Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming
- Department of Neuroscience, University of Copenhagen, Denmark
- Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Germany
- Center for Neuroscience and Cell Biology, University of Coimbra, Portugal
Figures
Figure 1 with 1 supplement
Syt-1 and Syt-7 are stand-alone calcium-sensors with different kinetics.
(A) Calcium uncaging experiment in Syt-1/Syt-7 DKO cells (orange) and in DKO cells overexpressing Syt-7 (black) or Syt-1 (blue). Top panel: [Ca2+] before (insert) and after calcium uncaging …
Figure 1—figure supplement 1
Amperometric charge quantification.
Integrated amperometric current 5 s after Ca2+-uncaging (mean ± SEM) of Syt-1/Syt-7 double KO (DKO), and DKO cells overexpressing Syt-1 (DKO + Syt-1) or Syt-7 (DKO + Syt-7). *: p<0.05; ***: p<0.001. …
Figure 2 with 2 supplements
Syt-7 potentiates primed vesicle pool sizes at higher prestimulation [Ca2+].
(A) Calcium uncaging experiment from low prestimulation [Ca2+] in WT cells (persian green), Syt-7 KO cells (vermilion) and in Syt-7 KO cells overexpressing Syt-7 (black traces). Panels are arranged …
Figure 2—figure supplement 1
Amperometric charge quantification.
(A) Integrated amperometry (mean ± SEM) of WT, Syt-7 KO, and Syt-7 KO overexpressing Syt-7 (KO + Syt-7) stimulated from low prestimulation [Ca2+]. **: p<0.01; ****: p<0.0001. Kruskal-Wallis test …
Figure 2—figure supplement 2
Mutation of Ca2+-binding sites in Syt-7 abolishes rescue function.
(A) Single confocal slices of new-born mouse chromaffin cells stained against Syt-1 (α-Syt-1, mouse monoclonal α-Syt1 Synaptic Systems 105011), Syt-7 (α-Syt-7, rabbit polyconal α-Syt7, Synaptic …
Figure 3
Calcium-dependent steady-state priming depends on Syt-7 expression.
Titration of burst of secretion (i.e. secretion within the first 0.5 s of the uncaging flash, approximately corresponding to the fusion of the RRP and SRP) against pre-stimulation [Ca2+]. Top panel: …
Figure 4 with 2 supplements
Syt-7 increases forward priming and decreases depriming.
(A) Simple model (Model I) featuring a single primed vesicle pool, a reversible priming reaction (forward rate: k1; reverse rate: k-1), and a fusion rate kf. (B) Recovery in WT cells (persian green) …
Figure 4—figure supplement 1
Differences in unpriming rate can be distinguished during pool recovery, if the Primed Pool is not limited by release sites.
(A) In Model I, we assume that vesicles from a very large Depot Pool prime reversibly (priming rate: k1; unpriming rate: k-1) into the Primed Pool, which is free to change is size. Fusion happens …
Figure 4—figure supplement 2
Double stimulation experiments in Syt-7 WT and KO cells.
(A) Mean [Ca2+]i and capacitance traces of Syt-7 WT cells during double uncaging experiments. Time intervals are indicated in panel B. For time intervals 4 and 8 s, the two sequential stimulations …
Figure 5 with 1 supplement
Blocking NSF-dependent de-priming occludes the effect of Syt-7 at low prestimulation [Ca2+].
(A,B) Calcium uncaging experiment from low prestimulation [Ca2+] in WT (A) and in Syt-7 KO (B) control cells (Control, gray) and in cells infused with 200 µM N-Ethylmaleimide (+NEM, magenta). Panels …
Figure 5—figure supplement 1
Blocking NSF had no effect in WT and Syt-7 KO cells when stimulated from high prestimulation [Ca2+].
(A,B) Calcium uncaging experiment from high prestimulation [Ca2+] in WT (A) and in Syt-7 KO (B) control cells (Control, gray) and in cells infused with 200 µM N-Ethylmaleimide (+NEM, magenta). …
Figure 6 with 3 supplements
Syt-7 stimulates PMA-induced potentiation of release at low prestimulation [Ca2+].
(A) Calcium uncaging experiment from low prestimulation [Ca2+] in Syt-7 WT cells (persian green) and in Syt-7 WT cells perfused with 100 nM phorbol 12-myristate 13-acetate (PMA) (WT + PMA) (gray). …
Figure 6—figure supplement 1
Integrated amperometry (mean ± SEM) of WT, WT cells treated with 100 nM phorbol 12-myristate 13-acetate (PMA) (WT + PMA), Syt-7 KO and Syt-7 KO with 100 nM PMA (Syt-7 KO + PMA) stimulated from low prestimulation [Ca2+].
