Why Move It When You Can Make It? Drosophila Neuronal Microtubule Polarity Requires Local Microtubule Nucleation

Open Access
Author:
Nguyen, Michelle Marie
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
August 14, 2013
Committee Members:
  • Melissa Rolls, Dissertation Advisor
  • Melissa Rolls, Committee Chair
  • Wendy Hanna Rose, Committee Member
  • Lorraine C Santy, Committee Member
  • Graham Hugh Thomas, Committee Member
  • Timothy J Jegla, Special Member
Keywords:
  • microtubules
  • neuronal polarity
  • Golgi
  • axon initial segment
  • Drosophila
  • centrosome
  • ankyrin
Abstract:
The polarization of neurons into axons and dendrites is essential in building functional neuronal circuits. The arrangement of microtubules is an important factor in neuronal polarity, as microtubules are used for long-range trafficking of vesicles and other cargo, cytoskeletal structure, and synaptic growth. In vertebrate neurons, axonal microtubules are primarily plus-end-out, while dendritic microtubules have mixed polarity. In contrast, Drosophila melanogaster has a simpler arrangement of neuronal microtubules. In Drosophila neurons, microtubules have opposite orientation in axons and dendrites: axons have microtubules with plus ends distal to the soma, while dendrites have minus ends distal to the cell body. Due to this simplicity, Drosophila provides a good model for the study of neuronal microtubule polarity. The commonly accepted model for how neuronal microtubules are organized is that microtubules are nucleated from the centrosome in the cell body, severed into small pieces, and transported down axons and dendrites via microtubule sliding. Using Drosophila, this model was investigated in dendritic arborization neurons. First, the role of the centrosome, an organelle that is the primary microtubule nucleator and organizer in dividing cells, was tested, since it is a key component of the microtubule sliding mechanism. Immunostaining experiments showed that the centrosome was inactive in neurons compared to mitotic cells. Additionally, the increase in microtubule nucleation after axon severing did not originate from the centriole, a core centrosomal component, and neither disruption nor ablation of the centriole yielded any changes in microtubule dynamics in the neuron. Second, the existence of an axon initial segment in Drosophila was directly assayed. Previous studies have found that the axon initial segment serves as a cue for axon recognition and sorting by molecular motors, and thus the axon initial segment could play a role in microtubule sliding. However, although FRAP experiments confirm the presence of an ankyrin-based axon initial segment in Drosophila, disruption of this plasma membrane diffusion barrier did not change microtubule orientation in axons. Since the centrosome is not a major microtubule-organizing center in neurons, it stands to reason that local microtubule nucleation outside of the soma likely drives microtubule organization in neurons. A likely candidate involved in this mechanism is gamma-tubulin, as it is the core nucleation protein that is essential for microtubule nucleation. To examine the function of gamma-tubulin, microtubule dynamics were analyzed in gamma-tubulin mutants using the plus end-tracking protein EB1. gamma-tubulin mutant animals displayed disrupted microtubule dynamics as well as altered microtubule orientation in axons and dendrites. These results fit with the model of local microtubule nucleation, as mixed microtubule polarity would not be expected if microtubules were nucleated in the cell body. Localization experiments further support the local microtubule nucleation model, as immunostaining and fluorescence localization experiments showed specific localization of gamma-tubulin in axons and dendrites, such as at dendrite branch points. This local nucleation does not involve the Golgi, because Golgi disruption and localization experiments did not correspond to gamma-tubulin results. The work presented here demonstrates that local microtubule nucleation is the predominant mechanism contributing to neuronal microtubule polarity in Drosophila. This type of nucleation requires tight regulation of gamma-tubulin and is independent from the centrosome and the Golgi. Future work elucidating the factors that localize and regulate gamma-tubulin will be crucial in determining how correct microtubule polarity is established and maintained in neurons.