Inactivation of Hsc70 ATPase activity blocks slow axonal transport and presynaptic accumulation of synapsin. (A) Schematic of assay to determine slow axonal transport of synapsin using photoactivatable synapsin (PAGFP:synapsin; ; ). In brief, PAGFP:synapsin was photoactivated in a discrete axonal ROI, and the fluorescence was tracked over time by live imaging. (B) Kymographs from photoactivation experiments in A. Note the anterogradely biased dispersion of PAGFP:synapsin fluorescence in control axons, thought to represent slow transport (midpoint of photoactivated zone marked by red arrowhead and dashed line; top). Also note that the Hsc70 inhibitor (100 µM VER155008; bottom) eliminated the anterograde bias. (C) Quantification of the transport experiments. In brief, the centroid of the photoactivated zone was quantified in each image of a given time-lapse video, and the displacement of the centroid was quantified over time (intensity center shift; ). Note that although the population of photoactivated synapsin was transported anterogradely, pharmacologic (VER155008) or genetic (DN-Hsc70) inactivation of Hsc70 ATPase activity prevented the biased transit. (D) Attenuation of endogenous synapsin levels at presynaptic boutons upon selective inactivation of Hsc70 in axons. Axons and presynaptic boutons were isolated from somatodendritic compartments using a triple-chamber microfluidic device (see Fig. S1 for design of device). Thereafter, the Hsc70 inhibitor VER155008 (or DMSO control) was selectively added to the axonal/presynaptic chamber, and the neurons were fixed and immunostained with anti-VAMP2 (to detect all presynapses; green) and anti-synapsin (red) antibodies. Representative images from axonal/presynaptic chambers are shown. (E) Quantification of colocalization between synapsin and VAMP2. Note that Hsc70 inactivation significantly attenuated the presynaptic localization of synapsin. Error bars show means ± SEM. **, P < 0.01.