INVESTIGATIONS OF THE FUNCTIONS OF GAMMA-TUBULIN IN CELL CYCLE REGULATION IN ASPERGILLUS NIDULANS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Tania Nayak, M.Sc. ***** The Ohio State University 2008 Dissertation Committee: Approved by Professor Berl R. Oakley, Advisor Professor Stephen A. Osmani _________________________________ Professor Harold A. Fisk Advisor Graduate Program in Molecular Genetics Professor Hay-Oak Park ABSTRACT Published data from several labs, including ours, have revealed that γ-tubulin has essential, but incompletely understood functions in addition to its established role in microtubule nucleation. Our lab has isolated several conditional γ-tubulin alleles in which γ-tubulin localization is normal, microtubules are abundant and mitotic spindle assembly is not inhibited, but growth is strongly inhibited at restrictive temperatures. Extensive studies on one such allele, the cold-sensitive allele mipAD159, revealed that, at restrictive temperatures, mitotic spindles assemble with normal kinetics; that anaphase spindle elongation rates are the same as controls; and that the mitotic spindle can exert considerable force on chromosomes. However, there were defects in the coordination of chromosomal disjunction, anaphase A, anaphase B and exit from mitosis (Prigozhina et al., 2004), indicating that γ-tubulin plays a role in the regulation of mitosis. Most mitotic regulatory proteins localize at the spindle pole body during mitosis, as does γ-tubulin, and it is possible that interactions between γ-tubulin and mitotic regulatory proteins are important for coordinating the events of mitosis. In any case, understanding how the localizations of mitotic regulatory proteins are altered in γ-tubulin mutants is likely to help clarify the role of γ-tubulin in mitotic regulation. One focus of my research has, consequently, been to study the distribution of mitotic regulatory proteins in vivo by ii fusing them to green fluorescent protein (GFP). In order to facilitate GFP tagging, I have generated a strain in which the nkuA (A. nidulans homolog of Ku70) gene is deleted. We have found that this deletion, along with the use of heterologous markers from A. fumigatus, increases the correct gene targeting frequency to >90% (Nayak et al., 2006, Genetics). Using this system, I have made GFP fusions of many mitotic regulatory proteins and studied their distribution over time by time-lapse live cell imaging in control cells and in cells carrying mipAD159. One of the pathways that ensure proper completion of mitosis is the mitotic exit network (MEN), which is described in S. cerevisiae. I have identified and either deleted or down-regulated all the A. nidulans homologs of the MEN and localized many of them. The results reveal that this pathway is not required for mitotic exit in A. nidulans, but many of the genes are required for septation (cytokinesis). Deletions of the MEN/SIN pathway do not interact synthetically with mipAD159, and were therefore not useful for determining functions of γ-tubulin. However, characterization of the SIN pathway in A. nidulans has revealed interesting functions of some of the genes and may be useful in understanding septum initiation, timing and positioning in A. nidulans. I have found that three critical mitotic regulatory proteins that are mislocalized in mipAD159 are cyclin B, cyclin dependent kinase 1 and the S. cerevisiae cdc14 homolog. Studies on cyclin B localization in mipAD159 indicate that the mitotic defects of γ- tubulin are at least partially caused by misregulation of the APC/C, and in particular, that γ-tubulin plays an essential role in the inactivation of the APC/C at the end of mitosis or in G1. In addition, I have determined that this role of γ-tubulin in APC/C inactivation is independent of its role in microtubule nucleation. iii DEDICATED TO MY FATHER AND MY MOTHER iv ACKNOWLEDGMENTS I would like to acknowledge all my teachers, family and friends who have supported and guided me over the years. I am especially grateful to my mentor and advisor, Dr. Berl R. Oakley for his scientific guidance and supervision through the years that I have worked in his laboratory. He has trained me to be careful and rigorous in both learning and implementing the scientific process and challenged me at every step with probing questions, and for this, I will always be grateful. I would also like to thank my committee members, Drs. Stephen Osmani, Harold Fisk, and Hay-Oak Park for their insights, helpful suggestions, and time. The Oakley lab has been a great place to work, and I would like to thank Liz Oakley, Edyta Szewczyk, Heather Edgerton and Yi Xiong for their countless helpful discussions. I feel fortunate to have found such sincere friends and I am grateful for their generosity and support through tough times. I also wish to thank all my friends who have provided me with emotional and moral support through the last six years of my graduate studies, and for making Ohio State a wonderful place to pursue my degree. Finally, I would like to thanks my parents who have taught me, among other things, the value of a good education, and my husband and sister who continue to provide me with the strength to pursue my goals with integrity, honesty and a sense of humor. v VITA June 11, 1979………………………………….. Born – Jaipur, India. 