Dendritic Microtubule Polarity is Maintained by Kinesin-2 Steering and Positioning of Nucleation Sites by Endosomal Wnt Signaling Proteins
Open Access
- Author:
- Weiner, Alexis
- Graduate Program:
- Molecular, Cellular and Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 13, 2019
- Committee Members:
- Melissa Rolls, Dissertation Advisor/Co-Advisor
Melissa Rolls, Committee Chair/Co-Chair
Timothy J Jegla, Committee Member
Lorraine C Santy, Committee Member
William O Hancock, Outside Member
Melissa Rolls, Program Head/Chair - Keywords:
- neurons
microtubules
nucleation
APC2
gTub
dendrite - Abstract:
- Cells rely heavily on an intact and tightly organized microtubule cytoskeleton to facilitate vital functions including cell division, structural support and transport of cargoes. These cargoes include mRNAs, proteins, and organelles. Unfortunately, inefficient transport of cargoes due to a disorganized cytoskeletal network can lead to various diseases and pathologies. This reason alone emphasizes the importance of an organized cytoskeletal network to cellular health over an organism’s lifetime. Due to their size and shape neurons experience challenges in organizing their microtubule network. Neurons rely on acentrosomal microtubule organization and how this network is supported remains poorly understood. It is known that within the dendritic compartment microtubule polarity is maintained by a few mechanisms. Previously, our lab discovered a microtubule polarity maintenance mechanism controlled by Kinesin-2. The proposed mechanism involves a complex that includes two members of the Adenomatous polyposis coli (Apc) protein family of tumor suppressors, Apc and Apc2. Apc2 recruits Apc to dendrite branch points where Apc is proposed to function as a linker between growing microtubule plus-ends via End binding protein 1 (Eb1) and Kinesin-2. Protein-protein interactions strengthened the proposal that these complex members work together and it was found that neuronal specific loss of complex members resulted in mixed microtubule polarity in dendrites. However, it was left unclear whether this mechanism functioned specifically at dendrite branch points. We came up with two hypotheses that could explain the steering phenomena. Microtubules could be pre-bundled to stable microtubule tracks before entering branch points or they could be steered at branch points only after encountering a stable microtubule track there. Therefore, we tested both models by examining precise moments when microtubules encountered branch points. By reducing Kinesin-2 subunits we determined that Kinesin-2 mediates microtubule steering not by bundling microtubules but by resolving microtubule collisions within branch points at pre-existing stable microtubule tracks. Due to the ability of Apc2 to target Apc to branch points we sought to investigate upstream regulators of Apc2 positioning to further understand requirements of the steering complex components. We determined through a targeted screen that mitochondrial energy, branched actin, submembrane cytoskeletal elements such as Ankyrin, and members of Wnt signaling contribute to the localization of Apc2 at dendrite branch points. We also visualized that many of the proteins involved in Apc2 targeting were themselves localized to branch points. To follow up on this we determined which molecular players were involved in their own targeting to branch points. This pinpointed the branch point as a region of microtubule control. Intriguingly, it has also been suggested that local microtubule nucleation occurs at dendritic branch points and contributes to dendritic polarity. Because gTub seems to act specifically at branch points we asked how gTub was targeted to branch points to better understand how local nucleation sites are positioned in dendrites. To determine how gTub enriches at its specific site of action we adopted the same candidate proteins screened for Apc2 since both proteins localize to the same region. We discovered that gTub shares some of the machinery required to localize Apc2. The candidates that overlapped between the two microtubule regulators involved members of the canonical Wnt signaling pathway. These proteins include G-protein coupled receptors frizzled and frizzled2, heterotrimeric G-protein alpha subunit, casein kinase 1y, and scaffolding proteins disheveled and Axin. Ultimately, the pathway converges on Axin and we were able to show that Axin was sufficient to recruit gTub to ectopic cellular sites confirming Axin as a major regulator of nucleation machinery. To test the functional significance of loss of gTub enrichment due to targeted depletion of Wnt signaling proteins we used two established assays in the lab. One measures the overall polarity of the dendrite by using Eb1 as a proxy for overall microtubule directionality. By severely reducing gTub levels we have observed mixed microtubule polarity in the dendrite. The second functional assay specifically tests the ability of gTub to nucleate microtubules. By injuring the axon with a UV pulse laser, we have shown that there is an upregulation of microtubules which relies on gTub nucleation. Both of these assays have been previously tested by reducing gTub levels. Therefore, we hypothesized that by reducing Wnt signaling proteins required for gTub enrichment at branch points we would phenocopy the loss of gTub. In fact, depletion of Wnt proteins required for gTub localization resulted in disrupted microtubule polarity and blocked the neuronal ability to upregulate dendritic microtubules in response to axon injury. To examine at which subcellular location this pathway organizes nucleation sites, we investigated membrane bound organelles due to fact that the receptors in the pathway are embedded in membranes at either the cell surface or on internal vesicles. Previously it has been reported that the small Golgi fragments function to organize nucleation sites but we have shown Golgi are dispensable for gTub organization in dendrites. We looked to endosomes which have previously been implicated in regulating Wnt signaling and identified a sub population of early endosomes that house the Wnt signaling proteins necessary to localize gTub to branch points. From these endosomes we witnessed microtubule polymerization events providing evidence that early endosomes housing Wnt Signaling proteins function as the microtubule organizing centers at dendrite branch points. These investigations solidify how two mechanisms work at dendritic branch points. Steering utilizes a complex of precisely targeted proteins to guide microtubules along pre-existing microtubule tracks. Additionally, microtubule nucleation occurs off endosomes that house Wnt signaling proteins to target nucleation machinery at branch points. Both of these pathways describe mechanisms that contribute to an overall understanding of microtubule polarity.