Neuroplasticity is the ability of the brain to change structure at the cellular level in response to stimulus; this dynamic remodeling of neuronal morphology is the basis of learning and memory. Regulating neuronal morphology requires tight coordination between the dynamic microtubule and actin cytoskeletons. In neurons, the dynamic plus-ends of microtubules have been shown to invade dendritic spines and stimulate actin- dependent spine remodeling in response to synaptic activity. This indicates that microtubule plus-ends deliver and/or activate factors that locally induce actin assembly in the spine. However, the mechanisms underlying this microtubule-actin crosstalk has remained elusive. The tumor suppressor protein adenomatous polyposis coli (APC) binds to microtubules and actin, and recent work from the Goode lab has shown that APC potently nucleates actin assembly and is required to coordinates actin and microtubule dynamics and allow directional cell migration. However, the role of APC in promoting actin assembly and microtubule-actin coordination in the nervous system has not been investigated. APC is enriched in dendritic spines and is trafficked in neurons on the growing plus ends of microtubule by the end-binding protein EB3. My preliminary data show that EB3 directly inhibits APC-mediated actin assembly by binding to the APC `Basic domain'. Other studies have shown that depleting either APC or EB3 diminishes the number of mature dendritic spines, consistent with APC and EB3 working together to regulate actin-dependent spine remodeling. The goals of this proposal are to elucidate the role of APC-mediated actin assembly, and its negative regulation by EB3, in neuronal branching and dendritic spine morphogenesis. This will be achieved by live-imaging in cultured primary mouse hippocampal neurons using an existing dominant mutant (APCm4) that abolishes APC- mediated actin assembly, and newly generated APC mutants refractive to EB3 inhibition. Changes in neuronal morphology, as well as the levels and dynamics of actin and microtubules will be correlated with APC localization by fixed- and live-cell microscopy. These efforts will be facilitated by a strong collaboration with Prof. Erik Dent (University of Wisconsin Madison), whose lab will be visited for hands-on training in live imaging of cytoskeleton dynamics in neurons. In parallel, single-molecule biochemistry will be used to define the mechanism by which EB3 inhibits APC-mediated actin assembly in vitro. This information will be used to test two models in vivo for how EB3 regulation of APC-mediated actin assembly influences dendritic spines: (1) The `sponge model', which postulates that invading microtubules soak up EB3, releasing/activating APC to promote actin assembly and spine remodeling; (2) The `delivery model', which postulates that APC-mediated actin assembly helps to recruit EB3-rich microtubules to spines, leading to APC inhibition, and an accompanying shift in the available actin monomer pool to a branched-actin nucleator system (Arp2/3 complex) underlying spine remodeling. This work will uncover fundamental mechanisms of neuronal cell biology, which are highly relevant to our mechanistic understanding of human cognition and neurological diseases in which these pathways are altered.
The adenomatous polyposis coli (APC) gene/protein plays a critical role in neuronal morphogenesis and in learning and memory, with mutations in APC leading to cognitive disorders, infantile spasms, epilepsy and seizures. The work described here will elucidate the molecular and cellular mechanisms by which APC influences microtubule and actin dynamics in neurons to control neuronal morphogenesis and dendritic spine remodeling ? events that are essential for normal function in the human brain.