1997-2000……………………………………... B.Sc. (Honors) Biochemistry Delhi University, New Delhi, India. 2000- 2002…………………………………….. M.Sc. Biotechnology Madurai Kamaraj University, Tamilnadu, India. 2002 – 2004…………………………………….Graduate Teaching Associate The Ohio State University. 2002-present……………………………………Graduate Research Associate The Ohio State University. PUBLICATIONS 1. Prigozhina NL, Oakley CE, Lewis AM, Nayak T, Osmani SA, Oakley BR. g-Tubulin plays an essential role in the coordination of mitotic events. Mol. Biol. Cell. 2004 Mar:15:1374–1386 . 2. Nayak T, Szewczyk E, Oakley CE, Osmani A, Ukil L, Murray SL, Hynes MJ, Osmani SA, Oakley BR. A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics. 2006 Mar;172;1557-66. 3. Szewczyk E, Nayak T, Oakley CE, Edgerton H, Xiong Y, Taheri-Talesh N, Osmani SA, Oakley BR. Fusion PCR and gene targeting in Aspergillus nidulans. Nature Protocols. 2006 1(6):3111-3120. vi 4. Chiang YM, Szewczyk E, Nayak T, Davidson AD, Sanchez JF, Lo HC, Ho WY, Simityan H, Kuo E, Praseuth A, Watanabe K, Oakley BR, Wang CCC.; Molecular genetic mining of the Aspergillus secondary metabolome: Discovery of the emericellamide biosynthetic pathway. Chemistry and Biology. 2008 June;15:1-6. FIELDS OF STUDY Major Field: Molecular Genetics, Cell biology vii TABLE OF CONTENTS Page Abstract…………………………………………………………………………….. ii Dedication……………………………………………………………………….….. iv Acknowledgments………………………………………………………………… v Vita…………………………………………………………………………………. vi List of Tables……………………………………………………………………….. xii List of Figures……………………………………………………………………… xiii List of Abbreviations……………...……………………………………………….. xvi Chapters: 1. INTRODUCTION………………………………………………………... 1 1.1 Overview…………………………………………………………………… 1 1.2 Microtubules and Microtubule Organizing Centers……….………………. 3 1.2.1 Microtubules………………………………………………………… 3 1.2.2 Centrosomes…………………………………………………………. 5 1.2.3 Spindle pole bodies………………………………………………….. 6 1.3 γ-Tubulin…………………………………………………………………... 7 1.3.1 Discovery……………………………………………………………. 7 1.3.2 Localization………………………………………………………….. 8 1.3.3 γ-Tubulin complexes……………………….………………………… 8 1.4 Functions of γ-Tubulin………………………………………….………….. 10 1.4.1 Microtubule nucleation………………………………………………. 10 1.4.2 Other functions of γ-tubulin and γ-tubulin complex proteins………... 11 1.5 γ-Tubulin Mutants in A. nidulans………………………………………….. 12 1.5.1 MipAD159…………………………………………………………… 12 1.6 Regulation of the Cell Cycle……………………………………………….. 14 viii 1.6.1 Cell cycle stages in A. nidulans……………………………………… 14 1.6.2 Checkpoints………………………………………………………….. 14 1.6.3 Spindle assembly checkpoint (SAC) ……………………………….. 15 1.6.4 Mitotic exit and G1…………………………………………………… 17 1.6.5 Cytokinesis, MEN and SIN………………………………………….. 19 1.7 A. nidulans as a Model System……………………………………………. 21 1.7.1 General description………………………………………………….. 21 1.7.2 Asexual life cycle……………………………………………………. 22 1.7.3 Sexual life cycle……………………………………………………… 23 1.7.4 A. nidulans as a model system for genetic and cell cycle research….. 25 1.8 Aims of Dissertation Research…………………………………………….. 26 2. MATERIALS AND METHODS………….……………………………... 38 2.1 Strains and Media………………………………………………………….. 38 2.2 Strain Construction………………………………………………………… 40 2.2.1 Gene Identification…………………………………………………... 40 2.2.2 Construction of transforming DNA molecules by fusion PCR……… 40 2.2.2.1 C-terminal tagging of genes…………………………………… 41 2.2.2.2 Gene deletion…………………………………………………... 42 2.2.2.3 Promoter replacement………………………………………….. 42 2.3 PCR and Amplification Conditions………………………….…………….. 43 2.3.1 Fragment amplification……………………………………………… 43 2.3.2 Cassette amplification……………………………………………….. 44 2.3.3 Fusion PCR………………………………………………………….. 44 2.4 Band Purification…………………………………………………………... 45 2.5 Transformation…………………………………………………………….. 46 2.5.1 Strain inoculation and growth………………………………………... 46 2.5.2 Enzyme preparations and protoplasting……………………………… 46 2.5.3 Purification of protoplasts……………………………………………. 47 2.5.4 Transformation of protoplasts………………………………………... 48 2.5.5 Selection……………………………………………………………... 48 2.6 DNA Extraction from A. nidulans Strains (miniprep) …………………….. 48 2.7 Confirmation of Correct Gene