Progress 10/01/13 to 09/30/14
Outputs
Target Audience: General scienfific community. Cell biologists in general and microbiologists with expertise in pathogenic fungi of plants. Oklahoma crops such as wheat and corn are affected by several fungal diseases. A key element of their pathogenecity is the hyphal invasion of the fungus into the host crop. Microtubules are required for this hyphal growth This project studies how microubules are controlled by microtubule assoicated proteins, Pac1p, Bik1p and Stu2p. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Four GRADUATE STUDENTS were trained on this project, Maliha Rahman, Annabel Alonso, Jake Kline, and Matthew Greenlee. Annabel Alonso is expected to graduate with her Ph.D. in May 2015. Maliha Rahman graduated with a M.S. degree in December 2012. She is now a Ph.D. graduate student at the University of Oklahoma. Jake Kline and Matt Greenlee earned M.S. degrees. Matt Greenlee is expected to complete his Ph.D. in 2017 in the Miller lab. Several UNDERGRADUATE STUDENTS have conducted hands-on molecular biology research on this project. They learned both research and presentation skills. Kayla Davis, 2009 - May 2014. Using two-hybrid analysis, Kayla showed that the Stu1p protein interacted with Pac1p. She also analyzed several phosphorylation-site mutants for their interaction. Kayla is now a Ph.D. graduate student in the life sciences at Harvard University. Brooklyn Ryan worked on the project from January 2014- May 2014. Brooklyn learned DNA mini-preps and assisted in several cloning projects. She also learned yeast two-hybrid technology. Brooklyn is an OK-LSAMP scholar. Elizabeth Williamson, Aug 2013-December 2013. Andrea Talley, Sept 2012- October 2013, is an OK-LSAMP scholar and she cloned both of the two domains of Pac1p into two-hybrid vectors for analysis, learning valuable molecular biology skills. Darren Figuerido January 2012- August 2013. Darren is an OK-LSAMP scholar. Nick Rackely from East Central University worked during the summer of 2012. Nick epitoped tagged several dynactin compontents with the HA-epitope. Nick is a McNair Scholar. River Crawford, August 2014-present. River is an OK-LSAMP scholar. Megan Ragsdale, August 2014-present. Megan is a CASNR undergraduate research scholar. One POSTDOCTORAL FELLOW trained on the project: Dr. Jessica Matts, February 2013 to May 2014. Four HIGH SCHOOL INTERNS participated in hands-on research during the summers: Kayla Davis 2009, 2010, Jessie High 2012, Iain Kirsche 2013 & 2014, and Cameron Jackson 2014. Cameron worked on identifying four clones by doing DNA mini-preps, restriction enzyme digestion, and analysis on ethidium bromide stained agarose gels. Cameron is now a freshman majoring in Marine Biology at the University of Texas, Galveston. TEACHING ACTIVITIES: Courses taught at Oklahoma State University include. BIOC 4523 BIochemistry of the Cell in Spring semester 2013, 2014, and BIOC 4990 independent study. PROFESSIONAL DEVELOPMENT: Annabel Alonso (graduate student) attended three national meetings. Jessi Matts (post doc) and the three graduate students (Annabel Alonso, Jake Kline and Matt Greenlee) attended the American Society for Cell Biology meeting. At each of these meetings, they gained additional knowledge about the field of microtubules, fungal cell biology, and fungal agents. They each learned additional presentation skills and networked with other scientists. How have the results been disseminated to communities of interest? The results from this project for this reporting period have been communicated at three national meetings, one regional meeting, and two invited seminars. INVITED SEMINARS: 1. Agricultural Institute of the Biosciences, O.S.U. Ardmore, OK. Sumoylation - a new post-translational modification of the dynein regulator, Pac1p/Lis1. June 2013, (host: Randy Allen). 