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Dissertation
Functional analyses of microtubule severing proteins in the central nervous system
Doctor of Philosophy (Ph.D.), Drexel University
Mar 2009
DOI:
https://doi.org/10.17918/00007775
Abstract
The configuration and behavior of microtubules are essential for the neuron to achieve its unique highly polarized cell shape. The mobility of microtubules renders a significant plasticity for their organization in developing neurons. However, only short microtubules are mobile, while long ones are stationary. To date, two microtubule-severing proteins, known as P60-katanin and spastin, have been identified to cut the long microtubules into short pieces in neurons. Despite the well-documented significance for microtubule organization during neuronal development, little evidence has been obtained regarding microtubule-severing. My dissertation investigates the functions of microtubule-severing proteins in developing neurons and the means by which they are regulated temporally and spatially. There are three specific aims. Aim 1: Investigate whether certain microtubule-associate proteins (MAPs) can provide microtubules protection against P60-katanin and identify the main protectors in the axon. Aim 2: Test whether spastin and P60-katanin participate differently in the formation of axonal branches. Aim 3: Directly test the "cut and run" model for microtubule reconfiguration during neuronal morphogenesis. Aim 1 includes two sets of studies. The first one utilized RFL-6 fibroblasts to investigate the capacity of individual MAPs to protect microtubules against P60-katanin. The second one utilized hippocampal neurons to identify the main axonal protectors. These studies revealed that tau, MAP2 and MAP4, but not MAP1b, provide strong protection for microtubules against P60-katanin. Tau was key protector in the axon. Aim 2 combined overexpression and knock-down strategies to compare the impact both of the severing-proteins on the formation of axonal branches and the microtubule array. We identified that there may be two different microtubule-severing modes ("P60-katanin-based" mode and "spastin-based" mode) that contribute to neuronal morphogenesis. Aim 3 utilized live-cell imaging to analyze the frequency of microtubule-transport corresponding to different levels of microtubule-severing. The microtubule-severing effect of extracellular signal inducing neuronal morphological changes was investigated via western blots. In addition, various spastin truncations were applied to show the microtubule-severing activity of the molecule is essential its impact on neuronal morphology. These studies validate the various predictions of microtubule "cut and run" and demonstrate the tight regulation of "cut and run" is critical to neuronal morphogenesis.
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Details
- Title
- Functional analyses of microtubule severing proteins in the central nervous system
- Creators
- Liang Qiang
- Contributors
- Peter W. Baas (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- ix, 167 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Neurobiology and Anatomy; College of Medicine; Drexel University
- Other Identifier
- 991021888956004721