2.1 Three-dimensional tracking of point-like features .