2. Upstate University, Syracuse, NY. “Sumoylation regulates the position of the mitotic spindle.” May 2013 (Host: David Pruyne) REGIONAL MEETING: 3. Rocky Mountain Yeast Club, University of Colorado, Boulder. The SUMO-like modifications of the dynein adaptor, Pac1 / Lis1, are regulated by osmotic stress and the cell cycle. Jan. 10, 2014. NATIONAL MEETINGS: 4. The Ubiquitin Family Meeting, Cold Spring Harbor Laboratory, NY May 2013. 5. The Cell Biology of Yeast meeting at Cold Spring Harbor , Nov. 2013. 6. The American Society for Cell Biology meeting, New Orleans, LA Dec 2013. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Previous annual progress reports describe the addition of another goal for this project. This goal is to increase our understanding of the regulation of the Bik1p-interacting protein, Pac1p/LIS1, which is a major regulator of the dynein motor protein that is important in the biology of fungal hyphae. Our studies of this regulation has focused extensively on the modification of MAPs by SUMO. We have made outstanding progress toward this goal. Pac1. We have made several advances in understanding the regulation of Pac1p by SUMO and ubiquitin modifications. We have found that the modifications of Pac1p-tagged with protein A (4Z) are most pronounced in the cell cycle during G1 and during S-phase. For G1 enrichment, the cells were grown to saturation which enriches for G0/G1 cells. For S-phase arrest, we arrested cells with the drug, hydroxyurea. We are working to confirm this finding for the HA-tagged Pac1p. We have identified two lysine residues that are required for the ubiquitin-like modification of Pac1p. These are lysine 20 and lysine 114. These lysines were mutated to arginine residues which prevents the addition of ubiquitin-like modifications. This generated pac1-K20R and pac1-K1124R. We are currrently analyzing these mutants to determine the extent to which they display a phenotype. Pac1-Stu2. We have identified a new protein-protein interaction for Pac1p. We have demonstrated by two-hybrid analysis and co-immunoprecipitation experiments that Pac1p interacts with the XMAP215 family member, Stu2p. Stu2p is a well known microtubule-binding protein that classically acts to promote and stabilize microtubule polymerization. This interaction has the potential to be a new mechanism for regulating Pac1p. Because Pac1p is sumolylated, we also analyzed Stu2p to characterize its SUMO status. We showed that Stu2p interacts with SUMO and the sumoylation machinery (Ubc9 and an E3, and Wss1p) by two -hybrid analysis. To confirm these findings, we analyzed Stu2p for the presence of upper molecular weight bands in the Ulp1-TS strain, which has an impaired allele of the protease that removes SUMO from target substrates. In this Ulp1-TS strain, we find that SUMO co-purifies with Stu2p. There are two forms of Stu2p that differ in size by 10kDa, as seen by previous researchers. The SUMO reactive band co-migrates with the larger form of Stu2p, suggesting that this larger sized Stu2p is sumoylated. Pac1 interacts with Stu2p and Stu2p is sumoylated. We then asked "With which form of Stu2p does Pac1p interact?" Immunoprecipations of Pac1p and western blotting analysis showed that Pac1p co-purifes with the smaller and putatively non-modified form of Stu2p. This suggests a model in which sumoylation of Stu2p blocks its interaction with Pac1p. This represents a new mode of regulation for the Stu2p MAP and Pac1p. These findings support our hypothesis that sumoylation is a general mechanism for the regulation of microtubule associated proteins. Because XMAP215/Stu2p is important for microtubule stability and because microtubules are critical for the growth of hyphae in filamentous fungi, our findings also should help to elucidate the molecular mechanism by which the microtuble motor dynein moves cargo along the hyphae of fungal pathogens, contributing to hyphal invasion of plants.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Rocky Mountain Yeast Club, University of Colorado, Boulder. The SUMO-like modifications of the dynein adaptor, Pac1 / Lis1, are regulated by osmotic stress and the cell cycle. Jan. 10, 2014.
