Reexamination of N-terminal domains of syntaxin-1 in vesicle fusion from central murine synapses
- Universität Berlin, Humboldt-Universität zu Berlin, Germany
- Berlin Institute of Health, Germany
- Einstein Center for Neurosciences Berlin, Germany
Figures
Figure 1 with 1 supplement
STX1A’s Habc-domain is essential and N-peptide is dispensable for neurotransmitter release.
(A) Domain structure of STX1A. The protein consists of a short N-peptide (aa 1–9 or 1–28), Habc domain (aa 29–144) formed by three helices, Ha, Hb, and Hc, followed by the H3 helix (aa 189–259; …
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Figure 1—source data 1
Quantification of the STX1AWT and mutant STX1A expression induced by lentiviral transduction of STX1-null neurons and the consequent neurotransmitter release properties.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
STX1A∆Habc expression cannot be detected.
(A) Example images (left) and quantification of immunofluorescence labeling for Bassoon and STX1A shown as red and green, respectively, in the corresponding composite pseudocolored images obtained …
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Figure 1—figure supplement 1—source data 1
Quantification of the expression of FLAG-tagged WT and mutant STX1A.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig2-data2-v3.xlsx
Figure 2
STX1’s Habc-domain is essential for the overall function of STX1A.
(A) Example images of high-density cultures of STX1-null, STX1AWT, and STX1A∆Habc hippocampal neurons at DIV 8, 15, 22, and 29 represented with immunofluorescent labeling of microtubule associated …
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Figure 2—source data 1
Quantification of the neuronal density at different time intervals and quantification of ultrastructural synaptic properties in high density cultures of STX1-null, STX1AWT, and STX1AΔΗabc neurons.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig2-data1-v3.xlsx
Figure 3 with 2 supplements
Deletion of the entire N-terminal stretch does not impair neurotransmitter release.
(A) Example images of immunofluorescence labeling for Bassoon, STX1A, and Munc18-1 shown as red, green, and blue, respectively, in the corresponding composite pseudocolored images obtained from …
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Figure 3—source data 1
Quantification of the lentiviral expression of STX1AWT and STX1AΔΝ mutants in STX1-null neurons and the consequent neurotransmitter release properties.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Phosphorylation of S14 of STX1 has no effect on synaptic transmission.
(A) Example images of immunofluorescence labeling for Bassoon, STX1A, and Munc18-1 shown as red, green, and blue, respectively, in the corresponding composite pseudocolored images obtained from …
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Figure 3—figure supplement 1—source data 1
Quantification of lentiviral expression of phosphonull and phosphomimetic STX1A mutants and the consequent neurotransmitter release properties.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig5-data5-v3.xlsx
Figure 3—figure supplement 2
Modifications of STX1’s N-peptide either by deletions or phosphorylation does not compromise neuronal viability.
(A) Example images of high-density cultures of STX1-null hippocampal neurons at DIV 8 and 29 represented with immunofluorescent labeling of microtubule associated protein 2 (MAP2). Red and green …
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Figure 3—figure supplement 2—source data 1
Quantification of neuronal density of STX1AΔΝ and phosphorylation mutant neurons in comparison to STX1AWT and STX1-null neurons at DIV 8 and DIV 29.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig6-data6-v3.xlsx
Figure 4 with 3 supplements
‘Opening’ of STX1A in combination with the deletion of its entire N-terminal stretch does not impair neurotransmitter release.
(A) Example images of immunofluorescence labeling for Bassoon, STX1A, and Munc18-1 shown as red, green, and blue, respectively, in the corresponding composite pseudocolored images obtained from …
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Figure 4—source data 1
Quantification of lentiviral expression of STX1ALEOpen and STX1ALEOpen + ΔΝ mutants in STX1-null neurons and the consequent neurotransmitter release properties.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Interruption of both Munc18-1 binding modes of STX1 ultimately leads to neuronal death.
(A) Example images of high-density cultures of STX1-null hippocampal neurons at DIV 8, 15, 22, 29, 36, and 43 represented with immunofluorescent labeling of microtubule associated protein 2 (MAP2). …
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Figure 4—figure supplement 1—source data 1
Quantification of neuronal density of neurons expressing STX1ALEOpen or STX1ALEOpen + ΔΝ mutants at different time intervals.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig8-data8-v3.xlsx
Figure 4—figure supplement 2
Reducing the expression levels of STX1AWT or STX1ALEOpen does not alter their synaptic release properties.
(A) Example images of immunofluorescence labeling for VGlut1, STX1A, Munc18-1, and NLS-GFP shown as red, green, blue, and gray, respectively, in autaptic STX1-null hippocampal neurons rescued with …
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Figure 4—figure supplement 2—source data 1
Quantification of the effects lentiviral downtitration of STX1AWT and STX1ALEOpen on STX1A's and Munc18-1's expression levels and on neurotransmitter release parameters.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig9-data9-v3.xlsx
Figure 4—figure supplement 3
Exogenous expression of STX1A using 1× volume of lentiviral particles is approximately threefold higher than endogenous STX1A expression.
