Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses
- Okinawa Institute of Science and Technology Graduate University, Japan
Figures
Figure 1 with 3 supplements
Autoregressive motion analysis reveals high and broad range of SV mobilites in cultured giant terminals.
(A) Confocal z-stack imaging of a giant presynaptic terminal expressing cytosolic GFP- (Green) and Q655-Syt2 (Red)-labeled vesicles; corresponding volume rendering of GFP terminal and SV detection …
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Figure 1—source data 1
Data and statistics for Figure 1E.
- https://doi.org/10.7554/eLife.24845.004
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Figure 1—source data 2
Data and statistics for Figure 1—figure supplement 1B.
- https://doi.org/10.7554/eLife.24845.005
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Figure 1—source data 3
Data and statistics for Figure 1—figure supplement 2D.
- https://doi.org/10.7554/eLife.24845.006
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Figure 1—source data 4
Data and statistics for Figure 1—figure supplement 3D.
- https://doi.org/10.7554/eLife.24845.007
Figure 1—figure supplement 1
Q655-Syt2 labels SV more efficiently than Q655 alone.
(A) Volume rendering of GFP terminal and SV detection in calyceal terminals loaded with Q655 only or Q655-Syt2 after 16 hr. (B) Comparison of the number of SVs detected in calyceal terminals loaded …
Figure 1—figure supplement 2
Labeling and tracking of SVs with C5E-Syt2.
(A) Confocal z-stack imaging of a giant presynaptic terminal expressing cytosolic GFP (Green) and C5E-Syt2 (Red)-labeled vesicles. (B) Visualization and quantification of exocytosis induced by bath …
Figure 1—figure supplement 3
Newly retrieved SVs have lower mobilities and restricted distributions in giant terminals.
(A) Confocal images showing the co-localization of SVs labeled overnight with Q655-Syt2 (Red) and for 1 hr or 3 hr with Q585-Syt2 (Green), co-localization (white). (B) Comparison of the Pearson …
Figure 2 with 3 supplements
Fast and heterogeneous SV movements occur at giant calyceal synapses.
(A) Live confocal imaging of a giant calyceal terminal expressing cytosolic GFP- and Q655-Syt2-labeled vesicles, with SV tracking, color-coded over time, or sorted according to trajectory lengths …
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Figure 2—source data 1
Data and statistics for Figure 2E.
- https://doi.org/10.7554/eLife.24845.014
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Figure 2—source data 2
Data and statistics for Figure 2—figure supplement 2E and H.
- https://doi.org/10.7554/eLife.24845.015
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Figure 2—source data 3
Data and statistics for Figure 2—figure supplement 3B.
- https://doi.org/10.7554/eLife.24845.016
Figure 2—figure supplement 1
Comparison of calyceal and hippocampal cultures.
(A) Maximum intensity projection image of confocal z-stack showing giant calyceal terminals (arrowheads) over-expressing cytosolic GFP after 18 days in culture (left panel) and hippocampal neurons …
Figure 2—figure supplement 2
Mobility and displacement modality of SVs in giant calyceal terminals.
(A) Live confocal imaging of a calyceal terminal expressing cytosolic GFP and Q655-Syt2-labeled vesicles, and individual SV tracking sorted according to trajectory lengths (Blue <2 µm, Green 2–4 µm …
Figure 2—figure supplement 3
Data acquisition rate does not affect SV tracking.
(A) Syt2-C5E-loaded SV tracking sorted according to trajectory lengths (Blue <2 µm, Green 2–4 µm and Red >4 µm) at three different image acquisition speed (0.5 s, 1 s and 2 s per image). Trajectory …
Figure 3
Small and homogeneous SV movements occur at small conventional synapses.
(A) Live confocal imaging of a hippocampal bouton expressing cytosolic GFP- and Q655-Syt2-labeled vesicles, with SV tracking color-coded over time, or sorted according to trajectory lengths (Blue <2 …
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Figure 3—source data 1
Data and statistics for Figure 3E.
- https://doi.org/10.7554/eLife.24845.023
Figure 4 with 1 supplement
SV mobility decreases after morphological maturation of giant terminals.
(A) Live confocal imaging of a giant immature terminal expressing cytosolic GFP and Q655-Syt2-labeled vesicles, with SV tracking color-coded over time, or sorted according to trajectory length (Blue …
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Figure 4—source data 1
Data and statistics for Figure 4D and E.
