Chris Lopez ’13: Dimensions Fellow in Pharmacology
On Sunday the membranes were cut into pieces and put into primary antibody and rocked overnight. Monday I washed these membranes and put them into secondary antibody. When these were done rocking I stained the AKAP1 bands with fast green. I then imaged the membranes.
I also ran eight SDS-PAGE gels. Four of the gels contained the input samples (the four tissues and their corresponding dilute samples). The other four were run like the ones last week; each tissue had its own gel with the NM samples. Once the gels were done running I transferred them onto nitrocellulose membranes. Afterwards I cut the membranes into three pieces; top, middle, and bottom. I stained the membranes with Ponceau S and imaged them before rocking them in primary antibody overnight.
Tuesday I put the membranes into secondary antibody and imaged them. I did band quantitation on the images and then I worked on the presentation I was to give on my project. The presentation is on Wednesday and I will present in front of the members of my lab.
Wednesday I presented to the members of my lab. I gave a powerpoint presentation and told them what my project was about. I also presented data and possible interpretations of what my results meant.
Thursday I made updates to my powerpoint and worked on the rest of the data I had to quantitate.
Friday I finished the quantitation on the data from the coomassie stained membranes. I made photocopies of all of my notes and printed all of the images I have scanned since the beginning of my project. I labeled all of the images and scanned them. I kept the notes and the images in a notebook for my use and made another notebook for the lab. I said goodbye to all of the lab members and thanked them for welcoming me into the lab and being great to work with.
The data I obtained was very interesting. I found that all of the tissues were able to pull-down PKA with the different regulatory subunits: RI, RIIalpha, and RIIβ. However, RI was bound much less. I also found that the ST-DE double mutation showed a significant decrease in regulatory subunit binding compared to the other samples. This information could be important for future experiments. If the phosphomimetic mutations that I made have this much of an impact, then actual phosphorylation at the 315, 322, and 328 positions (S315, T322, T328) could have an even greater impact. If there is a decrease in PKA binding to AKAP1, then there would be a decrease in the amount of Drp1 that could be phosphorylated (PKA can phosphorylate Drp1). Drp1 is a protein that becomes active if it is dephosphorylated. In the dephosphorylated state Drp1 causes mitochondrial fragmentation through GTP hydrolysis. Therefore, if we can influence the amount of PKA that can bind to AKAP1 on the outer mitochondrial membrane, then we could influence the amount of Drp1 phosphorylation and therefore mitochondrial fragmentation.
My research pretty much laid the groundwork for other research. More research will be done to see the affect that actual phosphorylation at the phosphosites (as opposed to the pseudophosphorylation mutations I created) has on mitochondrial morphology.
Overall, this fellowship has been one of the greatest experiences I’ve had in my time at Cornell College. I have made great strides in my lab techniques and lab knowledge. I have also learned a tremendous amount about mitochondria and protein-protein interactions. I have met some great people in the lab and I am very grateful that I was given this opportunity through the Dimensions program at Cornell. This has shown me that hard work really does pay off and I accredit much of my work ethic to taking classes at Cornell. This research experience has given me a lot of insight as to what graduate school is like and what research is like as a career. I have only really considered going to medical school after graduating for Cornell College but I am now considering graduate school as an option as well. My experiences at Cornell and the classes I have taken there really prepared me for lab work at the University of Iowa. I will keep in touch with Stefan and Ron about what steps will be taken next in regards to the research and what is to come next.
Here are some pictures of my final week of research. A couple of them are during my presentation and another is when some of the lab members and I had a nice lunch outing at Short’s in downtown Iowa City (the burgers were delicious!).