- The generation and characterization of the GFP-CENTRIN2 transgenic mouse line
- A study of centrosome dynamics in migrating neurons
- A study of migrating neurons from the Doublecortin knock out mice
The underlying theme of my research was: Being that many human mental disorders arise from a problem in neuron migration, an important step in brain development, how is neuron migration controlled in both the normal and diseased brain?
Cells have a certain structure that corresponds to their function. For example, neurons have an extensive network of branching dendrites and axons that allows them to communicate with other cells. In order to form and maintain this network, neurons, like other cells, need an internal framework or skeleton. Much like a whole organism, cells use their cytoskeletons to provide a physical structure that can keep the cell in proper form while still allowing enough flexibility for it to move around or remodel if it needs to. The cytoskeleton is made up of flexible tubular fibers called microtubules, which are formed from a structure called the centrosome. By analogy, if the microtubules were the spokes of a wheel, the centrosome would be the hub, and as the hub, the centrosome is key to the organization of the microtubules.
Because the centrosome plays such an important role in the cell, especially during cell movement, my lab wanted to create a tool that would allow us to see the centrosome in living cells so we could watch how its position changes as the cell moves. Seeing the centrosome in live cells would tell us a lot about how the cytoskeleton is organized.
To this end I and others in my lab created a transgenic mouse line that has inserted into its genome an artificial gene that causes the centrosome of every cell of the mouse to be a fluorescent green when viewed under a fluorescent microscope. The mice we made, called the GFP-CENTRIN2 mice, were perfectly healthy and indeed had a fluorescent green centrosome in every cell of the body. These animals should be a valuable tool for the cell biology community.
Higginbotham, H., Bielas, S., Tanaka, T., and Gleeson, J.G. (2004). Transgenic mouse line with green-fluorescent protein-labeled Centrin 2 allows visualization of the centrosome in living cells. Transgenic Res 13, 155-164. PDF
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As mentioned above, the cytoskeleton plays an important role in giving cells a shape that facilitates their function. Perhaps this is nowhere more evident than in neurons, which after being born from their mother cells, must travel over large distances within the developing brain to reach the locations where they will mature and establish connections. For efficient travel, or migration, the neurons need to adopt a slender, elongated shape, adapted for moving easily through a crowded environment, like this picture shows:
Not surprisingly, neurons migrate to very specific locations in the developing brain rather than wandering randomly. The path that the neurons take to their destination is marked by chemical cues, i.e. proteins that are secreted by other cells into the tissue and that act like attractants or repellants for the neurons. My studies focused on how the signals from these cues are translated into changes in the cytoskeleton that allow a neuron to move in the right direction, either toward the attractant or away from the repellant. Among other things, I discovered that as a neuron grows out a leading process, the centrosome moves into the process. If the centrosome fails to move into the process, a neuron’s ability to move away from a repellant is severely impaired.
Here is a movie of a neuron taken from a GFP-CENTRIN2 mouse. It’s migrating in a gel in a dish in this movie, but its behavior is similar to what one would see in the brain. The arrow points to the green fluorescent centrosome. The frames of the movie are taken every 5 minutes.
Higginbotham, H., Tanaka, T., Brinkman, B.C., and Gleeson, J.G. (2006). GSK3beta and PKCzeta function in centrosome localization and process stabilization during Slit-mediated neuronal repolarization. Mol Cell Neurosci 32, 118-132. PDF
My graduate mentor’s (Dr. Joseph Gleeson) lab studies brain development. Dr. Gleeson is a pediatric neurologist, and he works with children who have various genetic brain disorders, including autism, Joubert syndrome and lissencephaly, among others. One of the goals of his lab is to discover the genetic basis for these disorders so that we can create the best treatments for the children and possibly even develop cures.
As mentioned, one of the disorders the Gleeson lab is interested in is lissencephaly, a disease marked by a brain surface that lacks the normal gyrations and folds. Children with lissencephaly can have severe developmental problems, including mental retardation. Lissencephaly (smooth brain) is caused by a defect in neuron migration.
One of the genes that has been implicated in this disease has been named Doublecortin, for reasons too complicated to explain here, but you can read more about it here.
When I arrived in Dr. Gleeson’s lab, one thing that was still unknown was how mutations in Doublecortin caused neurons to be unable to migrate. A key insight into this question came with Dr. Gleeson’s discovery that the doublecortin protein helps to stabilize microtubules, the core components of the cytoskeleton. Using a knockout mouse where Doublecortin was deleted, I analyzed the migration of neurons that lacked the doublecortin protein and found that the mutant neurons grew excessive branches off of their leading processes, causing them to stall in their forward movement.
Hopefully, this discovery together with many others will get us closer to a full understanding of how these diseases work and what interventions we can use to correct them.
Koizumi, H., Higginbotham, H., Poon, T., Tanaka, T., Brinkman, B.C., and Gleeson, J.G. (2006). Doublecortin maintains bipolar shape and nuclear translocation during migration in the adult forebrain. Nat Neurosci 9, 779-786. PDF
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