PMC

(A) Sample track of a vesicle that disappeared during observation. Track is color-coded by instantaneous vesicle speed. (B) Parameters of vesicle image from (A) before and after vesicle disappearance indicated by a dotted line. Amplitude of the vesicle image (PSF) normalized to image bit-depth (2¹⁴-1) (Top), and localization error of vesicle position as a function of time (Bottom). (C) Half-width of a Gaussian fit to the whole-synapse image (σ, Top) and whole-synapse integrated intensity (Bottom) before (Black) and after (Red) vesicle detection was lost, for the same vesicle as in A. Raw images (insets) of vesicle image at different time points show that vesicle is still present in the synapse after detection is lost. (D) Sample track of a vesicle that undergoes multiple disappearance/reappearance events. Track is color-coded with initial track shown in black, the first reappearance in red, the second reappearance in blue. (E) Parameters of vesicle track from (D). Vesicle position over time plotted as a distance from center of the bouton (Top). Amplitude of the vesicle image (Middle) and localization error of vesicle position (Bottom) as a function of time. Periods of vesicle disappearance are highlighted in grey. The color coding is the same as in (D).

For simulation, the time constants for endocytosis and for reacidification were modeled to be variable to the stimulus condition. (A) Relative size of the alkaline vesicle population depending on the action potential number according to the “ideal” model. Endocytosis time constants were derived by deconvolution. The relative alkaline vesicle population size decreased with rising number of action potentials. The stimulus 50ap20Hz was omitted, as it was not possible to calculate an endocytosis time constant by deconvolution due to low signal-to-noise ratio. (B) Relative size of the alkaline vesicle population depending on the action potential frequency according to the “ideal” model. Endocytosis time constants were derived by deconvolution. The relative alkaline vesicle population size increased with rising number of action potential frequency. (C) Relative size of the alkaline vesicle population depending on the action potential number according to the “ideal” model. Endocytosis time constants were derived by monoexponential curve fitting from the decaying surface pool time course. The calculated relative alkaline vesicle population size reflects the experimentally defined alkaline vesicle population dynamics () except for stimulation with 50ap20Hz. (D) Relative size of the alkaline vesicle population depending on the action potential frequency according to the “ideal” model. Endocytosis time constants were derived by monoexponential curve fitting from the decaying surface pool time course. The calculated relative alkaline vesicle population size reflects the experimentally defined alkaline vesicle population dynamics () except for stimulation with 200ap80Hz.

Step-by-step illustration of the vesicle structure removal from a micrograph: (a) Vesicle in micrograph, (b) normalised vesicle, (c) windowed, normalised vesicle, (d) projection onto the windowed vesicle subspace, (e) vesicle subtraction as the projection onto the orthogonal complement of the windowed vesicle subspace.

Shown is a dendrogram illustrating the relatedness of SV2, SVOP and homologous proteins in invertebrates. To generate the dendrogram, Human SV2A, SV2B, SV2C and SVOP protein sequences were used in a series of NCBI BLASTP queries. The best BLASTP hit (lowest E-value) per paralog for each species was taken as a representative sequence. Protein sequences were aligned using the sequence alignment tool MUSCLE []. Sequence alignment was further refined using MAFFT under the Li-NSI option []. The phylogenetic tree was generated using the Maximum Likelihood method as implemented in ETE 3 []. Schematic drawings of paralogs were generated using Protter []. Protein sequences used for tree construction were: synaptic vesicle glycoprotein 2A [Homo sapiens] (NP_001315603); synaptic vesicle glycoprotein 2A [Mus musculus] (NP_071313); synaptic vesicle glycoprotein 2A [Rattus norvegicus] (NP_476558); synaptic vesicle glycoprotein 2A [Danio rerio] (XP_696434); synaptic vesicle glycoprotein 2A [Xenopus tropicalis] (XP_012825002); synaptic vesicle glycoprotein 2B [Homo sapiens] (NP_001309966); synaptic vesicle glycoprotein 2B [Mus musculus] (NP_001347503); synaptic vesicle glycoprotein 2B [Rattus norvegicus] (NP_476555); synaptic vesicle glycoprotein 2B [Danio rerio] (XP_005161575); synaptic vesicle glycoprotein 2B [Xenopus tropicalis] (XP_004912713); synaptic vesicle glycoprotein 2C [Homo sapiens] (NP_055794); synaptic vesicle glycoprotein 2C [Mus musculus] (NP_083486); synaptic vesicle glycoprotein 2C [Rattus norvegicus] (NP_113781); synaptic vesicle glycoprotein 2C [Danio rerio] (NP_001121811); synaptic vesicle glycoprotein 2C [Xenopus tropicalis] (XP_002934873); synaptic vesicle glycoprotein 2C isoform X1 [Strongylocentrotus purpuratus] (XP_003723431); synaptic vesicle glycoprotein 2C-like [Aplysia californica] (XP_012946265); synaptic vesicle 2-related protein isoform X2 [Strongylocentrotus purpuratus] (XP_003726207); synaptic vesicle 2-related protein isoform X3 [Xenopus tropicalis] (XP_002932106); synaptic vesicle 2-related protein isoform 1 [Homo sapiens] (NP_061181); synaptic vesicle 2-related protein isoform 1 [Mus musculus] (NP_081081); synaptic vesicle 2-related protein isoform 1 [Rattus norvegicus] (NP_599231); synaptic vesicle 2-related protein [Danio rerio] (XP_005165254); CG3168, isoform A [Drosophila melanogaster] (NP_572345); CG4324, isoform A [Drosophila melanogaster] (NP_611868); Dmel_CG31272 [Drosophila melanogaster] (NP_650013); svop-1 [Caenorhabditis elegans] (NP_498960.2).