ISOLATION OF COPY NUMBER SUPPRESSORS OF THE NIMA1 KINASE AND MITOTIC REGULATION OF NUCLEOLAR STRUCTURE 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 Leena Ukil ***** The Ohio State University 2007 Dissertation Committee: Approved by Dr. Stephen A. Osmani, Advisor Dr. Berl R. Oakley _________________________________ Dr. Hay-Oak Park Advisor Graduate Program in Molecular Genetics Dr. Harold A. Fisk ABSTRACT Regulation of the cell cycle is critical for normal development of multicellular organisms and an understanding of this process is crucial for studying cell proliferation and cancer. A number of cell cycle dependent protein kinases specifically control mitotic progression and chromosome segregation. The nimA gene in Aspergillus nidulans encodes one such protein kinase that is both required and sufficient for chromosome condensation, mitotic spindle formation and disassembly of the nuclear pore complex to allow tubulin and regulators to enter nuclei during mitosis. There exist protein kinases structurally similar to nimA in other organisms, including humans. In the filamentous fungus Aspergillus nidulans, the NIMA kinase is required for the regulation of mitosis along with CDK1/cyclin B. Levels of NIMA are regulated throughout the cell cycle, reaching a maximum at mitotic entry and falling dramatically at mitotic exit. Forced expression of the nimA gene can promote mitotic entry, even in human cells. The essential function of NIMA in A. nidulans and the growing recognition of its function in other eukaryotes, means that a study of NIMA function would reveal unique insights into cell cycle regulation among a broad range of organisms. I describe here the characterization of three novel genes mcnA, mcnB and mcnC, three multi-copy number suppressors of the nimA1 conditional mutant, identified in a copy number suppression screen of the nimA1 mutant, and describe the potential novel roles they may play in mitotic regulation. Characterization of MCNC suggests that it is involved in mitotic regulation. First, over expressed mcnC suppresses the G2 arrest caused by nimA1. Second, MCNC over expression leads to the dispersion of the nuclear pore protein SONB suggesting its potential role in regulating NIMA localization to the nucleus during mitosis. Third, mcnC genetically interacts with nimA. Additionally, the deletion of mcnC causes polarization defects due to delayed germtube emergence. Over expression of MCNC concomitantly ii leads to multiple germ tube formation suggesting a positive regulatory role of mcnA in controlling polarized growth of A. nidulans. The other two nimA1 suppressing genes, mcnB and mcnA both lead to up regulation of NIMA protein levels when over expressed. mcnB is nuclear during G2/M and carries a prominent transcription factor domain called the forkhead domain. MCNB begins to accumulate in the nucleus during G2 and peaks at mitotic entry suggesting it may play a role as a nimA specific transcription factor. Its ortholog in Schizosaccharomyces pombe, Sep1, is known to specifically regulate transcription of a number of genes with roles in sister chromatid separation, septation and cytokinesis and shown to be required for the periodic accumulation of the nimA related kinase fin1. Therefore, a role for forkhead transcription factors regulating cell cycle specific nimA expression is conserved between S. pombe and A. nidulans. MCNA was found to have a fascinating and dynamic location within cells. Endogenously tagged mcnA appears as a single dot in the nucleus. Co-localization studies of MCNA with nucleolar markers show MCNA to locate in the vicinity of the nucleolus and to have a unique pattern of segregation during mitosis. During G2, MCNA is closely associated with nucleolar proteins and appears as an intranuclear body. During mitotic DNA segregation, the MCNA body localizes to the cytoplasm. When mitosis is completed, MCNA remains as a single body outside newly formed daughter nuclei which begins to appear within new daughter nuclei only during G1. At the same time, multiple smaller MCNA bodies are formed in the cytoplasm which finally disappears to completely re-accumulate into the two daughter nuclei by very late G1. Our studies of MCNA localization through the cell cycle shows a highly specific pattern for MCNA and puts forth suggests a possible function of mcnA in regulating nimA turnover during mitosis. The association of MCNA with the nucleolus prompted us to further study the nucleolus in A. nidulans and its segregation during mitosis. The nucleolus is a prominent nuclear structure whose mitotic segregation is poorly understood. During yeast mitosis the nucleolus segregates