When I tell people that I worked in a research laboratory during the summer, they tend to look at me as if I’m crazy. “You did what? You want to do that with your summer break? You enjoy that?” Most students who want to work in a science field, either research or medicine, need some type of laboratory experience in order to be competitive applicants for graduate schools. The truth is though, that’s not why I chose to spend my summer working in a neuroscience lab at the NYS Psychiatric Institute. I chose to because I do enjoy it. I love science and I love laboratory research. This summer was a chance to act on that love. Thanks to Stern’s Biology department, I was able to work for Dr. Zachary Freyberg of Columbia University exploring multiple projects relating to various fields.
One project I worked on consisted of analyzing images of mitochondria taken using electron microscopy. A technique called cryo electron microscopy was used, producing images of these organelles that were exceptionally clear and detailed. In order to be imaged cells are traditionally fixed in place using harsh chemicals that essentially kill the cells. Instead, these cells were plunge-frozen in liquid ethane, which freezes them in the exact state they were in when alive. The images we saw appeared as if we were looking at live cells, with amazing clarity. Many things that had not been previously imaged became visible. In order to see the structure of some of these structures, we took tomograms: Imaging many slices of the picture in order to piece together a 3-dimensional image of the organelles and their components.
My part of the project initially entailed looking through this data to identify large unknown structures in the mitochondria. There are many of these in each mitochondrion, and they seem to sit near or on the cristae. However, this goal was set aside when we noticed smaller structures dotting all of the cristae. They were small and round, sitting along the cristae like beads on a string. Dr. Freyberg hypothesized that these were ATP synthase dimers, which play a critical role in the production of ATP and cellular respiration. They sit on the cristae because in order to function, they need the difference in proton concentration that exists between the matrix of the mitochondrion and the intermembrane space.
Once we identified the position of enough of these, we obtained a sub-tomogram average, a computational device used to analyze the data and essentially account for missing data in the 3D structure. This gave a high- resolution image of the structure we are still attempting to determine. If all goes well, we will see the structure of ATP synthase. The hope is that by imaging the structure of these enzymes in healthy mitochondria, we can eventually do the same in mitochondria of cells with various diseases, which could give insight to their cause on a structural level.
Another aspect of my work in the lab involved genetic constructs that code for various molecules that fluoresce inside a cell, called reporters. These biosensors can report about a number of substances inside a cell, including insulin and calcium. I have been transforming bacterial cells to contain these sequences, and growing them so that they replicate the DNA. We now have stocks of DNA for many of these constructs. The calcium and insulin reporters, along with others, will be used in various projects that involve imaging cells. The insulin reporter is used when studying pancreatic cells, which secrete insulin.
By growing cells that contain this gene, we can expose them to different conditions and observe the secretion of insulin. The calcium reporters, which can be targeted to specific organelles, will help us observe how calcium works as a secondary signaling molecule inside the cell. For example, we can observe the location of calcium before and after a cell is depolarized, which happens in neurons and pancreatic cells in response to a stimulus.
These reporters can give us more insight into how these various molecules function inside cells.
Hopefully I will continue to work on these projects part-time during the coming year. I had a wonderful experience in the lab, and I can’t wait to see where it takes me. Despite what people think, I’m so glad this is how I decided to use the summer break.
Sources
Dobro, Megan J., Linda A. Melanson, Grant J. Jensen, and Alasdair W. McDowall. “Plunge Freezing for Electron Cryomicroscopy.” Methods in Enzymology. Vol. 481. N.p.: Academic, 2010. 63-82. ScienceDirect. Web. 11 Aug. 2015.
Strauss, Mike, Götz Hofhaus, Rasmus R. Schröder, and Werner Kühlbrandt. “Dimer Ribbons of ATP Synthase Shape the Inner Mitochondrial Membrane.” EMBO J The EMBO Journal 27.7 (2008): 1154-160. PubMed. Web. 11 Aug. 2015.
Bartesaghi, Alberto, and Sriram Subramaniam. “Membrane Protein Structure Determination Using Cryo-electron Tomography and 3D Image Averaging.” Current Opinion in Structural Biology 19.4 (2009): 402-07. ScienceDirect. Web. 11 Aug. 2015.