Barry M Willardson, Chemistry and Biochemistry

Students Involved

1. Devon Blake. Devon was the most productive student I have had in my 18 years at BYU. The MEG award helped support his work from January 2013 through May 2014. During this time, Devon co-authored two papers on the effects of cell-type specific deletion of phosducinlike protein 1 (PhLP1) in rod and cone photoreceptor cells (Lai et al. (2013) J. Neurosci. 33, 7941-7951, selected by the Faculty of 1000 as a high-impact paper and Tracy et al. (2014) PLOS One in press). He reported this work in poster form at an international meeting of the Federation of American Society of Experimental Biology on the Biology and Chemistry of Vision in Steamboat Springs, CO June 2013. He also was acknowledged for his contributions to a study of the structures of chaperone-bound intermediates in the process of G protein complex formation (Plimpton et al. 2014 Proc. Natl. Acad. Sci. under final review). He applied to many of the best graduate schools including Harvard, Yale, NYU, Duke, Univ. of Pennsylvania and the Univ. of North Carolina and was accepted to them all. He chose to attend North Carolina because of their cell signaling expertise and because his wife was also accepted in a graduate program there.
2. Tanner Shaw. Tanner also had a productive time in my lab. The MEG award helped support him from January 2013 to Dec. 2013. He co-authored one paper on the role of programmed cell death protein 5 (PDCD5) in protein folding by the cytosolic chaperonin CCT which was selected as paper of the week by the Journal of Biological Chemistry (Tracy et al. 2014 J. Biol. Chem. 289, 4490-4502). Tanner applied to medical school but was not accepted in the programs he wanted. As a result, he took a laboratory technician position at Baylor University School of Medicine and reapplied to medical schools this year. His work in my lab was very instrumental in his landing the job at Baylor.
3. Grant Ludlam. Grant has been partially supported by the MEG award from March 2014 to present. He is working on the original project of the MEG award to determine the structures of the Bardet-Biedl syndrome complex (called the BBSome) and the chaperonin complex responsible for assembly of the BBSome. He has shown very good lab skills and has purified the BBSome core complex and is in the process of purifying the second complex. He has helped a graduate student, Takuma Aoba, to probe the structure of the BBSome complex with chemical crosslinking coupled to mass spectrometry. The structure of the complex is also being examined using single particle cryo-electron microscopy by our collaborators in Madrid. Together these two structural techniques should give us a good view of the structure of the BBSome core complex. This work will produce a high quality publication, like those of Devon and Tanner, in which Grant will be a co-author.
4. Nick Taylor. Nick has been partially supported by the MEG award from March 2014 to present. He is working on a project to understand the chaperone role of the protein Ric8A in the folding of the G protein α subunit in vivo. Using the Cre-lox system for conditional gene deletion, he is specifically removing the Ric8A gene from rod photoreceptor cells in the retina, and he will measure the effects on the G protein signaling pathway responsible for vision. He is in the final breeding stages and will have mice with the correct genotypes in February. This study will determine in vivo if Ric8A is required for the folding and function of the G protein α subunit.

Findings of the project

1. Using Cre-lox methods, we deleted the PhLP1 gene specifically from rod photoreceptor cells in mice and characterized the effects on the G protein pathway of vision. The effects were profound. There was a 50-fold decrease in formation of the G protein βγ dimer and a 10-fold decrease in light sensitivity in the rod cells of these mice. There was also a 20-fold reduction in the complex between the regulator of G protein signaling 9 (RGS9) and the G protein β5 subunit (Gβ5) that is responsible for turning off the light response. Accordingly, there was a 15-fold decrease in the rate of photoresponse recovery. These results show that PhLP1 is required for Gβγ and Gβ5-RGS9 dimer formation in vivo. This work was published in the Journal of Neuroscience and was selected as a Faculty of 1000 high impact paper. Devon Blake contributed significantly to this work.
2. Using these same Cre-lox techniques, we deleted the PhLP1 gene specifically from cone photoreceptor cells in mice and observed similar losses in cone-mediated vision at high light intensities. Cone Gβγ and Gβ5-RGS9 dimers were reduced and there was a 27-fold decrease in light sensitivity and a 38-fold delay in photoresponse recovery. These findings confirm that PhLP1 is required for G protein complex assembly in cones as well as in rods. The paper describing this work is now in press in the journal PLOS ONE. Devon Blake contributed significantly to this work.
3. We discovered and characterized the interaction between PDCD5 and CCT, showing how PDCD5 interacts with CCT. These findings suggest that PDCD5 is a regulator of protein folding by CCT and suggest a link between the tumor suppressor function of PDCD5 and protein folding. This work was published in the Journal of Biological Chemistry and was selected as paper of the week. Tanner Shaw contributed significantly to this work.
4. We have isolated the BBSome core complex consisting of BBS2, BBS7 and BBS9. We have sent the complex to our cryo-EM collaborators in Madrid for a structural analysis. Initial images looked good with distinct homogeneous particles. We will have a reconstruction available soon. We have performed chemical crosslinking of the complex and identified multiple crosslinks between BBS2 and BBS7 in a region that is predicted to form a coiled-coil between the two subunits, indicating a region of contact between these two subunits. This study will be ready for publication in a major scientific journal by the end of summer 2015 and Grant Ludlam will be a co-author.
5. One of the goals of the project was to determine the structure of the chaperonin-like protein complex responsible for assembly of the BBSome. We have struggled to isolate the complex, but we have now succeeded in expressing the entire complex consisting of the BBS6, BBS10 and BBS 12 subunits in HEK-293T cells and we are purifying the complex. Once the purification is complete, we will begin the structural analysis. We anticipate publishing this study in 2016 and Grant Ludlam will be a co-author.
6. We are breeding mice for cell type specific deletion of Ric8A from rod photoreceptor cells. We predict that Ric8A will be needed for the folding and cellular expression of the G protein α subunit. If this hypothesis is correct, we will see a loss of light sensitivity in Ric8A depleted rod cells. Such a result would show that Ric8A is required for G protein α subunit folding in vivo. Nick Taylor has contributed significantly to this work and will be a co-author when the work is published.

Achievement of the academic goals

We were able to publish three high quality studies involving the undergraduate students supported by the MEG award. This level of productivity must be considered a success for this type of biochemical and physiological work. Still we have made less progress toward the structural characterization of the BBSome than we would have liked. However, we have laid the necessary groundwork and are now ready to finish the BBSome projects. In fact, the BBS studies are the basis of a major NIH grant submission in March 2015.

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. Devon Blake is a perfect example. He was a bright and motivated student with very little experience when he joined the lab. He left the lab having coauthored two quality publications and contributed to a third. He had an opportunity to present his work at a respected scientific meeting. He was accepted to some of the best graduate programs in the nation and was very well prepared to enter graduate school. This is exactly what the MEG program was intended to accomplish. The program also worked for Tanner Shaw even though he has not yet achieved his goal of attending medical 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 idea 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 big part of these projects. 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.