PMC

Synapsin-mCherry (n = 10 cells) was used as a marker for SVs, whereas NPY (n = 11 cells) is a DCV cargo. Bars represent mean ± s.e.m.

A) Wild type and mutant EYFP-tomosyn-1m constructs were co-expressed with synapsin-mCherry using lentiviral vectors. B) Representative examples of EYFP-tomosyn-1m (Tom1) and synapsin-mCherry (Syn) dynamics, depicted as kymographs for each construct. None of the constructs analyzed showed a loss of vesicular co-migration. C) Quantitation of the percentage of immobile and mobile synapsin-mCherry puncta that co-labeled EYFP-tomosyn fluorescence. Statistical tests were performed for mobile synapsin-containing puncta. Data show mean ± s.e.m from N = 9 cells and 290–396 puncta.

Cultured hippocampal neurons were fixed at DIV14. Local accumulation of (A) endogenous tomosyn-1 (detected with a tomosyn-1 specific antibody) as well as (B) EYFP-tomosyn-m1 was observed in axons (open arrowheads) and dendrites (closed arrowheads). (C) Typical example of Western blot analysis confirming EYFP-tomosyn-m1 expression in these preparations. Asterisks indicate endogenous (*) and overexpressed (**) tomosyn-1. (D) Endogenous tomosyn-1 (red) and EYFP-tomosyn-xb2 (green) showed similar subcellular distributions.

Co-localization of EYFP-tomosyn-xb-2 (‘Tom-2’; green) puncta with antibodies recognizing endogenous (A) VAMP2 and (B) chromogranin B (red) was observed. Fluorescence intensity profiles along the depicted neurites are given below the images. (C) Expression of EYFP-tomosyn-xb2 in these preparations was verified by Western blotting. Asterisks indicate endogenous (*) and overexpressed (**) tomosyn-2. Co-localization was quantified using (D) Pearson’s correlation and (E) Manders’ overlap M1 and (F) M2. Co-migration of EYFP-tomosyn-xb2 puncta (arrowheads) with (G) synapsin-mCherry and (H) NPY-mCherry was observed in living neurons, as seen in these kymograph examples.

(A) Time-lapse images show bidirectional movement of EYFP-tomosyn-m1 puncta (solid arrowheads). During recording, additional puncta occasionally emerged from stable or moving tomosyn puncta (open arrowheads). After 30 s, the cells were stimulated with 16x 50 action potentials at 50 Hz. (B) DIC image of the same region. (C) Puncta movement along the neurite during the same time-lapse is depicted as a kymograph. The stimulation period is represented by inverted tones. (D) The velocity of moving tomosyn puncta reduced significantly during stimulation. Error bars depict s.e.m. and the number of analyzed vesicles is depicted in the bars. **, p<0.01.

(A-B) EYFP-tomosyn-m1 (‘Tom-1’; green) puncta co-localized with (A) synaptic vesicle marker VAMP2 and (B) DCV marker chromogranin B (both depicted in red) in DIV14 Syt-1^KO/KO neurons. Fluorescence intensity profiles along the neurites are shown below the images. (C-E) Overall co-localization of VAMP2 vs. Tom-1 (‘VAMP2’) or of chromogranin B vs. Tom-1 (‘Chromogr B’) was quantified using (C) Pearson’s correlation, (D) Manders’ overlap M1 and (E) M2, which were similar in wild type and Syt-1^KO/KO neurons. Furthermore, mobile EYFP-tomosyn-m1 puncta (green) co-migrated with (F, arrowheads) synapsin-mCherry (‘Synapsin’; red) and (G, arrowheads #1–3) NPY-mCherry (‘NPY’; red) in Syt-1^KO/KO neurons.

(A) Typical time-lapse images and (B) the corresponding kymograph show co-migration (arrowhead #1) of synapsin-mCherry (red) and EYFP-tomosyn-m1 (green) in hippocampal neurons. Also, synapsin-mCherry-negative tomosyn puncta (arrowhead #2) and EYFP-tomosyn-m1-negative synapsin puncta were observed. Similarly, (C) typical time-lapse images and (D) the corresponding kymograph are shown for NPY-mCherry co-migration. Two moving NPY-mCherry positive tomosyn puncta (arrowheads #1 and #2) and an EYFP-tomosyn-m1-negative NPY punctum (arrowhead #3) are seen in this example. Quantification of the amount of overlap is shown in . (E) While the mean velocity of moving tomosyn puncta without synapsin-mCherry was not significantly higher than the velocity of puncta containing this SV marker, (F) puncta without NPY-mCherry moved on average faster than puncta with NPY-mCherry in the same cells. (G) Vesicular EYFP-tomosyn-1 did not affect the velocity of NPY puncta. Error bars depict s.e.m. and the number of analyzed vesicles is depicted in the bars. ***, p<0.001.

A) Wild type and mutant EYFP-tomosyn-1m constructs were co-expressed with synapsin-mCherry or NPY-mCherry using lentiviral vectors. mCherry-labelled puncta were observed by live imaging during 60s at 1 frame/s. Previously mapped interaction domains with Syt-1, Rab3, SNAP25 and syntaxin-1 are indicated by solid black lines below the constructs [,,,–]. B) Representative examples of EYFP-tomosyn-1m (Tom1) and synapsin-mCherry (Syn) dynamics in neurites depicted as kymographs for each construct. In all groups, co-migration of EYFP and mCherry was observed in mobile puncta (some examples are indicated by closed arrowheads). Open arrowheads indicate mobile mCherry puncta with no detectable EYFP-tomosyn fluorescence. Asterisks indicate immobile double-labelled structures. C) Quantitation of the percentage of synapsin-mCherry puncta that showed detectable EYFP-tomosyn fluorescence. Both mobile and immobile structures are displayed. Data are presented as mean ± s.e.m from n = 37–45 cells and 1940–3090 puncta. Statistical tests were performed for mobile synapsin-containing puncta. The strongest reduction in the percentage of tomosyn-labelled puncta was observed after deletion of the N-terminal domain (see constructs “Tail-CC” and “CC”, ***; p<0.001). D) Similar quantitation data for NPY-mCherry puncta calculated from n = 17–21 cells and 373–912 puncta).

(A-J) Images show expression of EYFP-tomosyn-m1 (‘Tom-1’; green) and endogenous synaptic and secretory vesicle markers (red). Fluorescence intensity profiles along depicted neurites are displayed below each image. Co-localization of EYFP-tomosyn-m1 puncta with antibodies recognizing endogenous (A) SNARE protein syntaxin, (B) the presynaptic SNARE-associated protein munc18 as well as the active zone protein bassoon was observed, confirming presynaptic localization. Moreover, tom-1 puncta co-localized with synaptic vesicle (SV) markers (D) synaptotagmin-1 (Syt-1), (E) VAMP2 and (F) synapsin, (G) vesicular glutamate transporter-1 (VGLUT1) and (H) the DCV marker chromogranin B. (I) CAPS, implicated in release from both SVs and DCVs, additionally co-localized with tomosyn-1 puncta. (J) Synapsin / VAMP2 co-localization was used as a positive control. (K-M) Co-localization was quantified using (K) Pearson’s correlation and Manders’ overlap (L) M1 and (M) M2. As a negative control, tomosyn images were rotated relative to syntaxin. (N-Q) Ultrastructural localization of endogenous tomosyn-1 in (N) presynaptic boutons (O) vesicles in neurites and (P) dense core vesicles. (Q) Overexpressed tomosyn-1 was predominantly localized to presynaptic boutons. Arrowheads indicate post-synaptic densities.