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Progress 01/01/12 to 12/30/12
Outputs
OUTPUTS: ACTIVITES: We have continued to make excellent progress in our experiments to understand the regulation of the dynein-regulating protein, Pac1/Lis1 by sumoylation. We have shown that Pac1p is modified by both SUMO and ubiquitin. It also interacts with the SUMO targeted ubiquitin ligase, Nis1p-Ris1p. TEACHING ACTIVITIES Courses taught at Oklahoma State University: BIOC 4523 Biochemistry of the Cell, Spring 2011 BIOC 6723 / BIOC 6820 Signal Transduction, Fall 2012 BIOC 4990 Independent Study INVITED SEMINARS I was an invited seminar speaker at the University of Memphis, Department of Biology, April 2012 and at Tulsa Community College, March 2012. PROFESSIONAL MEETINGS: I attended two professional meetings: The Yeast Genetics and Molecular Biology meeting in August 2012 and the American Society for Cell Biology meeting in San Francisco, CA in Dec 2012. I presented posters at both of these meetings. PARTICIPANTS: Ph.D. GRADUATE STUDENTS: 1. Annabel Alonso January 2010 - present Thesis project: The regulation of Pac1p and Bik1p by sumoylation. Annabel has shown that Pac1 is both sumoylated and ubiquitinated. 2. Jake Kline, June 2011-present Thesis project: The sumoylation of Kar9p 3. Matt Greenlee, December 2011-present. Matt is studying how sumoylation affects the interaction of Pac1 with microtubule motor dynein heavy chain. MASTER's GRADUATE STUDENTS: 1. Maliha Rahman, Master's received December 2012 Thesis project: The regulation of Stu2p by sumoylation UNDERGRADUATE RESEARCHERS: Jacob Keeling, Niblack Scholar 2009, Wentz Scholar 2010, 2011- 2012 . Demonstrated that Pac1 interacts with the STUbL enzyme, Nis1p, Ris1p. Kayla Davis, OK-LSAMP Scholar 2009-present, Wentz Scholar 2012-2013. She is testing how phosphorylation sites regulate the interaction of Pac1p with SUMO. Carrie Tillett Sept 2011- May 2012. Kortney Flanagan Sept 2011-present. Andrea Talley, Sept 2012- present. Andrea is an OK-LSAMP Scholar 2012-2013 CASNR Undergraduate Research Scholarship, awarded December 2012. She is using the yeast two-hybrid system to map the domains of Pac1p that interact with the sumoylation enzymes. HIGH SCHOOL INTERN: Jessica High, Summer 2012. HIGH SCHOOL TEACHER TRAINING: Becky Hammack, (Middle School science teacher, Stillwater Public Schools), Summer 2012 TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: This project is focussing on the post-translational modifications of Pac1, which is a binding partner protein of the Bik1p described in the original project.
Impacts
This project is significant to MICROTUBULE BIOLOGY in several ways: DYNEIN MOTOR REGULATION. Dynein is the major motor protein that walks toward the minus end of microtubules. Dynein carries a variety of different cargoes. However, regulation of the attachment of cargo to dynein is poorly understood. The movement of dynein's cargo is central to the biology of several diseases and their therapies. For instance, adenovirus vectors are increasingly being used for gene therapy and dynein is important for the movement of adenovirus particles toward the nucleus. Therefore, the work of this project could have implications for the treatment of a large number of diseases. Dynein plays several roles in the mitotic spindle and at the kinetochore. Dynein is important for gathering the poles of the mitotic spindle into a tight focus. It is important in chromosome capture and for silencing of the spindle assembly checkpoint, which monitors bi-orientation of sister chromatid attachment by kinetochore microtubules. Errors in any of these processes can lead to increases in abnormally segregated chromosomes, aneuploidy. Aneuploidy results in a much worse outcome for victims of cancer. BRAIN DEVELOPMENT DISEASE: Mutations in Lis1 cause type I Lissencephaly, a devastating disease of abnormal development of the brain cortex. In Lissencephaly, neuronal cells display abnormal mitoses and incorrect cell migrations. Understanding the regulation of Lis1/Pac1p by SUMO in yeast will provide insight into how this modification regulates microtubules in neurons and its role in Lissencephaly. Lis1 is a critical regulator of dynein and modifies several aspects of its function. Lis1 binds near the tail of dynein and promote its interaction with cargo. Lis1 also competes with the dynactin adaptor