Control of activity-dependent bulk endocytosis by signalling cascades

Activity-dependent bulk endocytosis (ADBE) is the dominant mechanism by which central nerve terminals retrieve synaptic vesicle (SV) membrane during high intensity stimulation. We have identified a key role for the enzyme glycogen synthase kinase (GSK3) in this process due to its phosphorylation of the large GTPase dynamin I. GSK3 activity is tightly regulated by a number of different signaling cascades, suggesting that these cascades may also control ADBE. The project will test this hypothesis using a combination of molecular and cell biology in concert with live cell imaging of SV recycling in primary neuronal cultures. Specifically, the project will determine the impact of different signaling cascades on GSK3-dependent dynamin I phosphorylation. Cascades that modulate this event will then be examined for their role in ADBE using a range of live single cell fluorescence imaging techniques and quantitative electron microscopy of nerve terminals. Signalling cascades will be manipulated using either overexpression of dominant negative mutants, silencing of expression with shRNA or via pharmacological agonists and antagonists. Cascades which modulate ADBE should be key targets for strategies to alter either learning/memory generation or epileptic seizures, since ADBE will be the dominant SV retrieval mode during these conditions.