Non-canonical role for ATPase HSC70 in driving Clathrin remodeling during compensatory endocytosis in synapse

Neurons use Clathrin-mediated endocytosis to retrieve synaptic vesicle (SV) proteins during compensatory endocytosis after presynaptic SV fusion. We have shown SV cargo to be re-sorted and pre-assembled outside the active zone into Clathrin-coated structures (CCS) of variable size and curvature, constituting a readily retrievable pool. During compensatory endocytosis CCS of the readily retrievable pool must be remodeled swiftly into spherical vesicles within 10 seconds at physiological temperature. How this is achieved remains elusive. Here we performed live-cell imaging on intact as well as scanning electron microscopy on unroofed hippocampal Xenapses, TIRFM-amenable presynaptic boutons formed en-face on microstructured and functionalized coverslips. While CCS can slowly remodel into spherical pits in unroofed Xenapses within tens of minutes, this process is highly accelerated in intact Xenapses, as evidenced by the rapid (< 10 seconds) exchange of EGFP-labelled Clathrin and AP2 adaptor after photo-bleaching. This fast remodeling of CCS was observed in the absence of stimulation and cannot be explained by constitutive endocytosis. Hence, this process must be driven by cytosolic factors which are lost during unroofing. Using membrane-permeant interfering peptides we identify Hsc70, the ATPase which along with Auxilin drives uncoating of endocytosed Clathrin-coated vesicles, to have an additional role in driving curvature of invaginating CCS.