for dynein. Lis1 also increases the time of dynein's attachment to microtubules. The Miller lab recently demonstrated that Lis1/Pac1p is sumoylated. Understanding how the SUMO modification controls Lis1/Pac1p will increase our understanding of many aspects of dynein's functions, including new insights for how cargo-binding to dynein is controlled. This knowledge is expected to lead to a better understanding of how dynein contributes to aneuploidy, which is found in cancers and several types of birth defects. This knowledge may lead to new therapeutic targets for cancer and to new ways to combat viral infections. PARADIGM FOR THE REGULATION OF OTHER MICROTUBULE BINDING PROTEINS. Microtubules are dynamic structures that are controlled by MAPs, yet it is not understand how the regulation of MAPs contribute to changes in microtubule dynamics. Seven distinct classes of MAPs are known to be sumoylated. Astonishingly little is known about how sumoylation alters the function of any of these MAPs. Thus, the discoveries that are made in this project for Pac1p/Lis1 will have ramifications for a large number of functionally distinct classes of regulatory MAPs and a variety of cellular processes. These findings will be especially relevant in diseases in which cytoskeletal elements accumulate to high levels, like Alzheimer's disease.
Publications
- Alonso, A., S. D. Silva, S., M. Rahman, P. B. Meluh, J. Keeling, N. Meednu, H. Hoops, and R.K. Miller. 2012. The yeast homolog of the microtubule-associated protein LIS1 interacts with the sumoylation machinery and a SUMO-targeted ubiquitin ligase. Molecular Biology of the Cell. 23: 4552- 4566.
- Alonso, A., S. DSilva, M. Rahman, J. Keeling, N. Meednu, R.K. Miller. 2012. The dynein regulator, Pac1p/Lis1, interacts with a STUbL. 2012. ---Abstract at the American Society for Cell Biology meeting. San Francisco, CA, Dec. 2012 ---Abstract at the Yeast Genetics and Molecular Biology meeting. Princeton University, August 2012.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: OUTPUTS: The goal of this project is to better understand the molecular mechanisms by which microtubules are regulated. Microtubules are important for mitosis, and they also serve as tracks on which various cargo are moved to specific destinations within the cell. Microtubule binding proteins regulate microtubules. This project investigates how the Bik1p microtubule binding protein is regulated, which in turn controls microtubule properties and spindle positioning. This will be valuable for a wide range of different systems and organisms that use microtubules. ACTIVITIES: Conducting Experiments: As noted in the progress report for 2010, we have added a new area of research that focuses on the sumoylation of microtubule binding proteins. In 2011, we have been very successful with this, showing that the Bik1p-interacting protein and dynein regulator, Pac1p/Lis1, is modified by SUMO. TEACHING ACTIVIITES: My teaching activities in 2011 were in two areas, teaching in the laboratory and teaching two lecture courses: Biochemistry of the Cell BIOC 4523 (Spring 2011) and Signal Transduction, BIOC 6723 (Fall 2011). For laboratory teaching, I have directly supervised the research of three graduate students (Annabel Alonso, Maliha Rahman, Jake Kline), three first year rotation students (Maurie Balch, Matt Greenlee, and Junho Cho), two undergraduates (Jacob Keeling and Kayla Davis), and three lab assistants (Taylor Reed, Carrie Tillet, and Kortney Flanagan). Three graduate students: --Annabel Alonso, Ph.D. student. Her project this year has been to understand the post-translational modifications of the Bik1p interacting protein, Pac1p/Lis1, by ubiquitin and the ubiquitin-like moiety, SUMO. --Maliha Rahman, Ph.D. student. Maliha studies the post-translational modification, sumoylation, of the microtubule binding protein, Stu2p. --Jake Kline, Ph.D. student. Jake joined the lab in June 2011. His project is to purify SUMO to make an anti-SUMO antibody. He will use this to characterize the sumoylation of Pac1p and other microtubule binding proteins. DISSEMiNATION: During 2010, Dr. Miller collaborated with several other researchers, including Dr. Holly Goodson at Notre Dame Univ. and Dr. Harold Hoops at SUNY-Geneseo, and Dr. Jeanmarie Verchot of O.S.U. Dr. Miller gave three invited seminars and presented at one professional meeting: INVITED