Progress 10/01/05 to 09/30/09
Outputs
OUTPUTS: The outputs for this project include conducting experiments, giving poster presentations at scientific meetings and presenting seminars, as well as a collection of yeast strains and reagents that are available to the scientific community upon request. Another major output associated with this project is the training of a number of undergraduate students in molecular techniques. PARTICIPANTS: The following individuals worked on this project between 2005 and 2009: Christiane Wiese (PI), Lingling Liu (graduate student), Sarah Slauson (graduate student), Jae Jang (undergraduate student), Chris Zahm (undergraduate student), Sona Son (undergraduate student), Allison Riederer (undergraduate student), Mitch Brey (undergraduate student), James Driver (undergraduate student). TARGET AUDIENCES: The target audience is not restricted to a particular group of people, as we all benefit from a deeper understanding of basic cell biological processes. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our research objective for this project was to contribute to our understanding of how cells are organized and what roles gamma-tubulin and its associated proteins play in this process. Our research activities contributed to the general knowledge regarding the role of XNedd1 in recruitment of gamma-tubulin to the centrosome.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: During this reporting period, we continued to perform experiments and to generate a panel of yeast (S. pombe) mutants that can be used to analyze gamma-tubulin in vivo. We involved graduate and undergraduate students in this project and taught them the basics of molecular biology and cloning as well as how to analyze proteins using sucrose gradients. PARTICIPANTS: Christiane Wiese (PI), Sarah Slauson (graduate student), Jae Jang (undergraduate student), Chris Zahm (undergraduate student). TARGET AUDIENCES: The target audience is not restricted to a particular group of people, as we all benefit from a deeper understanding of basic cell biological processes. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We extended our previous analysis of XNEDD to examine its binding partners in Xenopus egg extracts. This work revealed a very interesting connection between XNEDD and a protein involved in vesicular trafficking. Both proteins appear to be involved in the formation or maintenance of kinetochore microtubules, a special subset of microtubules that move chromosomes during cell division. We also began our analysis of gamma-tubulin in the yeast, S. pombe.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The activities on this project during the reporting period were limited to conducting and analyzing experiments, teaching, and mentoring of undergraduate students. Knowledge gained was disseminated at the annual conference of the American Society for Cell Biology. The product of this project was new fundamental knowledge, which was reported in the publication listed below.
PARTICIPANTS: Individuals who worked on the project: Christiane Wiese (project director), Lingling Liu (graduate student), Sarah Slauson (graduate student).
TARGET AUDIENCES: Everyone because we all benefit from increased knowledge of basic biological processes.
Impacts
The goals and objectives of this work are to gain a better understanding of the roles of proteins that interact with gamma-tubulin in the function of the gamma-tubulin complex and in the function of the centrosome. During the present reporting period, we focused our attention on one of the subunits (GripWD/NEDD1) whose overexpression appeared to reduce the amount of gamma-tubulin at nucleating centers. Truncation mutants of NEDD1 showed that both the N-terminus and the C-terminus interact with the centrosome. We found that depletion of NEDD1 from Xenopus egg extracts resulted in defective mitotic spindles. Importantly, we found that NEDD1 is most liely not a canonical subunit of the gamma-tubulin complex, as has previously been reported. Instead, NEDD1 is involved in microtubule organization independent of centrosomes, and the reduction in gamma-tubulin at the centrosome upon NEDD1 disruption might be caused by indirect effects.
Publications
- Liu, L., and C. Wiese. (2008). XNEDD1 is required for microtubule organization in Xenopus egg extracts. J. Cell Sci., in press.
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Progress 01/01/06 to 12/31/06
Outputs
In this study, we are using biochemical and cell biological approaches to determine how the gamma-tubulin ring complex associates with the centrosome. We have identified a peripheral subunit of the complex (XNedd) that aids in the recruitment of the complex to the centrosome. This subunit also has additional roles in the organization of microtubule structures. We are currently identifying its interacting proteins in an attempt to further understand and define the role of XNedd in centrosome function.
Impacts
Gamma-tubulin and its associated proteins are involved in the function of the centrosome, which is required for proper cellular organization. It is important for us to understand centrosme function in molecular detail, and what the role is of individual proteins in this process. Understanding the mechanism by which gamma-tubulin and its interacting proteins contribute to centrosome function may provide the means to correct centrosomal defects that result in infertility, cell death, cancer, or developmental defects
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Gamma-tubulin is an essential component of the centrosome that exists as a multiprotein complex. Little is known to date about the contribution of the gamma-tubulin interacting proteins to microtubule nucleation or organization. The goal of this work is to determine the roles of the gamma-tubulin interacting proteins in (1) the function of the gamma-tubulin complex and (2) the function of the centrosome. Towards this end, we are analyzing the effects of overexpressing the components of the complex in tissue culture cells. We find that in most cases, subunit overexpression results in cell death and/or increased mitotic index. This suggests that all of the components of the complex that we have analyzed to date are required for the function of the complex. We have focused our attention on one of the subunits (GripWD) whose overexpression appears to reduce the amount of gamma-tubulin at nucleating centers, indicating that this subunit might be involved in recruiting the
complex to the centrosome. We are generating truncation mutants of this subunit to understand how it interacts with gamma-tubulin and with the centrosome. We will use the centrosome-binding domain as bait to identify centrosomal docking factors. We will also use the truncation mutants to map the protein's gamma-tubulin interacting domain. Thus, we are beginning to dissect the roles of gamma-tubulin interacting proteins in both the function of the complex and the function of the centrosome.
Impacts
Gamma-tubulin and its associated proteins are involved in the function of the centrosome, which is required for proper cellular organization. It is important for us to understand centrosme function in molecular detail, and what the role is of individual proteins in this process. Understanding the mechanism by which gamma-tubulin and its interacting proteins contribute to centrosome function may provide the means to correct centrosomal defects that result in infertility, cell death, cancer, or developmental defects.
Publications
- No publications reported this period
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