A synaptotagmin suppressor screen indicates SNARE binding controls the timing and Ca2+ cooperativity of vesicle fusion
- Massachusetts Institute of Technology, United States
- Wayne State University, United States
- Universidad Central del Caribe, Puerto Rico
- Texas Tech University Health Sciences Center, United States
Figures
Figure 1 with 1 supplement
An intragenic suppressor screen for mutations that disrupt the lethality induced by Syt1C2BD1,2N or Syt1C2BD3,4N expression.
(A) View of key residues in the Syt1 C2B Ca2+ binding pocket. The Ca2+ binding loops 1 and 3 are highlighted in blue, the negatively charged Ca2+-binding residues in green, and Ca2+ ions in red. …
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Figure 1—source data 1
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Sidak's multiple comparisons test are presented for the data in Figure 1C and Figure 2C,G,K,O.
- https://doi.org/10.7554/eLife.28409.004
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Figure 1—source data 2
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Sidak's multiple comparisons test are presented for the data in Figure 1E and Figure 2D,H,L and P.
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Figure 1—source data 3
Sample size (n), mean, SEM, and Student's t test are presented for the data in Figure 1—figure supplement 1B, C and E
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Figure 1—figure supplement 1
Summary of Western analysis of Syt1 alleles that suppress lethality following overexpression of Syt1C2BD1,2N or Syt1C2BD3,4N.
(A) Western of overexpressed Syt1 mutant proteins in head extracts of elavC155-GAL4; UAS-Syt1C2BD1,2N/+ (OE D1,2N SYT), control UAS-Syt1C2BD1,2N/+without a driver (D1,2N SYT), elavC155-GAL4; …
Figure 2 with 2 supplements
Characterization of suppressor mutation effects on DN Syt1C2BD1,2N physiology.
(A) Location of the R250 residue (yellow) in C2A at the Syt1 dimer interface. (B) Representative eEJCs recorded in 0.2 mM extracellular Ca2+ in control (black), elavC155-GAL4; UAS-Syt1C2BD1,2N (OE …
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Figure 2—source data 1
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Turkey's multiple comparisons test are presented for the data in Figure 2—figure supplement 2C,D,F,G,I,J,L,M
- https://doi.org/10.7554/eLife.28409.011
Figure 2—figure supplement 1
Postsynaptic current recordings of spontaneous release at third instar larval muscle 6 synapses in controls and in larvae overexpessing the indicated Syt1 transgenic protein by elav-GAL4 in HL3.1 saline.
Scale bar: 2 nA, 200 ms.
Figure 2—figure supplement 2
Characterization of the effects of mutant overexpression in the wildtype background.
(A) Western Blot of overexpressed Syt1 mutant proteins in head extracts of elavC155-GAL4; UAS-WT-Myc SYT (OE WT SYT), control elavC155-GAL4 without UAS (Control); elavC155-GAL4; UAS-R250H-Myc SYT …
Figure 3 with 3 supplements
Effects of the R250H mutation on synaptic vesicle fusion.
(A) Representative eEJCs recorded in 2.0 mM extracellular Ca2+ in syt1 -/- null larvae (magenta), and null mutants rescued with wildtype Syt1 (WT SYT rescue, black) or R250H Syt1 (R250H SYT rescue, …
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Figure 3—source data 1
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Sidak's multiple comparisons test are presented for the data in Figure 3B, Figure 6B and Figure 8B.
- https://doi.org/10.7554/eLife.28409.016
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Figure 3—source data 2
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Sidak's multiple comparisons test are presented for the data in Figure 3E, Figure 6E and Figure 8E.
- https://doi.org/10.7554/eLife.28409.017
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Figure 3—source data 3
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Sidak's multiple comparisons test are presented for the data in Figure 3F, Figure 6F and Figure 8F.
- https://doi.org/10.7554/eLife.28409.018
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Figure 3—source data 4
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Sidak's multiple comparisons test are presented for the data in Figure 3G, Figure 6G and Figure 8G.
- https://doi.org/10.7554/eLife.28409.019
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Figure 3—source data 5
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) multiple comparisons test are presented for the data in Figure 3—figure supplement 2
- https://doi.org/10.7554/eLife.28409.020
Figure 3—figure supplement 1
Syt1 mutant proteins normally target to the synapse.
(A) Representative NMJs stained with anti-Syt1 antiserum (magenta) in control syt1 null mutants (Null) versus null mutants rescued with wildtype Syt1 or R250H Syt1 driven by elavC155-GAL4 in the syt1…
Figure 3—figure supplement 2
Comparison of wildtype (white) animals and Syt1 transgenic rescue.