SEMINARS: East Central University, Oklahoma. "O.S.U. & B.M.B., a place for graduate school success." Oct. 2011. Czech Cytoskeletal Club, Prague, Czech Republic, "Sumoylation as a new post-translational mechanism for regulating the position of the mitotic spindle," May 10, 2011. Oklahoma State University, Department of Microbiology, "Sumoylation as a new post-translational mechanism for regulating the position of the mitotic spindle ...and possibly microtubules in general." April 4, 2011. PROFESSIONAL MEETINGS: A. Alonso, S. D'Silva, N. Meednu, H. Hoops, J. Keeling, M. Rahman, R.K. Miller 2011. Pac1p/Lis1 regulation via sumoylation. ---Poster presentation at the American Society for Cell Biology meeting. Denver, CO, Dec. 2011 PARTICIPANTS: INDIVIDUAL PARTICIPANTS IN THE PROJECT: GRADUATE STUDENTS: Annabel Alonso: She has worked to characterize the interaction between Pac1p and SUMO. Maliha Rahman: She has worked at characterizing the interaction between Kar9p and SUMO and Stu2p and SUMO. Jake Kline: Jake joined the lab in June 2011. He has engineered and cloned a GST-SUMO construct that will be used to make a SUMO antibody. This will also be used in binding studies of MAPs to SUMO. He has supervised an undergraduate in the lab, Kayla Davis. He has optimized several cloning protocols. UNDERGRADUATES: Kayla Davis: has been screening for additional MAPs that interact with SUMO. Jacob Keeling: has found that Pac1 interacts with a new class of enzyme, the SUMO-targeted ubiquitin ligase. PREFESSIONAL DEVELOPMENT: Annabel Alonso attended the national meeting of the American Society for Cell Biology in Denver, CO in December 2011, where she presented her findings on the sumoylation of Pac1p. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
OUTCOMES / IMPACTS We have made excellent progress in our project on the sumoylation of Pac1p. Pac1p is a microtubule-associated protein that physically interacts with Bik1p and it also regulates dynein. It is conserved throughout evolution and is found in mammals. This is a high impact area of research because when the human version of Pac1p is mutated, the developmental brain disorder, Lissencephaly results. This is caused by the failure of Lis1 to properly regulate dynein in its movement of the nuclei in neurons to the correct location in the brain for neuronal development and cell division. BACKGROUND ON PAC1P/ LIS1 AND ITS FUNCTION. Pac1p is important for the localization and regulation of dynein at the plus end of the microtubule in yeast. Pac1p/Lis1 helps dynein by allowing it to exert more force when carrying a heavy load. In epithelial cells, Lis1/Pac1 helps organize the cortical band of microtubules found in the upper layers of the skin. The molecular mechanisms that regulate Pac1p in these processes are not known. This year we showed that Pac1p can be modified by the small ubiquitin-like moiety, SUMO, which is a novel signal transduction pathway. Little is known about SUMO's role in regulating MT function. Because the Pac1/Lis1 in this study has homologues in higher eukaryotes and plants, this information will be transferable to other MT-based processes that will be important for biofuel production and other agricultural applications. Background on SUMO. SUMO/Smt3p is a conserved small ubiquitin-related modifier that is covalently attached to other proteins as a post-translational modification. SUMO regulates numerous basic cellular events, however little is known about the role that sumoylation plays in MT dependent processes. Target proteins regulated by SUMO are associated with many cellular processes, but very few microtubule-associated proteins are known to be modified by SUMO. The SUMO moiety can be removed from target proteins by deconjugating proteases, Ulp1p and Ulp2p. NEW RESULTS: Our previous work showed that Pac1p interacts by two-hybrid analysis with the ubiquitin-like moiety, SUMO, and with ubiquitin itself. To determine whether modification of Pac1p might be a normal phenomenon, in 2011 we tested for higher molecular weight forms of Pac1p in whole cell extracts made from wild-type cells. We found that there was a series of "smearey" bands that were easily visible as higher molecular weight bands larger than the regular Pac1p. Pac1p protein could be greatly shifted into many larger forms by inhibiting the sumo-protease Ulp1p that removes SUMO from target proteins. This suggests that sumoylation is involved in the shift of Pac1p. Blotting with anti-ubiquitin antibodies to ubiquitin suggests that Pac1p may be modified by both ubiquitin and SUMO. Our analysis of the eighteen Bik1p phosphorylation mutants for phenotypes is still in progress. We still plan to determine their sensitivity to the drug benomyl, genetic interactions including synthetic lethality with kar9 mutants, and MT length using fluorescently-tagged tubulin. This project has been slowed due to the success of the Pac1p sumoylation project.
