Investigating Mitotic Spindle Orientation

Jessica A. Lioy, a PhD Student in the Genetics program, takes us through her first rotation project.

My first rotation of three necessary for my PhD program was completed in Dr. Christopher Brownlee’s laboratory at the Center for Molecular Medicine at Stony Brook University. I had the amazing opportunity to work on a mechanism known to influence the development of cancer. Besides working on an incredible and promising field of cancer research, I had the opportunity to be mentored by Dr. Brownlee.  Dr. Brownlee received his doctorate in Cellular and Molecular Medicine from the University of Arizona College of Medicine. He then completed his postdoc at the University of California-Berkeley in Rebecca Heald’s laboratory. He has received numerous awards including National Science Foundation (NSF) funding which is a huge deal in STEM. I was honored to be accepted into his lab. I loved this opportunity so much I wanted to share my project I was blessed with, with you all! I hope to explain the overall importance of my project and why you should care that Dr. Brownlee is at Stony Brook University carrying out his work. 

Cells go through a process called cell division, this is how cells replicate to give rise to more cells. This happens during development but also when you get an injury such as a scrape. Your cells will divide to make more cells to fix the damage. Of course, there are many highly complicated mechanisms associated with development and tissue repair but I will focus on a brief overview of cell division.. There are two types of cell division: asymmetric division and symmetric division. Asymmetric division maintains the dividing pool in one cell while also generating a cell that can differentiate into some other cell (such as a skin or heart cell). Symmetric division is when a cell divides into two identical cells which is mostly used in development. Division of one cell into two cells is controlled by a complex called the mitotic spindle. The mitotic spindle is made up of hundreds of proteins.

Figure 1: Mitotic Spindle in Symmetric Division

Pictured in Figure 1, you can see the mitotic spindles (green cylinders) at each end of the cell. The spindles will pull apart the chromosomes/DNA (in blue) as the single cell divides and becomes two cells in the end. Each cell will have an equal amount of DNA at the end of division. 

During the cell cycle there are two mitotic spindles made and they migrate to opposite ends of the cell. Some questions in the lab are: what happens if there is only one mitotic spindle? What happens if one or two of the hundreds of proteins of the spindle complex are missing, broken or dysfunctional? Well, I study one of the many mechanisms known to be involved in orienting the mitotic spindle into the right place for division and how cells look when it is wrong! Why should you care if the mitotic spindle may or may not be in place? Well, if the mitotic spindle is not in the correct place, the DNA does not get separated properly into the two cells. This can encourage cancer development or cell death which gives rise to many known diseases. Likewise, if there is only one spindle, then the cell divides incorrectly and can lead to cancer or cell death. 

Shown in Figure 2 is a cancer cell I was able to image with a microscope where the DNA (blue) is being pulled into three different directions. Granted, cells have many mechanisms for regulating and checking that everything is all good before dividing. Those mechanisms are, however, not perfect. Another possibility is the cell may have mutations in genes that regulate those mechanisms. The cell can be cancerous where those incorrect mechanisms are turned on more/upregulated to help generate more cancer cells therefore encouraging cancer progression. It is all a game of survival of the fittest which you may have heard in your biology classes. Cancer is very good at making messed up cells masters of replicating quickly and taking over tissues. Cancer is very good at what it does. This is obviously the problem for humans and many species. The thing about cancer is that it is not one disease, it is many diseases as cancer is not the result of a single physiological dilema. Literally anything that happens in the cell can go wrong and result in cancer. If you’re ever wondering why there is no cure for cancer, well thats why. Cancer is a monster of a cluster of diseases that just keeps changing and surprising daily. And why/how things go wrong is the whole purpose of cancer research. 

Figure 2. Asymmetric Division in a Cancer Cell

For  my project, there were two parts:
In the first part, I wanted to determine the role of the protein (KPNA2) in the orientation of the mitotic spindle. I could tell you a whole bunch of science gibberish on how I planned to do that, but I will spare you. Overall, I added some small molecule inhibitors to block a process known to control KPNA2’s location in the cell. Based on the location of KPNA2, the spindle should or should not form. Or the spindle will form in the wrong places. We are showing the importance of KPNA2 in spindle orientation. I then got to visualize this all with a microscope and pretty colors that come from using antibodies that have fluorescent molecules attached to them.

Figure 3. Antibody Labeling of Normal Cells

In Figure 3 I show two ordinary cells about to divide. The DNA is lined up in the center of the cells (blue) and the spindle poles (end of the red lines) are in place and you can see the outline of the cell based on where the green is located (green was labeling another protein). A whole bunch more went into moving KPNA2 and some other associated proteins of KPNA2 around the cell. We planned to do what I’ll call a rescue mission to recover the correct orientation of the spindles after messing it up. Unfortunately, I did not make it that far in my ten week rotation. 

Figure 4. African Clawed Frog

In the second part of my project, I used the African clawed frog, Xenopus laevis. It is simply some frog that I must admit is quite large and hard to handle. No worries here though, no frogs were harmed or killed! This part of my project is incredible, at least I think so. The goal here was to control the location of the mitotic spindle and its components in artificial cells made from the frogs eggs. Using a microfluidic approach, one can stick lipids together to make an enclosed cell-like structure. We call this an artificial cell because like normal cells, our artificial cell uses lipids to make its walls. We can essentially print out a cell with nothing in it. This is exactly what we want! We then take out the proteins we want from the eggs via protein purification and inject those into the artificial cells. The artificial cells allow us to remove all other proteins present in a normal cell thereby allowing us to only look at the proteins we want to understand how they interact. This method can clearly support our idea that the proteins we are investigating (ex. KPNA2) are the only ones necessary for mitotic spindle construction and function. Once the proteins are injected into the artificial cell, we can control the location of the proteins and thereby the assembly of the spindle using a blue light technology developed by Dr. Brownlee and his peers. We use this technology to move proteins of interest around the artificial cell.  We can then directly show the necessary proteins for both the spindle formation and orientation. We are able to choose the location of the mitotic spindle based on what proteins we add to the artificial cell and where we put them thanks to this blue light technology. This method of control is an incredibly important concept in this project. I believe this technology developed by Dr. Brownlee and his peers to be revolutionary in the field of molecular biology.

References:
IGCiencia. (2009, July 01). Cartoon of Chromosomes on Mitotic Spindle. Retrieved January 12, 2021, from https://www.eurekalert.org/multimedia/pub/15050.php?from=140060
African Clawed Frog. (2020, December 30). Retrieved January 22, 2021, from https://upload.wikimedia.org/wikipedia/commons/b/b4/Xenopus_laevis_02.jpg

Author: sbugwise

We are the Graduate Women in Science and Engineering group at Stony Brook University and we are dedicated to supporting women in STEM fields.

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