(A) Western analysis of head extracts from adults expressing the wildtype Syt1 transgenic protein driven by elavC155-GAL4 in the syt1 null background compared to controls (white) expressing …
Figure 3—figure supplement 3
Postsynaptic current recordings of spontaneous release at third instar larval muscle 6 NMJs of the indicated genotypes in HL3.1 saline with 2 mM Ca2+.
Scale bar: 1 nA, 200 ms.
Figure 4
The R199H mutation (homologous to Drosophila R250H) drastically enhances Syt1 conformational flexibility.
(A) RMSD computed for backbone atoms of mammalian Syt1 along the MD trajectory. Note a region of instability between 0.5 and 1 µs, which is followed by a conformational transition. Arrows indicate …
Figure 5
The R199H mutation destabilizes the Syt1 dimer.
(A) The dimer structure of native Syt1 is stable in the water/ion environment at the microsecond time scale. Note similar arrangements of monomers and dimers in the initial and final states. (B) The …
Figure 6
Physiological analysis of the K379E and P363S mutants on neurotransmitter release.
(A) Representative eEJCs recorded in 2.0 mM extracellular Ca2+ in syt1 -/- null larvae (magenta) and null mutants rescued with WT Syt1 (black), P363S Syt1 (purple) or K379E Syt1 (grey). (B) …
Figure 7
The Syt1-SNARE interaction surface (Zhou et al., 2015) reveals that residues S332 (S279 in mammalian Syt1), R334 (R281), Y391 (Y338), E348 (E295), and A455 (A402) are positioned at the Syt1-SNARE interface.
All five residues were identified as suppressors in our genetic screen. (A,B) Two perpendicular views of the Syt1-SNARE complex are shown. The residues listed above are shown in yellow. Orange – …
Figure 8
Effects of the S332L and R334H mutations on neurotransmitter release.
(A) Representative eEJCs recorded in 2.0 mM extracellular Ca2+ in syt1 -/- null larvae (magenta) and null mutants rescued with WT Syt1 (black), S332L Syt1 (light green) or R334H Syt1 (dark green). (B…
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Figure 8—source data 1
Sample size (n), mean, SEM are presented for the data in Figure 8H.
- https://doi.org/10.7554/eLife.28409.026
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Figure 8—source data 2
Sample size (n), mean, SEM, and Hill Fit of Null are presented for the data in Figure 8H.
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Figure 8—source data 3
Sample size (n), mean, SEM, and Hill Fit of WT SYT Rescue are presented for the data in Figure 8H.
- https://doi.org/10.7554/eLife.28409.028
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Figure 8—source data 4
Sample size (n), mean, SEM, and Hill Fit of R250H SYT Rescue are presented for the data in Figure 8H.
- https://doi.org/10.7554/eLife.28409.029
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Figure 8—source data 5
Sample size (n), mean, SEM, and Hill Fit of K379E SYT Rescue are presented for the data in Figure 8H.
- https://doi.org/10.7554/eLife.28409.030
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Figure 8—source data 6
Sample size (n), mean, SEM, and Hill Fit of S332L SYT Rescue are presented for the data in Figure 8H.
- https://doi.org/10.7554/eLife.28409.031
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Figure 8—source data 7
Sample size (n), mean, SEM, and Hill Fit of R334H SYT Rescue are presented for the data in Figure 8H.
- https://doi.org/10.7554/eLife.28409.032
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Figure 8—source data 8
Regression Analysis are presented for the data in Figure 8H.
- https://doi.org/10.7554/eLife.28409.033
Figure 9 with 1 supplement
Ultrastructural synaptic defects in syt1 mutations.
(A) Representative electron micrographs of third instar synaptic boutons from the NMJ of syt1 null mutants rescued with the indicated transgenic lines. A 2X-magnified view of the T-bar is shown in …
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Figure 9—source data 1
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Sidak's multiple comparisons test are presented for the data in Figure 9B.
- https://doi.org/10.7554/eLife.28409.036
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Figure 9—source data 2
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Sidak's multiple comparisons test are presented for the data in Figure 9C.
- https://doi.org/10.7554/eLife.28409.037
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Figure 9—source data 3
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Sidak's multiple comparisons test are presented for the data in Figure 9—figure supplement 1.
- https://doi.org/10.7554/eLife.28409.038
Figure 9—figure supplement 1
Characterization of FM 1–43 endocytosis in R250H and K379E rescue lines.
