Studying the significance of neuronal microtubule polarity and the influence of support cells on microtubule nucleation in dendrites
Restricted (Penn State Only)
- Author:
- Thyagarajan, Pankajam
- Graduate Program:
- Molecular, Cellular, and Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 05, 2024
- Committee Members:
- Melissa Rolls, Program Head/Chair
Lorraine Santy, Major Field Member
William Hancock, Outside Unit & Field Member
Melissa Rolls, Chair & Dissertation Advisor
Santhosh Girirajan, Major Field Member - Keywords:
- microtubule
cytoskeleton
neuron
dendrite
microtubule nucleation
microtubule polarity
significance of microtubule polarity
regulation of microtubule nucleation
sites of nucleation
endosomes
endocytosis
clathrin
support cells
skin cells
glia
Wnt
Wnt signaling
frizzled
AP2
sites of endocytosis
dendrite branchpoint
dsh
axin
Frizzled
g-tubulin
EB1-GFP - Abstract:
- The neuron has specialized compartments including the cell body, axon, and dendrites. The microtubule cytoskeleton is essential for the neuron’s structure and proper functioning at its different stages including development, maintenance, and regeneration. Defects in the cytoskeleton have been implicated in several neurodegenerative diseases. The microtubules have a distinct arrangement in axons (uniformly plus-end-out) while dendrites exhibit mixed or minus-end-out polarity. Dendrites of different model organisms share a common feature: the presence of minus-end-out microtubules. To test whether minus-end-out microtubules contribute to the dendritic identity, we used an in vivo model that has neurites that contain both plus-end-out and minus-end-out microtubules. We used this model to study the inter-relationship of microtubule polarity with the different aspects of neuronal polarity (Chapter 2). We find that changes in microtubule polarity can affect cargo trafficking like previous studies. We studied microtubule stability by counting the number of growing microtubule ends. The neurites with plus-end-out polarity had a lesser number of growing ends, suggesting more stability. This suggested that microtubule polarity underlies microtubule stability. The microtubule-dependent response to injury also differed between these two types of neurites. A surprising finding was the formation of ectopic diffusion barriers in the plus-end-out neurites. They were identified using fluorescence recovery after photobleaching (FRAP) assays. The change in polarity did not have a major effect on the morphology of the neurite. Overall, Chapter 2 gives a comprehensive picture of the interactions between microtubule polarity and other different mechanisms that maintain polarity. After addressing the significance of microtubule polarity in Chapter 2, the next research chapter focuses on one of the mechanisms that maintain microtubule polarity, microtubule nucleation. Microtubule nucleation is the de novo formation of microtubules typically mediated by the γTubulin ring complex. γTubulin is the key player in microtubule nucleation that forms the template for the growth of tubulin heterodimers to form protofilaments organized to form hollow tubular structures. γTubulin is typically housed in centrosomes that organize microtubules in a cell. However, in neurons, microtubule nucleation does not depend on centrosomes and local microtubule nucleation takes place. The location of acentrosomal microtubule nucleation sites has recently been identified through studies in invertebrates – both studies suggest that endosomes are capable of housing γTubulin. In flies, early endosomes at dendrite branchpoints are essential for microtubule nucleation as they help in recruiting γTubulin. γTubulin recruitment in the fly system is known to be regulated by a subset of Wnt signaling proteins that are housed on the endosomes. The involvement of multiple transmembrane receptors like Frizzled, Frizzled-2, and coreceptors (Arrow and Ror) let us hypothesize that surrounding cells can be the source of the Wnt ligand that activates this signaling pathway. We used a combination of genetics and live imaging of fluorescently tagged proteins in Chapter 3 to identify that skin cells are the source of the Wnt ligand. Through RNAi screening, we further identified the specific Wnt ligands involved (Wnt4 and wntD) and confirmed their role in γTubulin localization using mutants. These Wnt ligands also affected the nucleation-dependent increase in microtubule dynamics post-injury. The overexpression of these Wnt ligands caused an increase in microtubule dynamics in the dendrites. This is the first evidence of non-cell-autonomous control of microtubule nucleation. Based on the essential role of endosomes at branchpoints, we hypothesized that endocytosis would be involved in this model where Wnt is secreted from skin cells, and it needs to be internalized for active signaling. We visualized the localization of endocytic markers in the dendrites and observed that they form stationary patches at the dendrite branchpoints. We studied their dynamics and performed FRAP assays to characterize their exchange to confirm they are sites of endocytosis. We studied the colocalization of clathrin with other Wnt signaling machinery – we identified that Wnt signaling puncta can localize both with clathrin and Rab5 early endosome. This led us to study the effect of reducing clathrin on the localization of Wnt signaling machinery. Our data suggests that clathrin is essential for the localization of Dishevelled protein. We also blocked endocytosis by affecting dynamin, and this caused an accumulation of Frizzled at clathrin patches. This suggests that endocytosis could play a role in internalizing the receptor-ligand complex at branchpoints. Reduction of clathrin also affected the microtubule-nucleation-dependent response in dendrites post-axonal injury. This suggested that clathrin is essential for microtubule nucleation. Based on all the results in Chapter 3, we propose a model of Wnt secretion from skin cells that depends on clathrin for its internalization along with receptors and other Wnt proteins and subsequent recruitment to Rab5 endosomes for sustained signaling that regulates microtubule nucleation.