Disruption of dendritic spine stability is a hallmark of neurological and neurodegenerative disorders. Dendritic spines are supported by an underlying actin framework consisting of at least two distinct actin pools, with stable filaments concentrated in the spine core and dynamic branched filaments in the outer shell. How these distinct populations are regulated to maintain spine stability while allowing ongoing structural plasticity is unclear. The Abl2 nonreceptor tyrosine kinase is essential for dendritic spine stability. Previous work and my preliminary data show that Abl2 binding to actin both regulates actin filament stability and promotes Arp2/3 complex-mediated actin branching. These functions to not appear to require Abl2 kinase activity, suggesting that Abl2 modulates the cytoskeleton via direct interactions with actin. In this proposal, I will test the hypothesis that direct interactions of Abl2 with actin filaments, to control filament stability and actin branching, regulate dendritic spine morphology and stability.
My first aim i s to determine how Abl2 stabilizes filaments. In this aim, I will use single filament TIRF microscopy (TIRFm) assays to determine the minimal fragment of Abl2 capable of stabilizing filaments. I will then determine key features of Abl2 decoration of actin filaments required for stabilization using 2-color TIRFm with Abl2-GFP and Rhodamine-actin. This will reveal if filament stabilization requires a threshold of global Abl2 decoration or local Abl2 binding at the site of depolymerization.
My second aim i s to elucidate how Abl2 activates the Arp2/3 complex. It is not known what parts of Abl2 are required for Arp2/3 activation or which step of the Arp2/3 complex branching mechanism is impacted by Abl2. I will use TIRFm actin-branching assays to determine the minimal fragment of Abl2 capable of promoting actin branching. I will also use 2-color TIRFm to study the effects of Abl2 actin decoration on actin branching, testing if branches form preferentially on Abl2 decorated regions of filament.
My third aim i s to determine how Abl2 cytoskeletal regulation impacts dendritic spine stability and morphology. Knockdown (KD) of Abl2 in primary cultured neurons destabilizes dendritic spines, and alters spine shape, actin dynamics, and filamentous actin levels within the spines that remain. To test the functions of Abl2 required to restore these disruptions, I will rescue Abl2KD neurons with different mutants of Abl2 known to possess specific actin-regulating functions, including any found in Aims 1 and 2. I will use fluorescence recovery after photobleaching (FRAP) of GFP-actin and immunofluorescence to determine Abl2 functions sufficient to restore proper spine actin dynamics and filamentous actin levels in spines. I will then identify which functions of Abl2 are sufficient to support normal spine shape and long-term stability.
The disruption of proper neuronal structure is a hallmark of neurological and neurodegenerative disorders. My proposed studies will elucidate a mechanism by which Abl2 maintains proper neuronal structure in the brain. In doing so, I will uncover a mechanism responsible for a pathology common to psychiatric illnesses that burden millions of patients worldwide.
