Filopodia are dynamic, actin-rich structures that extend outward from the cell and explore the local environment. In neurons, filopodia are critical for numerous stages of development, including neuritogenesis, axon guidance, and dendritic spine formation. Defects in any of these stages of neuronal development can result in improper synaptic connectivity, neurodevelopmental disorders, and psychiatric syndromes. The Ena/VASP of actin polymerizes is well appreciated to localize to the filopodial tip complex and influence actin dynamics. Recently, the Gupton lab showed VASP transiently co-localizes with the E3 ubiquitin ligase TRIM9 at the filopodial tip. TRIM9 was required for the reversible, non-degradative ubiquitination of VASP and this modification was associated with decreases in filopodia stability and number. Appropriate control of filopodial dynamics is critical for several neuronal processes, yet we still do not understand how VASP is regulated at the filopodial tip. My central hypothesis that VASP-Ub negatively regulates actin dynamics in filopodia and dendritic spines. To test this hypothesis, I will chemically ubiquitinate and fluorescently label purified VASP protein. Using various biochemical assays, I will define the impact of ubiquitination on VASP tetramerization and VASP-mediated regulation of actin dynamics. Mechanistically understanding the impact of VASP ubiquitination in vitro is critical in understanding the cellular role of ubiquitinated VASP (VASP-Ub). Furthermore, I will examine the localization and function of VASP-Ub in dendritic spines. Previous work has shown that VASP is required for proper dendritic spine formation and my preliminary data shows that VASP-Ub localizes to the post-synaptic density. I will examine VASP and VASP-Ub abundance and localization during dendritic spine maturation and chemically induced plasticity will be analyzed in forebrains and cultured cortical and hippocampal neurons isolated from wild-type and Trim9-/- mice using both microscopy-based and biochemical assays. These experiments will determine how ubiquitination regulates VASP activity, and consequently actin polymerization and synaptic plasticity, in dendritic spines. Appropriate control of the cytoskeleton is critical for dendritic spine formation, and synaptic transmission, yet it is still unknown how actin dynamics are controlled in these specialized neuronal structures. Working at the interface of biochemistry, cell biology, and neuroscience, I will determine how non- degradative ubiquitination regulates the actin polymerase VASP and shapes neuronal morphology and function.
Appropriate control of filopodial dynamics is critical for numerous stages of neuronal development, including dendritic spine formation and maturation. The Gupton lab has shown that non- degradative ubiquitination of the actin polymerase VASP is associated with decreased filopodial number, yet the mechanistic impact of this modification is unknown. The proposed work seeks to determine how ubiquitination impairs VASP-mediated regulation of actin dynamics and how VASP ubiquitination influences dendritic spine morphology and synaptic function.
