Isolation of synapsin-associated protein complexes from mouse brains. (A) Whole-brain lysates from WT or synapsin TKO mice were differentially fractionated to obtain synaptosomal (P2) and synaptosome-depleted fractions (S2). The latter were further subfractionated into P100 and S100 fractions by high-speed centrifugation (red box, putative SCb fractions; see the Isolation of synapsin complexes… section of Results). (B) Synaptosomal (P2) and nonsynaptosomal fractions (P100 and S100) from A were coimmunoprecipitated with an anti-synapsin antibody under nondenaturing conditions. CoIP fractions were subjected to MudPIT-MS to identify peptides. TKO brain lysates were used as negative controls. (C) Silver-stained gels of synapsin coIP fractions from WT brain lysates (P2, P100, and S100) suggest the presence of polypeptides associated with synapsin. Single arrowheads represent synapsin-Ia, double arrowheads represent synapsin-Ib (note IgG controls, right lanes). (D) Raw spectrum counts of synapsins-I and -II from various coIP fractions as detected by MudPIT-MS. Note spectrum counts are high in WT fractions but undetectable in TKO lysates.

Criteria for including synapsin-interacting peptides identified by MudPIT-MS analysis. Raw spectrum counts for a given protein from WT and TKO lysates were normalized and averaged to obtain final spectral counts using conventional strategies as detailed in the Isolation of synapsin complexes… section of Results. The following inclusion criteria were then applied to determine synapsin-associated proteins. First, peptides detected in WT but not in the TKO lysates (spectrum count = 0) were included. Second, peptides with final spectrum counts of twofold or more than TKO fractions were also included in the list. The final numbers of proteins are indicated in the black boxes. IF, immunofluorescence.

Characterization of the synapsin proteome from the synaptosomal fraction. (A) Peptides meeting inclusion criteria from the synaptosomal fraction (P2) were classified based on the molecular function. Note that metabolic enzymes, transcription factors, and cytoskeletal elements constitute a large fraction of the identified peptides as expected (“%” represents the number of proteins in the functional group per total number proteins in the fraction × 100). (B) Table of known synapsin interactors detected in the synaptosomal fraction (also see Table S4).

Characterization of synapsin protein complexes from nonsynaptosomal fractions. (A) Functional categorization of peptides identified in the nonsynaptosomal (P100 and S100) fractions (“%” represents number of proteins in the functional group per total number proteins in the fraction × 100). (B) Bar graphs showing raw spectrum counts for selected chaperones detected in the S100 fraction. Error bars show means ± SEM. (C) Protein–protein interaction map of the chaperones identified in the S100 fraction. Individual proteins are highlighted by red dots, and protein–protein interactions are indicated by light gray lines. The dashed circle highlights the Hsp peptides. (D) CoIP of synapsin and Hsc70 in HEK293T cells. HEK cells were either transfected with the full-length GFP-tagged synapsin or various GFP-tagged deletion mutants containing only some of the domains that make up the protein (a schematic of various synapsin domains is on top). The transfected synapsin and endogenous Hsc70 was detected by anti-GFP and anti-Hsc70 antibodies, respectively. Note that only the synapsin deletion lacking the d-domain failed to associate with Hsc70 (fifth lane from left, bottom gel). The asterisk indicates IgG heavy chain. Molecular mass is indicated in kilodaltons.

Coclustering of endogenous synapsin and Hsc70 in axons visualized by two-color superresolution microscopy. (A) Principle of DNA-PAINT. Note weak hybridization between DNA strands leads to fluorophore “blinking,” allowing superresolution imaging. (B and C) Widefield and corresponding two-color superresolution image of axonal synapsin and Hsc70. Progressively zoomed insets from C are shown in C’ and C”. Note coclusters of synapsin and Hsc70 (some highlighted with dashed circles in C”). (D and E) Quantifications of axonal superresolution data. (D) Size of particles was ∼40 nm; and (E) ∼60% of the synapsin particles were within 100 nm of Hsc70 particles, suggesting coclustering.

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.

Inactivation of Hsc70 ATPase activity leads to disruption of axonal synapsin. (A) Strategy to analyze the fluorescence decay of PAGFP:synapsin in an axon ROI over time. PAGFP:synapsin was photoactivated in a discrete axonal ROI, and the dispersion of fluorescence was quantified over time. (B) Fluorescence decay of PAGFP:synapsin in axon ROIs. Note faster decay of fluorescence upon Hsc70 inactivation. (C) Representative images showing endogenous distribution of synapsin in axons upon pharmacologic and genetic Hsc70 inactivation (VER155008 and Hsc70-DN). Note that the punctate distribution is disrupted upon Hsc70 inhibition. (D) Quantification of the periodicity of axonal synapsin particles with or without Hsc70 inactivation. Periodicity of endogenous synapsin particles (i.e., distances between adjacent puncta) was determined from axonal line scans, as shown in the example (top left; red boxed region from C). Cumulative data from control axons is shown on the top right, represented by FFTs (see the Colocalization analysis of widefield images… section of Materials and methods for details). Note the major peak at ∼2.77 µm, representing the typical spacing of synapsin puncta in control axons (dashed line over minor peaks). Also note loss of the major peak upon Hsc70 inhibition, reflecting disruption of the periodic synapsin distribution in axons. (E) HEK293 cells were transfected with GFP:synapsin, immunoprecipitated with an anti-GFP antibody, and blotted with anti-GFP and anti-Hsc70 antibodies (with or without VER155008). Note that addition of the Hsc70 inhibitor led to a significant reduction in the amount of immunoprecipitated Hsc70. Molecular mass is shown in kilodaltons. (F) Quantification of blots in E. n = 3 separate experiments. Error bars show means ± SEM. **, P < 0.01.