Publications
- A. Alonso, S. DSilva, N. Meednu, H. Hoops, J. Keeling, M. Rahman, R.K. Miller 2011. Pac1p/Lis1 regulation via sumoylation. Abstract 1412, American Society for Cell Biology meeting. Denver, CO, Dec. 2011
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The goal of this project is to increase our understanding of how the regulation of Bik1p function, which in turn regulates microtubule properties and spindle positioning. This research will lead to a better understanding of how various signal transduction pathways control microtubule function in a variety of cellular processes. Understanding how Bik1p is regulated will lead to insights into how microtubules are regulated in higher organisms, which include regulation of mitosis and the mitotic spindle and cytoplasmic microtubules. ACTIVITIES: Conducting Experiments: Previously in the Miller laboratory, nine phosphorylation sites on Bik1p were identified by mass spectrometry. These nine phosphorylation sites were mutated by site-directed mutagenesis to either alanine or glutamic acid. This generated eighteen different mutant alleles that will be used in our future analysis. Identification of phenotypes for these alleles has proven to be more difficult and progressed more slowly than we initially thought. We have also added a second line of investigation, which studies the sumoylation of Stu2p and Bik1p. This new line of investigation has been very successful. Teaching Activities: My teaching activities in 2010 were largely in the area of teaching in the laboratory. I directly supervised the research of two graduate students, one first year rotation student (Mukta Dutta), four undergraduates (Jacob Keeling, Kayla Davis, Anthony Jones, Natalia Soulages), and three lab assistants (Taylor Reed, Madison Donica, Cassandra Camp). Two graduate students: --Annabel Alonso, Ph.D. student. She had two projects: Bik1p phosphorylation and modification of Bik1p by ubiquitin and the ubiquitin-like moiety, SUMO. --Maliha Rahman, Ph.D. student. Maliha studied the post-translational modification, sumoylation, of the microtubule binding protein, Stu2p. Dissemination: During 2010, Dr. Miller collaborated with several other researchers, including Dr. Holly Goodson at Notre Dame Univ. and Dr. Harold Hoops at SUNY-Geneseo. Dr. Miller gave four invited seminars and presented at three professional meetings: Invited seminars: 1. Oklahoma State University, Dept. of Botany, Nov. 2, 2010. 2. Oklahoma Medical Research Foundation, May 23, 2010. 3. William and Mary College, Dept. of Biology, April 16, 2010. 4. Southwestern Oklahoma State University, Chemistry Club, April 2010. Professional Meetings: 1. Miller, R.K., N. Meednu, S. D'Silva , M. Rahmen, J. Keeling, H. Hoops. 2010. SUMO interacts with several microtubule-associated proteins involved in spindle positioning. Poster at the FASEB meeting on Yeast Chromosome Structure, Replication and Segregation. Aug. 2010, Carefree, AZ. 2. Jacob Keeling and Rita Miller, 2010. Novel interactions of the sumoylation pathway with microtubule associated proteins. Oral presentation by an undergraduate student at the Oklahoma Academy of Sciences, Northeastern State University, Nov 2010. 3. R. Miller, S.D'Silva, N. Meednu, M. Rahman, H. Hoops. A Alonso. 2010. SUMO interacts with several microtubule-associated proteins important for spindle positioning. Poster at the ASCB meeting. Philadelphia, PA, Dec. 2010. PARTICIPANTS: TWO GRADUATE STUDENTS: Annabel Alonso and Maliha Rahman. ONE FIRST YEAR ROTATION STUDENT: Mukta Dutta. FOUR UNDERGRADUATES: Jacob Keeling, Kayla Davis, Anthony Jones, Natalia Soulages. THREE LAB ASSISTANTS: Taylor Reed, Madison Donica, Cassandra Camp. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: We have added the investigation of the role of the sumoylation of Bik1p and Stu2p to the project.