(A) Example images of immunofluorescence labeling for VGlut1, STX1A, and Munc18-1. Scale bar: 10 µm. (B) Quantification of the immunofluorescence intensity of STX1A as normalized to the …
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Figure 4—figure supplement 3—source data 1
Comparison of the lentiviral expression of STX1A with its endogenous expression.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig10-data10-v3.xlsx
Figure 5 with 1 supplement
‘Opening’ of STX1A in combination with the deletion of its entire N-terminal stretch reduces the number of docked synaptic vesicles (Svs).
(A) Example high-pressure freezing fixation combined with electron microscopy (HPF-EM) images of nerve terminals from high-density cultures of STX1AWT, STX1A∆N2-28, STX1ALEOpen, and STX1ALEOpen + …
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Figure 5—source data 1
Quantification of the ultrastructural synaptic properties of STX1AWT, STX1AΔΝ2-28, STX1ALEOpen, and STX1ALEOpen + ΔΝ2-28 neurons.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Synapse number and area are not affected neither by LEOpen mutation nor by LEOpen + ΔN mutation.
(A) Quantification of VGlut1-positive puncta using the images exemplified in Figure 4—figure supplement 2 and obtained from STX1AWT, STX1ALEOpen, and STX1ALEOpen + ∆N2-28 neurons. (B) Quantification …
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Figure 5—figure supplement 1—source data 1
Quantification of the number the total area of VGlut1 positive puncta in autaptic neurons expressing STX1AWT, STX1ALEOpen, or STX1ALEOpen + ΔΝ2-28.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig12-data12-v3.xlsx
Figure 6 with 2 supplements
STX1A’s N-peptide has a modulatory function in short-term plasticity and Ca2+-sensitivity of synaptic transmission.
(A) Example traces (left) and quantification (right) of STP measured by 50 stimulations at 10 Hz from STX1AWT, STX1A∆N2-9, STX1A∆N2-19, or STX1A∆N2-28 neurons. The traces show the absolute values, …
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Figure 6—source data 1
Quantification of the STP, recovery of RRP, RRP fraction released by 250 mM sucrose solution application and Ca2+-sensitivity of the vesicles in neurons expressing STX1AWT, STX1AΔΝ- or STX1ALEOpen mutants.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
Deletion of N-peptide increases the paired-pulse ratio (PPR) both in closed and open conformation of STX1A in low extracellular Ca2+-concentration.
(A) Quantification of absolute values of 50 EPSCs elicited at 10 Hz recorded from neurons expressing either STX1AWT, STX1A∆N2-9, STX1A∆N2-19, or STX1A∆N2-28. (B) Example traces (left) and …
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Figure 6—figure supplement 1—source data 1
Quantification of the absolute values of EPSCs during STP and Ca2+-sensitivity of the vesicles in neurons expressing STX1AWT, STX1AΔΝ- or STX1ALEOpen mutants.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig14-data14-v3.xlsx
Figure 6—figure supplement 2
Zoomed-in example traces of STP.
(A) Zoomed-in example traces of the first and last five EPSCs recorded by 50 stimulations at 10 Hz from STX1AWT, STX1A∆N2-9, STX1A∆N2-19, or STX1A∆N2-28 neurons. The traces show the absolute values. …
Figure 7 with 1 supplement
Ca2+-influx is reduced in STX1-null and in STX1LEOpen neurons.
(A, B) Example images and average of SynGCaMP6f fluorescence as (ΔF/F0) in STX1-null neurons either not rescued or rescued with STX1AWT, STX1A∆N2-9, STX1A∆N2-19, STX1ALEOpen, or STX1ALEOpen + ∆N2-28.…
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Figure 7—source data 1
Quantification of the increase in SynGCaMP6f signal recorded at baseline or different numbers of APs in neurons expressing STX1AWT, STX1AΔΝ- or STX1ALEOpen mutants.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig7-data1-v3.xlsx
Figure 7—figure supplement 1
Reducing the expression level of STX1AWT does not alter Ca2+-influx.
(A, B) Example images and average of SynGCaMP6f fluorescence as (ΔF/F0) in STX1-null neurons either not rescued or rescued with 1× or 1/12× viral volume for STX1AWT. The images were recorded at …
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Figure 7—figure supplement 1—source data 1
Quantification of the increase in SynGCaMP6f signal recorded at baseline or different numbers of APs in neurons expressing STX1AWT at low level.
- https://cdn.elifesciences.org/articles/69498/elife-69498-fig16-data16-v3.xlsx
Figure 8
Speculative model of effects of N-peptide deletions and LEOpen mutation on vesicular release.
(A) Native state of STX1A. (B) N-peptide deletion of STX1A leads to a decrease in Ca2+-sensitivity of vesicular release and short-term depression (STD) upon 10 Hz stimulation potentially through …
