PI: Barry Willardson

Students Involved

1. Grant Ludlam. The MEG award helped support Grant’s work from January 2015 through August 2016. During this time Grant has worked on three projects. He investigated the role of Ric8A in the folding of the G protein a subunit and its assembly into the G protein heterotrimeric complex. Unfortunately, his results showed that deletion of Ric8A in the photoreceptor cells of mice did not affect G protein signaling. As a result, he changed his research focus to determining the structure of the BBSome core complex. He assisted a graduate student, Takuma Aoba, in this work. They used a combination of chemical cross-linking coupled with mass spectrometry, unnatural amino acid cross-linking and electron microscopy to probe the structure. This work is now being prepared for publication and Grant will be a coauthor. He also worked with Takuma Aoba to determine the role of the chaperonin CCT and its co-chaperone phosducin-like protein in the assembly of the mTOR kinase complex. He reported this work in poster form at the Experimental Biology annual meeting in San Diego April 2016 and a paper is also being prepared for publication. Thus, Grant will be a co-author on two quality publications as a result of his undergraduate research. In August 2016, Grant joined our graduate program and is continuing his work on these project. He had also been accepted to the graduate program at Texas A&M university, but chose to remain at BYU to remain close to his fiancé who is working in Salt Lake.
2. Nicole Tensmeyer. The MEG award helped support Nicole’s work from January 2015 through December 2015. Nicole spent this time developing CRISPR gene editing methods for use in our lab. She subsequently decided to remain in the lab for a Master’s degree, while her husband finished his PhD in computer science. She has since used CRISPR to delete PhLP1 from cells and measure the effect on mTOR complex assembly and she is working to conditionally delete CCT from cells. Her work will be part of a publication on the role of PhLP1 and CCT in mTOR complex assembly.
3. Chance Clinger. The MEG award helped support Chance’s resear h from January 2016 through December 2016. Chance has worked closely with Nicole to determine the role of PhLP1 and CCT in mTOR complex assembly. He has been accepted to dental school and will begin in the fall of 2017.

Findings of the Project

1. Using Cre-lox methods, we deleted the Ric8A gene specifically from rod photoreceptor cells in mice and measured the effects on the G protein pathway of vision. Unfortunately, we saw no change in electroretinogram measurements of the light response in Ric8A knockout mice compared to controls, indicating that the G protein cascade of vision was not dependent on Ric8A. This result led us to surmise that Ric8A was not required for folding of the visual G protein, transducing. As a result, we have dropped our work on this project. Grant Ludlam contributed significantly to this effort along with a graduate student Madhura Dhavale.
2. We have used a combination of chemical cross-linking coupled with mass spectrometry (CLMS, unnatural amino acid cross-linking and electron microscopy to probe the structure of the Bardet Biedl syndrome (BBS) core protein complex consisting of BBS proteins 2, 7 and 9. A structural model generated from these results is shown in Fig. 1. This model places the R632P mutation in BBS2, that has been linked to BBS disease, at the interface between BBS2 and BBS9 (Fig. 2A). Moreover, binding measurement show that the interaction between BBS2 and BBS9 is disrupted by the R632P mutation (Fig. 2B). These findings suggest that BBS disease caused by the BBS2 R632P mutation stems from the inability of BBS2 to interact with BBS9 and form the BBSome core complex. Grant Ludlam contributed significantly to this effort along with a graduate student Takuma Aoba. Grant will be a co-author on this study, which will be published in 2017.

3. We have investigated the contributions of the chaperonin CCT and its co-chaperone PhLP1 in the folding of the mLST8 subunit of the mTOR complexes and found that both CCT and PhLP1 assist in the folding of mLST8. We have used CL-MS and cryo-EM to determine the structure of mLST8 in the CCT folding cavity (Figure 3). We found that mLST8 sits deep within the folding cavity near the CCTg subunit in a near native conformation. These results show that CCT traps mLST8 and folds it into a conformation that can interact with the mTOR kinase and form mTOR complexes. Both Grant and Nicole have contributed significantly to this work and will be coauthors on a publication in 2017.

Achievement of the academic goals

We have two studies ready for publication that involve undergraduates supported by the MEG award. These studies should have significant impact on their respective fields. This level of productivity must be considered a success for this type of challenging biochemical and structural work. The BBSome studies were the basis of a major NIH grant that was awarded January 2016, and the mTOR complex assembly studies are the basis of another NIH grant to be submitted in June 2017.

Evaluation of the mentoring environment

Each student supported by the MEG award has learned a great deal of science and has contributed in a meaningful way to the success of the projects. This experience has and will help them achieve their career goals. Grant Ludlam made major strides toward becoming an excellent scientist during this period. He will have two major publications to his name from his undergraduate work, and the experience has brought him to the point that he can direct major projects even though he is a new graduate student. Nicole Tensmeyer has learned important CRISPR techniques and will be a co-author on an impactful publication. Combining this experience with her Master’s work here will allow her to be accepted to one of the top PhD programs in the nation. No less importantly, Chance Clinger has learned many laboratory skills and has achieved his goal to attend dental school. From my perspective, I have thoroughly enjoyed working with these students and appreciate their efforts to contribute to our work. I consider them friends and look forward to their future successes. Our mentoring environment is good and it is achieving its goals.

Use of budgeted funds

Of the $20K, approximately 80% went to student salaries, allowing the students to commit the time to research that they would have otherwise committed to a part-time job to support their education. This is an ideal situation for them. They gain valuable experience in their academic field while being paid. The other 20% of the funds went to animal care costs that were a part of the Ric8A project. Additional funds from external grants, several times greater than the amount of the MEG funds, also supported this work by paying for supplies and the stipends of the graduate students who mentored the MEG undergraduates.