All ETDs from UAB

Advisory Committee Chair

Guillermo Marques

Advisory Committee Members

James Collawn

Kai Jiao

Lucas Pozzo-Miller

Rosa Serra

Bradley Yoder

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The Drosophila Bone Morphogenetic Protein (BMP) Glass bottom boat (Gbb) is a ligand for the BMP type II receptor, Wishful thinking (Wit). Mutations in either of these proteins impair synaptic growth at the neuromuscular junction (NMJ). Gbb is a retrograde signal (from target to innervating neuron) that is essential for proper NMJ development and function. Tissue-specific rescue of pathway mutants and immunolocalization experiments indicate that the receptor-ligand interaction occurs at the NMJ, and results in the nuclear accumulation of the phosphorylated form of the transcription factor Mad, indicating that retrograde transport along the axon is critical for pathway activation. We have investigated the mechanism of retrograde axonal transport of the BMP using a signaling endosome model of signal propagation. In this model, the receptor complex undergoes internalization at the synaptic terminal and is transported along axon microtubules to the cell body where it then activates down-stream molecules. Tools that we have utilized to characterize our pathway include fluorescent fusion proteins of our BMP receptors and Mad transcription factor as well as high resolution live imaging techniques. From our studies, we find that the receptors Wit and Tkv are being transported in a bi-directional manner along the motoneuron axon. Mad along the axon, however, appears diffuse and does not travel with the receptors. We see p-Mad in the nucleus and at the synaptic terminal, however we do not see p-Mad along the axon. A known inhibitor of the pathway, DN-Glued, dramatically decreases the amount of iv moving receptor vesicles, linking receptor transport to pathway activity. Additionally, we have found that Tkv-YFP and Wit-CFP colocalize along the axon in predominately retrograde moving particles and lose this directionality in gbb ligand mutants, consistent with our hypothesis of a signaling endosome. In further support of the signaling endosome model, manipulation of the endocytic pathway results in alterations in p-Mad levels and alterations in receptor vesicle transport. Our data suggests that, to propagate the BMP pathway along the motoneuron axon, the receptors are traveling in a signaling endosome that phosphorylates Mad in separate events at the synaptic terminal and proximal cell body.

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