Impacts
In a closely related work, very good progress was made on understanding the signal transduction systems that regulate Stu2p, a microtubule (MT) binding protein that physically interacts with Bik1p. Background on Stu2p and its function. Stu2p is a plus-end MT-associated protein that is part of the kinetochore attachment complex. This complex controls the dynamic attachment of MTs to the genetic material in the mitotic spindle. Stu2p stabilizes MTs. Stu2p is essential for life. How Stu2p is regulated is poorly understood. This year we showed that Stu2p can be modified by a small ubiquitin-like moiety, SUMO, which is a novel signal transduction pathway for regulating MT function. Because the Stu2p in this study has homologues in higher eukaryotes and plants, this information will be transferable to other MT-based processes that will be important for biofuel production and other agricultural applications. Background on SUMO. SUMO/Smt3p is a conserved small ubiquitin-related modifier that is covalently attached to other proteins as a post-translational modification. SUMO regulates numerous basic cellular events, however little is known about the role that sumoylation plays in MT dependent processes. Target proteins regulated by SUMO are associated with many cellular processes, but little is known about its role in MT function. The SUMO moiety can be removed from target proteins by deconjugating proteases, Ulp1p and Ulp2p. New Results: We knew from our previous work that Stu2p can interact by two-hybrid analysis with the ubiquitin-like moiety, SUMO. To determine whether modification of Stu2p might be a normal phenomenon, in 2010 we tested for higher molecular weight forms of Stu2p in whole cell extracts made from WT cells. We found that there was a band that was easily visible about 10-15 kDa larger than the regular Stu2p. We also found that the Stu2p protein could be greatly shifted to many very larger forms by inhibiting the enzyme Ulp1 that removes SUMO from target proteins. This suggests that sumoylation is involved in the shift of Stu2p. Our analysis also suggests that this modification does not occur during the S-phase of the cell cycle. Our analysis of the eighteen Bik1p phosphorylation mutants for phenotypes is still in progress. We still plan to determine their sensitivity to the drug benomyl, genetic interactions including synthetic lethality with kar9 mutants, and MT length using fluorescently-tagged tubulin. This project has been slowed due to the success of the Stu2-sumoylation project.
Publications
- In Press/Published: Keeling, J.W.P. and R.K. Miller. 2010. Indirect immunofluorescence for monitoring spindle assembly and disassembly in yeast. In Cell cycle checkpoint control protocols. W. Lee. Editor. Humana Press, USA, Totowa, NJ. Methods in Molecular Biology series. In press.
- Submitted: Muralla, R., R.K. Miller, and D. Meinke. The heterodimeric enzyme that modifies the wobble position of cytosolic tRNAs is required for seed development in Arabidopsis and is a member of a diverse family of zinc-dependent deaminases. Plant Journal, submitted May 2010, revisions submitted Nov. 2010.
