The Effects of Chromosome Number on Mitotic Fidelity and Karyotype Stability Joshua M. Nicholson Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Sciences Daniela Cimini, Chair John Robertson Roderick Jensen Richard Walker May 4th, 2015 Blacksburg, Virginia Keywords: aneuploidy, cancer, chromosome mis-segregation, tetraploidy, cytokinesis failure The Effects of Chromosome Number on Mitotic Fidelity and Karyotype Stability Joshua M. Nicholson Abstract The correct number of chromosomes is important for the maintenance of healthy cells and organisms. Maintenance of a correct chromosome number depends on the accurate distribution of chromosomes to the daughter cells during cell division, and errors in chromosome segregation result in abnormal chromosome numbers, or aneuploidy. Aneuploidy is typically associated with deleterious effects on organismal and cellular fitness; however, aneuploidy has also been associated with enhanced cellular growth in certain contexts, such as cancer. Another type of deviation from the normal chromosome number can occur when entire sets of chromosomes are added to the normal (diploid) chromosome number, resulting in polyploidy. Whereas polyploidy is found in certain normal tissues and organisms, tetraploidy (four sets of chromosomes) is associated with a number of precancerous lesions and is believed to promote aneuploidy and tumorigenesis. While it is clear that chromosome mis-segregation causes aneuploidy, the effect of aneuploidy on chromosome segregation is less clear. Similarly, it is unclear whether and how tetraploidy may affect chromosome segregation. The work described here shows that aneuploidy can cause chromosome mis- segregation and induces chromosome-specific phenotypic effects. In contrast, tetraploidy does not per se induce chromosome mis-segregation, but enables the accumulation of aneuploidy thanks to a “genetic buffer” effect that allows tetraploid cells to tolerate aneuploidy better than diploid cells. Dedication Dedicated to Professor Richard C. Strohman, my grandfather. iii Acknowledgements I would like to acknowledge my family for all the support they have given me throughout my life and especially throughout my time at Virginia Tech. Words cannot do justice to the gratitude that I feel, so I will simply say thank you and that I love you. I would also like to thank Daniela Cimini and the entire Cimini lab for pushing me to be a better scientist and person. I learned from each and all of our interactions and this work would not have been possible without you. iv Attribution Chapter 2: Chromosome mis-segregation and cytokinesis failure in trisomic human cells Chapter 2 was published in eLife 2015;10.7554/eLife.05068 Joshua Nicholson: Conception and design; Acquisition of data; Analysis and interpretation of data; Drafting or revising the article Joana Macedo: Acquisition of data; Analysis and interpretation of data Aaron Mattingly: Acquisition of data; Analysis and interpretation of data Darawalee Wangsa: Acquisition of data; Analysis and interpretation of data Jordi Camps: Acquisition of data; Analysis and interpretation of data Vera Lima: Contributed unpublished essential data or reagents Ana Gomes: Acquisition of data Sofia Dória: Contributed unpublished essential data or reagents Thomas Ried: Contributed unpublished essential data or reagents Elsa Logarinho: Conception and design; Analysis and interpretation of data; Drafting or revising the article; Contributed unpublished essential data or reagents Daniela Cimini: Conception and design; Analysis and interpretation of data; Drafting or revising the article; Contributed unpublished essential data or reagents Chapter 3: Polyploidy as a buffer for aneuploidy in human cells Chapter 3 has not been submitted for publication. Joshua Nicholson: Conception and design; Acquisition of data; Analysis and interpretation of data; Drafting or revising the article Nicolaas Baudoin: Conception and design; Acquisition of data; Analysis and interpretation of data; Drafting or revising the article Daniela Cimini: Conception and design; Analysis and interpretation of data; Drafting or revising the article; Contributed unpublished essential data or reagents v Table of Contents Abstract .............................................................................................................................. iii Dedication .......................................................................................................................... iii Acknowledgements ............................................................................................................ iv Attribution ............................................................................................................................ v List of Figures .................................................................................................................... vii Chapter 1. Introduction and literature review ............................................................. 1 Overview ................................................................................................................................ 2 Pre-neoplastic Aneuploidy .................................................................................................. 4 Chromosome Instability and the Cancer Karyotype – between Order and Disorder ............ 6 The effects of aneuploidy on the transcriptome and proteome ..........................................10 The adaptive potential of aneuploidy .................................................................................11 How mitotic errors contribute to the karyotypic diversity of cancer cells .................................13 Kinetochore-Microtubule Attachments ...............................................................................14 Spindle Assembly Checkpoint (SAC).................................................................................16 Multipolarity and Spindle Geometry ...................................................................................20 Microtubule Dynamics .......................................................................................................23 Polyploidization .................................................................................................................24 Knowledge gaps and open questions ....................................................................................27 Chapter 2. Chromosome mis-segregation and cytokinesis failure in trisomic human cells. ................................................................................................................ 37 Abstract .............................................................................................................................38 Introduction .......................................................................................................................39 Results ..............................................................................................................................42 Discussion .........................................................................................................................50 Materials and Methods ......................................................................................................58 Chapter 3. Polyploidy as a buffer for aneuploidy in human cells ........................... 83 Abstract .............................................................................................................................84 Introduction .......................................................................................................................85 Results ..............................................................................................................................87 Discussion .........................................................................................................................93 Materials and Methods ......................................................................................................95 Chapter 4. Conclusions and perspectives .............................................................. 105 Conclusions and Perspectives ............................................................................................ 106 References ................................................................................................................. 113 vi List of Figures Figure 1.1 Visualization of the karyotypic stemline in a karyograph ...................... 31 Figure 1.2 The pleiotropic effects of aneuploidy ...................................................... 32 Figure 1.3 Diagrammatic representation of the stages of mitosis .......................... 33 Figure 1.4 Types of kinetochore-microtubule attachments ..................................... 34 Figure 1.5 Possible fates of merotellically attached anaphase lagging chromosomes....................................................................................................... 35 Figure 1.6 Multipolarity, centrosome clustering and chromosome mis-segregation ............................................................................................................................... 36 Figure 2.1. Trisomy 7 and 13 in DLD1 and AF cells. ................................................ 69 Figure 2.1-figure supplement 1. aCGH in DLD1, DLD1+7, and DLD1+13 cells. ...... 71