***: p<0.001. Student’s t-test.
Figure 6—figure supplement 2
Application of phorbol esters to Syt-7 WT and KO at higher prestimulation [Ca2+].
(A) Calcium uncaging experiment from high prestimulation [Ca2+] in Syt-7 WT cells (green traces) and in Syt-7 WT cells perfused with 100 nM PMA (WT + PMA) (gray traces). Panels are arranged as in Fig…
Figure 6—figure supplement 3
Integrated amperometry (mean ± SEM) of WT, WT cells perfused with 100 nM phorbol 12-myristate 13-acetate (PMA) (WT + PMA), Syt-7 KO and Syt-7 KO treated with 100 nM PMA (syt-7 KO + PMA) stimulated from high prestimulation [Ca2+].
*: p<0.05; **: p<0.01. Student’s t-test.
Figure 7 with 1 supplement
Syt-7 stimulates ubMunc13-2-dependent priming at low prestimulation [Ca2+].
(A) Calcium uncaging experiment in WT overexpressing ubMunc13-2 (WT + ubMunc13-2) (cyan traces), and Syt-7 KO overexpressing ubMunc13-2 (KO + ubMunc13-2) (purple traces) stimulated from a low …
Figure 7—figure supplement 1
Overexpressing ubMunc13-2 in Syt-7 WT and KO cells at higher prestimulation [Ca2+].
(A) Calcium uncaging experiment from high prestimulation [Ca2+] in WT cells (persian green), WT overexpressing ubMunc13-2 (WT + ubMunc13-2) (cyan), Syt-7 KO (vermilion) and Syt-7 KO overexpressing …
Figure 8
Syt-7 induces membrane-apposition of LDCVs.
(A, C) 2D-EM micrographs of ultrathin adrenal sections from WT (A) and Syt-7 KO (C) newborn mice. Nucleus is designated (N). Scale bar: 2 µm. (B, D) Magnification of selection in (A) and (C), …
Figure 9 with 1 supplement
Syt-1 and Syt-7 displays limited colocalization.
(A) Single confocal slices of new-born mouse chromaffin cells stained against Syt-1 (α-Syt-1) and Syt-7 (α-Syt-7) in WT cells and in Syt-7 KO cells, and merged images. Scale bar: 5 µm. (B) …
Figure 9—figure supplement 1
Syt-1 and Syt-7 limited colocalization.
(A–C) Immunostaining and quantification of Syt-1 and Syt-7 expression in the absence of glutaraldehyde during the fixation step. Antibodies are a α-Syt-7 rabbit polyclonal antibody and a α-Syt-1 …
Figure 10 with 1 supplement
Syt-1 and Syt-7 are found on the outside of Chromogranin-A-positive vesicles.
(A, E) Single optical slices of WT mouse chromaffin cells stained against CgA (α-CgA) and Syt-7 (α-Syt-7) or Syt-1 (α-Syt-1) acquired with 3D-structured illumination microscopy (3D-SIM). Scale bar: …
Figure 10—figure supplement 1
Proposed role of Syt-7 and ubMunc13-2 in dense-core vesicle priming.
Syt-7 recruits vesicles into a critical distance (6–10 nm), where ubMunc13-2 can bridge vesicle and plasma membrane and stimulate SNARE-complex formation. The ubMunc13-2-dependent priming-step is …
Tables
Table 1
Secretion parameters for Syt-7 WT and KO.
