Microtubules are critical elements in a wide variety of fundamental cellular processes. They serve as tracks for the motors that move vesicles and other cargo around in the cell. As essential structures of the mitotic spindle, microtubules are important for segregation of the genetic material during cell division. Cytoplasmic microtubules are vital for accurately positioning the mitotic spindle prior to cell division. Asymmetric positioning of the mitotic spindle is important for the development of all eukaryotes. In the yeast S. cerevisiae, spindle positioning requires two genetic systems, the Kar9p pathway and the dynein pathway. The molecular mechanisms that regulate and coordinate these two pathways are not well understood. The long-term goal of the Miller lab at Oklahoma State University (OSU) is to understand how signal transduction mechanisms control the microtubule-associated proteins that in turn control the vital functions of microtubules. Only a few microtubule-associated proteins are known to be regulated by the post-translational modification of sumoylation. Recent data demonstrated that spindle positioning is regulated by sumoylation. The objective of this project is to identify the mechanisms by which this modification regulates spindle positioning. To achieve the objective, this work focuses on two spindle positioning proteins, Bik1p and Kar9p. Bik1p is a microtubule-binding protein that functions in the dynein pathway. Kar9p is a protein that links the actin and microtubule cytoskeletal networks. The specific aims of this project are to determine the functional consequences of Kar9p and Bik1p sumoylation for spindle positioning and to identify molecular mechanisms that control Kar9p sumoylation. The methods used will include genetic, biochemical and cell biological assays to determine the functional consequences of Kar9p and Bik1p sumoylation. Mutations in Bik1p and Kar9p that disrupt this modification will be generated and characterized to determine the extent to which these modifications are important for different aspects of spindle positioning and microtubule function. The two microtubule-associated proteins that are the main focus of this study in yeast are conserved in many higher organisms, including mammals. Understanding the molecular mechanisms by which these proteins are regulated will provide insights into how microtubules are regulated in aberrant cell divisions and chromosome mis-segregation. Broader Impacts. The broader impacts of this study are substantial and integrate research and education in four main areas for society. The Miller lab is committed to graduate and undergraduate education, high school involvement in science, minority involvement in science, and a novel collaboration with a teaching college. First, excellent training will be provided in genetics and molecular biology for a post-doctoral fellow, one graduate student, and several undergraduates. Talented undergraduates will be recruited for independent research projects that will be included in publications. Second, the Miller lab will bring High School Interns into the lab for hands on research. The PI will recruit a high school teacher to work in the Miller lab to do cutting-edge research. Third, the PI and her team will actively recruit students under-represented in STEM fields. Four minority students already working in the Miller Lab will participate in the research. The PI also works with the OSU-led and NSF-funded Oklahoma Louis Stokes Alliance for Minority Participation in STEM fields to recruit student researchers. Fourth, this project will build on an innovative research and educational collaboration with Professor Harold Hoops at the State University of New York (SUNY) Geneseo. SUNY-Geneseo is a small, non-Ph.D.-granting institution. Dr. Hoops' undergraduates will continue to work on research related to this project, with the goal of additional co-publications. The two labs will continue to share strains, reagents, protocols, and advice, thus providing exceptional lab experience to the SUNY undergraduates. This project will enhance multiple interdisciplinary-research partnerships at Oklahoma State University. Other researchers will have access to the Miller lab's expertise in yeast, a model organism, thus supporting OSU's infrastructure and collaborative-research atmosphere. Project results will be broadly disseminated in research seminars, scientific conferences, and by publication in peer-reviewed journals. All student participants will be encouraged to publish & present research.
Intellectual Merit: Microtubules are protein polymers that are important for a wide variety of cellular processes, including the transport of cargoes within a cell and positioning the mitotic spindle, a structure that separates genetic information at cell division. The goal in this research project was to identify molecular mechanisms by which proteins used to control microtubules are regulated by sumoylation, a type of specialized protein modification. Dynein is a motor-like protein that can move along microtubules. In this research, several approaches showed for the first time that two regulatory proteins for dynein, Pac1p/Lis1 and Bik1p/CLIP170, are modified by sumoylation. A third microtubule-regulating protein, Stu2p/XMAP215 was also shown to interact with SUMO and other proteins in the SUMO conjugation pathway. The importance of these new sumoylations is related to another new finding of this research that Pac1p, Bik1p, and Stu2p also interact with an enzyme called a STUbL, a SUMO-targeted ubiquitin ligase. STUbLs add ubiquitin to proteins that already have a SUMO modification on them. The addition of ubiquitin will result in the ultimate destruction of the ubiquitinated protein by a garbage-disposal like apparatus in the cell called the proteasome. Experiments indicate that the proteasome is responsible for some Pac1p degradation. This provides a new signal transduction mechanism by which an important regulator of the dynein motor may itself be regulated. These findings provide insight into how microtubules might be regulated in aberrant cell division, syndromes of chromosome mis-segregation, a brain development disorder, and some types of motor neuron degeneration. Results were disseminated at several regional and national research conferences, at multiple invited seminars at major research institutions, and published in peer-reviewed journals. Broader Impacts: Research project activities were integrated with educational activities at all levels. This project provided training in molecular biology, genetics, cell biology, and biochemistry to one postdoctoral fellow, three Ph.D. graduate students, and one Master’s degree student. Seven undergraduates learned research skills while working on this project, three of whom were drawn from the NSF-funded OK-LSAMP program at OSU. One middle school science teacher spent the summer doing hands-on research in the lab. Four high school interns experienced research first hand during a summer rotation. This research project supported a collaboration with a small, non-Ph.D. granting institution. Combined, these research experiences have enhanced the science-readiness of a wide-ranging and diverse group of students and educators.
