Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Biochemistry

Supervisor

Dr. Susan Meakin

Abstract

The neuronal cytoskeleton is responsible for governing dynamics such as neurite extension and cortex development. In particular, microtubules (MTs) and their associated proteins, and molecular motors, have been shown to be critical in many neuronal processes such as intracellular molecular transport and neuron differentiation. The fibroblast growth factors (FGFs) act as powerful morphogens that have been shown to play a role in regulating cortical development. FGFs activate receptor tyrosine kinases, of which fibroblast growth factor receptor substrate 3(FRS3) has been shown to interact with, to mediate downstream signaling cascades (regulating cell proliferation and differentiation). In addition to FRS3’s role in signaling, preliminary evidence suggested that FRS3 is a novel MT binding protein, and also interacts with MT associated proteins and the molecular motor kinesin. Furthermore, FRS3’s expression has been shown to coincide with migrating cortical neurons from E12 cortical precursor cell cultures. This thesis hypothesized that FRS3 regulates the differentiation of neurons as well as post-mitotic neuron function by regulating MT stability and intracellular transport of cargo and organelles.The research presented here has addressed this hypothesis in a multi-disciplinary manner. First, FRS3’s MT binding properties and its interactions with various proteins involved in molecular transport within cortical and hippocampal neurons (including motors, adapters and cargo) were investigated. FRS3 was shown to interact with markers of migrating and post-mitotic neurons (acetylated alpha and Beta III tubulin, respectively), post-synaptic density scaffolding proteins (Shank 2 and 3), cargo (Flg/FGFR1), molecular motor components (Kinesin Heavy Chain and Kinesin Light Chain), synaptic vesicle proteins (Syntaxin I and Synaptophysin) and finally MT associated proteins (MAP2, Doublecortin and Tau). Next, loss of FRS3 expression in SN56 cells resulted in increased sensitivity to the MT depolymerizing agent nocodazole, suggesting FRS3 is necessary for proper MT dynamics. Furthermore, loss of FRS3, but not the related signaling adaptor FRS2, resulted in impaired neurite outgrowth in both hippocampal and cortical cell culture. These results, taken together, suggest that FRS3, in addition to its role in FGF signaling, also interacts with members of the molecular transport machinery and confers stability to microtubules.

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