Estimated parameters for Syt-7 WT and KO when secretion is measured 60 ms or 600 ms after Ca2+-uncaging, which corresponds approximately to the fusion of the RRP, or the RRP + SRP, respectively. …
| 60 ms (approx. RRP) | 600 ms (approx. RRP+SRP) | |||
|---|---|---|---|---|
| Syt-7 WT | Syt-7 KO | Syt-7 WT | Syt-7 KO | |
| Pool size (fF) | 74.2 ± 3.3 | 33.6 ± 0.77 | 145 ± 4 | 85.4 ± 2.7 |
| k-1 (s−1) | 0.043 ± 0.0083 | 0.091 ± 0.028 | 0.059 ± 0.0075 | 0.19 ± 0.038 |
| k1 (Before Stim) (fF/s) | 3.21 ± 0.63 | 3.07 ± 0.95 | 8.60 ± 1.12 | 15.8 ± 3.32 |
| k1 (After Stim) (fF/s) | 3.78 ± 0.78 | 1.90 ± 0.62 | 9.8 ± 1.3 | 11.29 ± 2.44 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | C57BL/6 | Experimental Medicine, Panum Stable, University of Copenhagen. | ||
| Strain, strain background (M. musculus) | CD1 | Experimental Medicine, Panum Stable, University of Copenhagen. | ||
| Genetic reagent (M. musculus) | Synaptotagmin-7 (syt7) null allele | Maximov A, Lao Y, Li H, Chen X, Rizo J, Sørensen JB, Südhof TC. Genetic analysis of synaptotagmins-7 function in synaptic vesicle exocytosis. Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3986–3991. | PMID:18308933 | |
| Genetic reagent (M. musculus) | Synaptotagmin-1 (syt1) null allele | Geppert M, Goda Y, Hammer RE, LI C, Rosahl TW, Stevens CF, Südhof TC. 1994. Synaptotagmins I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79(4): 717–727. | PMID:7954835 | |
| Transfected construct (Rattus norwegicus) | p156rrl-pCMV- pH(ecliptic GFP)-TEV-rnSyt1 | This paper, Syt-1 WT | Local reference: Lenti #94 | |
| Transfected construct (Rattus norwegicus) | p156rrl-pCMV- pH(ecliptic GFP)-TEV-rnSyt7S | This paper, Syt-7 WT | Local reference: Lenti #96 | |
| Transfected construct (Rattus norwegicus) | p156rrl-pCMV- pH(ecliptic GFP)-TEV-rnSyt7S-C2AB/D225,227,233,357,359A | This paper, Syt-7 C2AB* | Local reference: Lenti #133 | |
| Transfected construct (Rattus norwegicus) | p156rrl-pCMV- pH(ecliptic GFP)-TEV-rnSyt7S-C2B/D357,359A | This paper, Syt-7 C2B* | Local reference: Lenti #135 | |
| Transfected construct (Rattus norwegicus) | p156rrl-pCMV- pH (ecliptic GFP)-TEV-rnSyt7S-C2A/D225,227,233A | This paper, Syt-7 C2A* | Local reference: Lenti #136 | |
| Transfected construct (Rattus norwegicus) | pSFV1-EGFP-ubMunc13-2 | Zikich D, Mezer A, Varoqueaux F, Sheinin A, Junge HJ, Nachiel E, Melamed R, Brose N, Gutman M, Ashery U. 2008. J. Neurosci. 28:1949–1960. | PMID:18287511 | Local reference: Semliki #486 |
| Antibody | Chicken anti-GFP | Abcam | Ab13970 RRID:AB_300798 | 1:500; 2 hr at room temperature |
| Antibody | Rabbit anti-Chromogranin A | Abcam | Ab15160 RRID:AB_301704 | 1:500; Overnight at four degrees |
| Antibody | Rabbit anti-synaptotagmin-1 | Gift from T. C. Südhof, Stanford, CA | W855 | 1:2000; 2 hr at room temperature |
| Antibody | Mouse anti-synaptotagmin-1 | Synaptic System | SySy: 105011 RRID:AB_887832 | 1:500; Overnight at 4 degrees or 2 hr at room temperature |
| Antibody | Rabbit anti-synaptotagmin-7 | Synaptic System | SySy: 105173 RRID:AB_887838 | ICC: 1:500; Overnight at 4 degrees or 2 hr at room temperature WB: 1:500; Overnight at four degrees. |
| Antibody | Mouse anti-synaptotagmin-7 | Sigma-aldrich | MABN665 RRID:AB_2888943 | 1:200; Overnight/4 degrees |
| Antibody | anti-VCP | Abcam | Ab11433 RRID:AB_298039 | 1:10000; 1 hr at room temperature |
| Antibody | Goat anti-rabbit HRP | Agilent | Dako-P0448 RRID:AB_2617138 | 1:2000; 1 hr and30 min at room temperature |
