Exploring Mechanisms of Microtubule Polarity Establishment and Regulation in Drosophila Melanogaster
Open Access
- Author:
- Yanoshak, Emily
- Area of Honors:
- Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Melissa Rolls, Thesis Supervisor
Bernhard Luscher, Thesis Honors Advisor - Keywords:
- drosophila
neurons
neuroscience
microtubules
positive regulation
TRIM9
Klp61F
microtubule polarity - Abstract:
- As cells that must survive an organism’s entire lifetime, neurons’ distinct microtubule polarity helps maintain structural integrity. Axonal microtubules are oriented such that their plus end points outward towards the cell periphery, while dendritic microtubules in neurons of different animal species are either both plus and minus end out or minus end out only. This polarity of microtubules is critical to ensure proper shipment of cell cargoes throughout the cell through the use of motor proteins. Therefore, understanding mechanisms that maintain and establish microtubule polarity are of utmost importance. Previously discovered mechanisms such as branchpoint steering and nucleation show evidence for how microtubule polarity patterns are maintained in neural dendrites. However, the lack of growing plus ends towards dendritic cell body exit points provides evidence for the existence of checkpoints at cell body exit points. Thus, I hypothesized that there is a relationship between the cell body exit points and microtubule polarity maintenance mechanisms. To examine this hypothesis, several experiments were performed. Manipulation of gene expression by RNA interference and imaging of live neurons by fluorescent microscopy allowed for the screening of candidate proteins possibly involved in positive or negative regulation of microtubule polarity. These experiments provided evidence for a positive regulator at axonal exits as RNAi of Tripartite Motif Containing (TRIM) 9, a microtubule bundling E3 ubiquitin ligase, significantly decreased the amount of plus end microtubules that move past the axonal cell body exit point. Furthermore, neural injury assays were performed by severing the axon from the remainder of the cell, causing the cell to convert a dendrite into a new axon. The percentage of plus end microtubules that exited the new axon, a phenotype of a converted dendrite, was significantly reduced in the TRIM 9 knockdown neurons. Finally, fluorescence microscopy was used to determine that TRIM 9 localizes to the axon of uninjured neurons. Together, these findings suggest that TRIM 9 has a role as a positive regulator of microtubule polarity, and also functions to help maintain microtubule polarity patterns over the course of an organism’s life span.