August 21, 2011
Friends of UTokyo Inc. Internship Report
This summer, I spent 10 weeks in Tokyo, Japan working as a student researcher in the Ozawa Laboratory, located in the Department of Chemistry at the University of Tokyo. This lab studies various biological processes by fluorescence and bioluminescence imaging with the goal of establishing novel imaging methods and probes for applications in biochemical research. This internship was organized by MIT’s MISTI‐Japan Program and supported by Friends of UTokyo Inc.
My internship project aimed to characterize the membrane recruitment pattern of the protein Akt as a result of stimulation by grown factors. The entire project’s work would be equivalent to a doctoral program, so my summer work aimed to tackle the first portion of this project during my 10‐week stay. My direct supervisor was Dr. Yoshimura, a postdoctoral fellow in the Ozawa Laboratory.
Akt is a key protein in the Akt pathway. This pathway is responsible for regulating various cellular functions such as proliferation, cell division, and apoptosis. This pathway is of interest because mutations in the Akt pathway have been observed in various types of cancers and disease. A thorough understanding of how this pathway affects the cell is important for the creation of improved therapies to treat cancer and tumor cells. Receptors bind to growth factors and activate PI3 Kinase, which then phosphorylates PIP2 to PIP3. Akt, which resides in the cytoplasm, has a high affinity to bind to PIP3. Thus, upon stimulation, when a large amount of PIP3 is generated, Akt is recruited to the cell membrane and activated. The activated Akt then goes on to activate numerous downstream effects. Subsequent downstream cascades and resulting effect on the cell differs with each growth factor, and furthermore, even with the same growth factor, the strength of the stimulation results in differences in Akt activation. I suspected that Akt may be responsible for the differentiation of downstream signal transduction, so I decided to observe how Akt recruitment to cell membrane and activation differs according to the type of stimulation.
To do this, I planned to observe the movement of individual Akt molecules, made visible by fluorescent tags, as they localized to the cell membrane. I created five probes using Akt and the PH domain of Akt (the domain responsible for PIP3 binding) and EGFP as the fluorescent tag. I transfected CHO-PDGFR cells with the probes, and observed the cells with confocal microscopy to confirm fluorescence and targeting. I then used Total Internal Reflection Microscopy (TIRF) to observe individual Akt probes. Through this work, I noticed that the membrane localization time increases after stimulation, and that there is little difference in the number of probes before and after stimulation, and very little difference in behavior between Akt and PH. There is a suggestion that stimulation may cause a change in binding environment or conformation of PIP3, and that the PH domain may have a differentiation function in addition to PIP3 binding. Further investigations should provide more evidence to reveal a molecular explanation of Akt recruitment patterns. The Ozawa laboratory will continue to work on this project after I end my internship.
Overall, the internship was a great success, and I was able to gather a lot of data and move the project forward during the 10 weeks. The results I found were intriguing, and will be interesting to pursue further. Through this internship, I gained more research experience and had many opportunities to work with and interact with other researchers, both which will help me be a better researcher as I enter graduate school next year. I enjoyed being able to spend two and a half months in Japan. My most enjoyable time was spending my weekends and free time exploring Tokyo and all over Japan. The only part that I had to bear was the heat and humidity, but that was expected. Overall, this summer was a great experience, both personally and academically.
I would encourage anyone looking into doing research in Japan, either short-term or long-term, to seriously consider going for it. You will gain many skills, visit breathtaking places, and have a great time. Japan is an amazing country, and the University of Tokyo is one of the best research institutes in the country.