(A) Representative NMJ images for FM 1–43 internalization following 10 Hz stimulation at 0.5 min (upper panel) or 1 min (lower panel). The bouton area is outlined in white. Compared to controls …
Tables
Table 1
Summary of identified alleles that suppress lethality following overexpression of Syt1C2BD1,2N or Syt1C2BD3,4N.
The location of the residues is indicated for Drosophila melanogaster, and for the homologous rodent (Rattus norvegicus) residues. The number of independent suppressor hits for each residue is …
| Dros. AA# Change | (Rat AA#) | # Hits | Location | Stability |
|---|---|---|---|---|
| Q48 stop codon | 1 | Intravesicular | NT | |
| Q50 stop codon | 5 | Intravesicular | NT | |
| Q63 stop codon | 1 | Intravesicular | NT | |
| Q67 stop codon | 3 | Intravesicular | NT | |
| Q80 stop codon | 8 | Intravesicular | NT | |
| W113 stop codon | 5 | Transmembrane | NT | |
| R141 stop codon | 2 | Linker domain | NT | |
| K147 stop codon | 1 | Linker domain | NT | |
| Q158R/stop codon | 2 | Linker domain | NT | |
| Q191 stop codon | 1 | Linker domain | NT | |
| G194E | (G143) | 1 | C2A beta 1 | NT |
| N205 deletion | 2 | C2A beta 1 | NT | |
| V211E | (V160) | 1 | C2A beta 2 | D |
| Q215 stop codon | (Q164) | 1 | C2A beta 2 | NT |
| D229N | (D178) | 2 | C2A beta 3 | NT |
| Y231 stop codon | (Y180) | 1 | C2A beta 3 | NT |
| E245K | (E194) | 1 | C2A beta 4 | D |
| K247 stop codon | (K196) | 1 | C2A beta 4 | NT |
| R250H/C | (R199) | 3 | C2A beta 4 | S |
| P267 deletion | (P215) | 1 | C2A beta 5 | NT |
| G293D | (G241) | 1 | C2A beta 7 | NT |
| Q306 stop codon | 3 | C2A loop between beta 7 & 8 | NT | |
| W111 stop codon | (W259) | 3 | C2A beta 8 | NT |
| G321 deletion | 1 | Linker between C2A & C2B | NT | |
| Q323K/stop codon | (Q270) | 3 | Linker between C2A & C2B | NT |
| G327R | (G274) | 1 | C2B beta 1 | NT |
| S332L | (S279) | 2 | C2B beta 1 | S |
| R334H/C/L | (R281) | 6 | C2B beta 1 | S |
| Y335 stop codon | (Y282) | 1 | C2B beta 1 | NT |
| E348K | (E295) | 4 | C2B beta 2 | S |
| P363S | (P310) | 2 | C2B beta 3 | S |
| G373D | (G320) | 1 | C2B loop between beta 3 & 4 | S |
| R375 deletion | (R322) | 1 | C2B loop between beta 3 & 4 | NT |
| K379E | (K326) | 1 | C2B beta 4 | S |
| T381I/stop codon | (T328) | 3 | C2B beta 4 | D |
| P390L | (P337) | 2 | C2B beta 5 | D |
| Y391D/N/H | (Y338) | 4 | C2B beta 5 | S |
| Y392N | (Y339) | 1 | C2B beta 5 | S |
| Q404 deletion | (Q351) | 1 | C2B loop between beta 5 & 6 | NT |
| I405 stop codon(I352) | 1 | C2B loop between beta 5 & 6 | NT | |
| G421 deletion | (G368) | 1 | C2B loop between beta 6 & 7 | NT |
| G427D | (G374) | 5 | C2B beta 7 | Partial D |
| C429S | (V376) | 1 | C2B beta 7 | NT |
| W443 stop codon | (W390) | 2 | C2B loop between beta 7 & 8 | NT |
| A455T | (A402) | 3 | C2B beta 8 | S |
| Q456 stop codon | (Q403) | 2 | C2B beta 8 | NT |
| W457 stop codon | (W404) | 6 | C2B beta 8 | NT |
Additional files
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Source data 1
Sample size (n), mean, SEM, and One-way Anova (and nonparametric) Sidak's multiple comparisons test are presented for the data in Figure 9—figure supplement 1.
- https://doi.org/10.7554/eLife.28409.039
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Transparent reporting form
- https://doi.org/10.7554/eLife.28409.040