intact with rDNA (the nucleolar organizing region – NOR). In contrast, during open mitosis the nucleolus is disassembled then reassembled during iii mitosis. In A. nidulans nuclei, mitosis is a partially open process and I demonstrate that the nucleolus segregates through a completely novel mechanism. Unlike Saccharomyces cerevisiae, few A. nidulans nucleolar proteins segregate with DNA. Instead during DNA segregation, a double pinch of the NE occurs that results in the formation of two daughter nuclei and a central tertiary cytoplasmic structure we have termed the nuclear remnant that contains several nucleolar proteins. While the NOR segregates with the rest of the DNA, the bulk of the nucleolar proteins are seen to remain distinctly intact in the cytoplasm within this nuclear remnant structure. It is only during late telophase and early G1 that the nucleolar proteins from the remnant structure begin to undergo a sequential disassembly and reassembly into the daughter nuclei resulting in the formation of two functional daughter nucleoli in a step-wise manner. My study indicates that nucleolar disassembly-reassembly in A. nidulans is under the control of the spindle assembly checkpoint and that the step-wise process may be regulated by the preferential action of the mitotic kinase CDK1 towards certain nucleolar proteins. A potential role of the phosphatase BIMG is also suggested as BIMG is seen to be localized to the nuclear remnant structure during this process. This study also indicates that A. nidulans undergoes mitotic disassembly then reassembly of its nucleolus, as do higher eukaryotes, and that generation of daughter nuclei occurs via a double fission mechanism, not a single fission as occurs in yeasts. I suggest this novel mitotic nuclear remnant serves as a storage pool from which equal distribution of nucleolar proteins can occur. Mathematical modeling provides supportive evidence for this hypothesis. The nuclear remnant may also serve as a sink for unwanted cytoplasmic proteins or RNAs that gain access to the nucleoli during partially open mitosis. iv DEDICATION This work is dedicated to my parents, to my brother Shubha, and to my loving husband Bose. v ACKNOWLEDGMENTS I am deeply indebted to my advisor Dr. Steve Osmani for his continual guidance, training and support throughout the years of my graduate studies. This achievement would not have been possible without his extreme patience and motivation. I would also like to thank my committee members, Dr. Berl Oakley, Dr. Hay-Oak Park, Dr. Harold Fisk and Dr. Russell Hill for their time and guidance during my graduate studies at The Ohio State University. Lastly, I would like to thank all current and former members of the Osmani lab for their friendship and support thus making my experience in the lab enjoyable. I would also like to specially thank Aysha Osmani for her motherly care and guidance and for making me a part of her extended family. vi VITA 1997-2001……………………………….B.S. Biochemistry, University of Madras. 2002 – 2003……………………………..Teaching Asst. The Ohio State University. 2003 – 2007……………………………..Research Asst. The Ohio State University. PUBLICATIONS 1. Nayak, T., Szewczyk, E., Oakley, C.E., Osmani, A., Ukil, L., Murray, S.L., Hynes, M.J., Osmani, S.A., Oakley, B.R. (2006). A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics 172 : 1557- 66. 2. Yang, L., Ukil, L., Osmani, A., Nahm, F., Davies, J., De Souza, C.P., Dou, X., Perez-Balaguer. A., Osmani, S.A. (2004). Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans. Eukaryotic Cell. 3 : 1359-62. 3. Dou, X., Wu, D., An, W., Davies, J., Hashmi, S.B., Ukil, L., Osmani, S.A. (2003). The PHOA and PHOB cyclin-dependent kinases perform an essential function in Aspergillus nidulans. Genetics 165: 1105-15. FIELDS OF STUDY Major Field: Molecular Genetics vii TABLE OF CONTENTS Page ABSTRACT……………………………………………………………………………..ii DEDICATION…………………………………………………………………………v ACKNOWLEDGMENTS………………………………………………………………vi VITA……………………………………………………………….. …………………vii LIST OF FIGURES………………………………................…………………xiv LIST OF TABLES………………………………………………………………...…xvii CHAPTERS: 1. INTRODUCTION.........................................................................................................1 1.1. Project Goal......................................................................................................1 1.2 Aspergillus nidulans..........................................................................................2 1.2.1. General classification and description...............................................2 1.2.2. Life cycle of Aspergillus nidulans.....................................................3