| Antibody | Goat anti-mouse HRP | Agilent | Dako-P0447 RRID:AB_2617137 | 1:10000; 30 mins at room temperature |
| Antibody | Goat anti-mouse Alexa 546 | ThermoFisher Scientific | A11003 AB_2534071 | 1:500; 30 min at room temperature |
| Antibody | Goat anti-rabbit Alexa 647 | ThermoFisher Scientific | A21245 RRID:AB_2535813 | 1:500; 30 min at room temperature |
| Antibody | Goat anti-mouse Alexa 488 | Invitrogen | A11029 RRID:AB_2534088 | 1:500; 30 min at room temperature |
| Antibody | Goat anti-chicken Alexa 488 | Abcam | Ab150169 RRID:AB_2636803 | 1:500; 30 min at room temperature |
| Commercial assay or kit | BCA Protein assay kit | Pierce | Pierce: 23227 | |
| Chemical compound, drug | NaCl | Sigma-aldrich | Sigma-aldrich: S9888 | |
| Chemical compound, drug | KCl | Sigma-aldrich | Sigma-aldrich: P5405 | |
| Chemical compound, drug | NaH2PO4 | Sigma-aldrich | Sigma-aldrich: S8282 | |
| Chemical compound, drug | Glucose | Sigma-aldrich | Sigma-aldrich: G8270 | |
| Chemical compound, drug | DMEM | Gibco | Gibco: 31966047 | |
| Chemical compound, drug | L-cysteine | Sigma-aldrich | Sigma-aldrich: C7352 | |
| Chemical compound, drug | CaCl2 | Sigma-aldrich | Sigma-aldrich: 499609 | |
| Chemical compound, drug | EDTA | Sigma-aldrich | Sigma-aldrich: E5134 | |
| Chemical compound, drug | Papain | Worthington Biochemical | Worthington Biochemical: LS003126 | |
| Chemical compound, drug | Albumin | Sigma-aldrich | Sigma-aldrich: A3095 | |
| Chemical compound, drug | Trypsin-inhibitor | Sigma-aldrich | Sigma-aldrich: T9253 | |
| Chemical compound, drug | Penicillin/streptomycin | Invitrogen | Invitrogen: 15140122 | |
| Chemical compound, drug | Insulin-transferrin-selenium-X | Invitrogen | Invitrogen: 51500056 | |
| Chemical compound, drug | Fetal calf serum | Invitrogen | Invitrogen: 10500064 | |
| Chemical compound, drug | MgCl2 | Sigma-aldrich | Sigma-aldrich: 449172 | |
| Chemical compound, drug | HEPES | Sigma-aldrich | Sigma-aldrich: H3375 | |
| Chemical compound, drug | Nitrophenyl-EGTA (NPE) | Synthesized at the Max-Planck-Institut for biophysical chemistry, Göttingen. | ||
| Chemical compound, drug | Fura-4F | Invitrogen | Invitrogen: F14174 | |
| Chemical compound, drug | Furaptra | Invitrogen | Invitrogen: M1290 | |
| Chemical compound, drug | Mg-ATP | Sigma-aldrich | Sigma-aldrich: A9187 | |
| Chemical compound, drug | GTP | Sigma-aldrich | Sigma-aldrich: G8877 | |
| Chemical compound, drug | Ascorbic acid | Sigma-aldrich | Sigma-aldrich: A5960 | |
| Chemical compound, drug | EGTA | Sigma-aldrich | Sigma-aldrich: E4378 | |
| Chemical compound, drug | Ethylmaleimide (NEM) | Sigma-aldrich | Sigma-aldrich: 04259 | |
| Chemical compound, drug | Phorbol 12-myristate 13-acetate (PMA) | Sigma-aldrich | Sigma-aldrich: P8139 | |
| Chemical compound, drug | Paraformaldehyde | Sigma-aldrich | Sigma-aldrich: P6148 | |
| Chemical compound, drug | PIPES | Sigma-aldrich | Sigma-aldrich: 80635 | |
| Chemical compound, drug | Triton X-100 | Sigma-aldrich | Sigma-aldrich: T8787 | |
| Chemical compound, drug | BSA | Sigma-aldrich | Sigma-aldrich: A4503 | |
| Chemical compound, drug | Prolong Gold | Invitrogen | Invitrogen: P36934 | |
| Chemical compound, drug | Protease cocktail inhibitor | Invitrogen | Invitrogen: 87785 | |
| Chemical compound, drug | RIPA buffer | Invitrogen | Invitrogen: R0278 | |
| Chemical compound, drug | ECL plus western blotting substrate | Pierce | Pierce: 32132 | |
| Software, algorithm | Igor 8.0 | Wavemetrics | ||
| Software, algorithm | ImageJ | NIH software |