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Progress 12/15/08 to 12/15/09
Outputs
OUTPUTS: The goal of this project is to increase our understanding of how the phosphorylation of Bik1p regulates Bik1p function, which in turn regulates microtubule properties and spindle positioning. This research will lead to a better understanding of various signal transduction pathways control microtubule function in a variety of processes, including cytoplasmic processes and cell division which requires the mitotic spindle, a microtubule-based machine. This work is significant because Bik1p is a yeast protein that is conserved in many other higher organisms. Understanding how Bik1p is regulated will lead to insights into how microtubules are regulated in several cellular processes in higher organisms, which include regulation of mitosis and the mitotic spindle and cytoplasmic microtubules. Cytoplasmic microtubues serve as the tracks for delivery of cargo to various destinations within the cell. Activities: Conducting Experiments. Previously in the Miller laboratory, nine phosphorylation sites on Bik1p were identified by mass spectrometry. These nine phosphorylation sites were mutated by site-directed mutagenesis to either alanine or glutamic acid. This generated eighteen different mutant alleles that will be used in our future analysis. Teaching: During this period of the Hatch Grant, Dr. Miller developed a new graduate level course on signal transduction, BIOC6820. This course had 14 graduate students from departments across OSU. Several topics included in this course are relevant to this Hatch Grant. These topics include 1. The role that phosphorylation plays in modulating cellular processes. 2, Other post-translational modifications. 3. An introduction to microtubules. and 4. Experimental methods used to investigate cellular phosphorylation and signal transduction pathways. Many of the experimental methods discussed are applicable to the individual research projects that the graduate students are conducting. Dr. Miller supervised the independent research of four undergraduates in 2009. These students were Natalia Souglages, Cassie Camp, Jacob Keeling, and Baxton Nottingham. Dr. Miller mentored five graduate students who rotated in her lab during the Fall semester 2009. During their rotations they studied laboratory techniques related to this project. These rotations are beneficial because they broaden the background and training of the graduate student. The students learned a variety of techniques, including site directed mutagenesis and protein purification on a micro-scale. Two graduate students joined the Miller laboratory in December 2009, Annabel Alonso and Maliha Rahman. Annabel will work directly on the work described in this Hatch Grant. Maliha will work on a closely related, synergistic project. Dissemination: Dr. Miller continues to collaborate with several other researchers, including Dr. Holly Goodson at Notre Dame University, and Dr. Harold Hoops at SUNY-Geneseo. During this reporting period, Dr. Miller made presentations at the Yeast Cell Biology Meeting at Cold Spring Harbor, Cold Spring Harbor New York (August), and at the national meeting of the American Society for Cell Biology in San Diego, CA (December). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge. In a closely related work, progress was made on optimizing the procedures for purification of the 40 amino-acid cargo-binding domain of Bik1p. This construct consisted of fusion to the maltose binding protein (MBP), which was tagged with six histidines. A detail protocol was developed for purifying this construct from bacteria using nickel affinity resin. A single MBP-Bik1p was purified that contained only one contaminating band. When this Bik1p-fusion is finally purified with labeled amino acids, it will be used for structural studies by NMR in collaboration with Dr. Michael Massiah, who is located here in the Biochemistry Department at OSU. The analysis of the eighteen Bik1p phosphorylation mutants for phenotypes is currently in progress. For this analysis, we will include tests that determine their sensitivity to the drug benomyl, genetic interactions including synthetic lethality with kar9 mutants, and microtubule length using fluorescently-tagged tubulin. Preliminary data on these is expected in April 2010.
Publications
- Miller, R., S. D'Silva, S. and H. Hoops, 2009. Regulation of Bik1p. ---Poster/abstract presentation #265. Amer. Soc. Cell Biol. Meeting, San Diego, CA.
- Balla, K. S. D'Silva, H. Hoops, and R.K. Miller, 2009. Regulation of Bik1, a microtubule binding protein. ---Poster/abstract presentation, #106. Yeast Cell Biology Meeting, CSH Laboratory, Cold Spring Harbor, NY.
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