CAPS-1 promotes fusion competence of stationary dense-core vesicles in presynaptic terminals of mammalian neurons
- VU University Amsterdam, Netherlands
- VU University Medical Center, Netherlands
Figures
Figure 1 with 2 supplements
CAPS-1 clusters are present at synaptic and extra-synaptic sites and partly co-localize with DCVs.
(A) Example image of a hippocampal neuron (DIV 14) stained for endogenous CAPS-1 (green), dendrite marker MAP2 (blue) and synapse marker VGLUT1 (red). Scale bar 10 µm. (B) Zoom of a neurite stained …
Figure 1—figure supplement 1
Specificity of CAPS-1 antibody.
(A) CAPS-1 immunoreactivity (green) and co-localization with synaptobrevin (VAMP, red) in CAPS-2 KO neurons stained for the dendritic marker MAP2 (blue) (top panel). Bottom panel: absence of …
Figure 1—figure supplement 2
Endogenous DCV marker (chromogranin B) distribution in isolated single neurons.
(A) Confocal image of a WT hippocampal neuron (DIV 14) labeled with antibodies for the dendritic marker MAP2 (green), endogenous DCV cargo protein ChgB (red) and the synaptic marker VGLUT1 (blue). A …
Figure 2
CAPS deletion does not influence DCV peptide loading or DCV biogenesis in hippocampal neurons.
(A) Frequency distribution of fluorescence intensity increase (ΔF) of individual DCV fusion events in WT and CAPS DKO neurons. No major differences are observed in ΔF of fusion events between WT and …
Figure 3 with 1 supplement
CAPS deletion reduces DCV fusion in hippocampal neurons.
(A) Schematic drawing of a neuronal mass culture used in these experiments in which one neuron expresses a fluorescently tagged morphology marker (green) and a DCV marker (red). Non-labeled neurons …
Figure 3—figure supplement 1
Different fusion events reported by SemapHluorin.
Cartoon depicting the different fusion events reported by SemapHluorin and analyzed in depth in de Wit et al., 2009. Electrical stimulation triggers calcium and SNARE protein-dependent membrane …
Figure 4
CAPS-1 deletion impairs DCV fusion in isolated neurons.
(A) Schematic drawing of a single isolated neuron grown on a micro island of astrocytes and expressing fluorescently tagged morphology marker (green) and a synapse marker (red) in addition to either …
Figure 5 with 1 supplement
Deletion of CAPS-1 affects fusion of stationary DCVs.
(A) Stills from DCV fusion assay. Electrical stimulation starts at second 10. Yellow arrowheads indicate stationary vesicles that fuse, red arrowhead indicates a stationary vesicle that does not …
Figure 5—figure supplement 1
DCV docking definition and zooms of Figure 5F.
Zooms of three example DCVs showing the classification of docked versus undocked vesicles. We define a docked DCV as a vesicle containing a dense core that has no detectable distance between the …
Figure 6
CAPS-1-EYFP fusion protein replaces CAPS-1 in DCV secretion and synaptic transmission.
(A) Isolated single CAPS DKO neuron grown on glia micro-island expressing CAPS-1-EYFP (green) and synapsin-ECFP (blue). Top panels: maximum projection of the entire neuron, Scale bar 10 µm. Bottom …
Figure 7
Localization of CAPS-1 at synapses is calcium dependent.
(A and B) Grey scale images of synapsin-ECFP (left) and CAPS-1-EYFP (right) labeled synapses showing the same region before (pre-15s) during (stim-22s) and after (post-150s) electrical stimulation …
Figure 8
Presence of CAPS-1 increases DCV release probability at single synapses.
(A) Left top and bottom panels: kymographs of CAPS-1-EYFP and NPY-mCherry imaged simultaneously (acquisition frequency 2 Hz) for 90 s and stimulated at second 30 (dashed box, 16×50 AP at 50 Hz). …