- https://doi.org/10.7554/eLife.24845.025
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Figure 4—source data 2
Data and statistics for Figure 4—figure supplement 1D.
- https://doi.org/10.7554/eLife.24845.026
Figure 4—figure supplement 1
Morphological maturation of giant terminals involves a developmental switch in SV mobility.
(A) Confocal image showing GFP-over-expressing presynaptic neurons overlaid with 3D rendering of giant terminals from different stages of maturation (stages 1–2: immature terminals; Stages 3–4: …
Figure 5 with 1 supplement
Vesicular glutamate transporter isoforms regulate SV dynamic properties.
(A) Confocal z-stack imaging of giant presynaptic terminal expressing Venus-VGLUT1 (Green). (B) Confocal z-stack imaging of giant presynaptic terminal expressing Venus-VGLUT2 (Green). (C) …
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Figure 5—source data 1
Data and statistics for Figure 5C, F and G.
- https://doi.org/10.7554/eLife.24845.029
Figure 5—figure supplement 1
Localization of endogenous vesicular glutamate transporter isoforms in cultured giant calyceal terminals.
Confocal imaging of calyceal terminals labeled with antibodies against VGLUT1 or VGLUT2.
Figure 6 with 2 supplements
Presynaptic MT network regulates long and rapid directional SV movements.
(A) Confocal z-stack imaging of a calyceal terminal labelled with antibodies against de-tyrosinated α-tubulin (Red), VGLUT1 (Green) and DAPI (Blue). (B) Live confocal imaging of a calyceal terminal …
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Figure 6—source data 1
Data and statistics for Figure 6F and G.
- https://doi.org/10.7554/eLife.24845.032
Figure 6—figure supplement 1
Microtubules and kinesins localize in giant calyceal terminals.
(A) Upper panels: confocal z-stack imaging of giant terminals in culture for 18 days and labeled with antibodies against de-tyrosinated α-tubulin (Red), VGLUT1 (Green) and DAPI (Blue). Lower panels: …
Figure 6—figure supplement 2
Actin network localizes in presynaptic swellings.
(A) Live confocal imaging of SiR-Actin in giant cultured terminals over-expressing cytosolic GFP. (B) Volume rendering of SiR-Actin and SiR-Tubulin in GFP-overexpressing giant terminals. (C) …
Figure 7 with 1 supplement
Synaptic stimulation does not increase SV mobility.
Analysis of C5E-Syt2-labeled SVs in giant calyceal terminals. (A) KCl stimulation: Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces in control …
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Figure 7—source data 1
Data and statistics for Figure 7D, E and F.
- https://doi.org/10.7554/eLife.24845.036
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Figure 7—source data 2
Data and statistics for Figure 7—figure supplement 1C.
- https://doi.org/10.7554/eLife.24845.037
Figure 7—figure supplement 1
SV mobility does not change after spontaneous or stimulated uptake.
(A) Upper panels: confocal images of C5E-Syt2 labeled vesicles loaded during spontaneous activity for 1 hr or during train of 1 Hz electrical stimulation. Lower panel: Number (N) and fluorescence …
Figure 8
Electrical stimulation does not affect SV mobility within or outside of active zone.
(A) Live confocal imaging of surface GluR1/2-Cy3 and Syt2-C5E-loaded SVs in GFP over-expressing giant calyceal terminal. (B) SV tracking color-coded according to trajectory length (Blue < 2 µm, …
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Figure 8—source data 1
Data and statistics for Figure 8C and D.
- https://doi.org/10.7554/eLife.24845.040
Videos
Video 1
Volume rendering of GFP-expressing giant calyceal terminal and spot detection of Q655-Syt2-labeled SVs (color-coded according to their z position).
https://doi.org/10.7554/eLife.24845.011
Video 2
Tracking of Q655-Syt2-labeled SVs using autoregressive (Red) or Brownian (Blue) motion algorithm.
https://doi.org/10.7554/eLife.24845.012
Video 3
Tracking of Q655-Syt2-labeled SVs color-coded according to SV trajectory lengths (Blue: short, Green: intermediate and Red: long trajectories).
https://doi.org/10.7554/eLife.24845.020
Video 4
