Measurement of inner and outer synaptic vesicle diameter.

<p>In order to get an approximate value of the discrepancies between vesicle diameters of manual and automated measurement we applied the Fiji measurement tool. Fig 3 (A) shows the inner diameter of a vesicle that was annotated by 3D ART VeSElecT, (B) shows the outer diameter. Fig 3 (C) gives the results of the discrepancy of inner and outer diameter of all measured vesicles shown as a histogram (number of measurements = 80).</p

Workflow of vesicle annotation using 3D ART VeSElecT.

<p>First, the automated registration macro is used, which scales the tomogram via user input of the pixel size and applies various filters in the preprocessing step. Afterwards the foreground is separated, the user semi-automatically selects an area of interest, and the macro applies the watershed algorithm for vesicle segmentation and registration. Second, an optional manual proof-reading step can be applied here, if necessary. Finally, the automatic measurement macro is used to extract results using certain characteristics. All manual steps are colored in yellow, semi-automated steps are in turquoise, automated steps are in blue.</p

Tabular schedule showing required time for semi-automated reconstruction.

<p>Tabular schedule showing required time for semi-automated reconstruction.</p

Error rate of zebrafish tomograms.

<p>Error rate of zebrafish tomograms.</p

Analysis of embryonic zebrafish NMJ using 3D ART VeSElecT in comparison to manual analysis using IMOD.

<p>We show in Fig 2 A) the original tomogram of 4dpf zebrafish NMJ, in Fig 2 B) the manual reconstruction is included in the tomogram of A), in B') the 3D reconstruction of the manual annotation (vesicles are colored in light blue) is shown. This is compared to Fig 2 C) which shows the semi-automated vesicle recognition overlaid with the original tomogram, and C') which shows the vesicle pool of the semi-automated annotation as 3D reconstruction (vesicles are in arbitrary colors). In D) boxplots show the results of the comparison of 4dpf and 8dpf zebrafish embryos using manual annotation (left) and semi-automated annotation (right). The box of the box plots shows the mid-50% of data. The line in the box represents the median of all data. Whiskers end at lowest value within 1.5 interquartile range (IQR) of the lower quartile and at the highest value within 1.5 IQR of the upper quartile. Data that is not included in between both whiskers are plotted as outliers with a dot.</p

Tissue organisation.

<p>A - Tissue produced in a pore made of 4 adjacent circles and stained for actin stress fibres and myosin IIb. Actin fibres colocalised with myosin IIb are present on the whole surface but their higher density on concave interfaces suggests a local higher stress state of the cells. B - Tissue is made of cells and collagen. Nuclei (<i>red</i>), actin stress fibres (<i>green</i>) and collagen fibres (<i>visualized by polarized microscopy</i>) are oriented parallel to the interface. The white arrows show polarisation direction. C - The homogeneous distribution of nuclei shows that cell density is independent of geometry and suggests a local dependence of cell proliferation on the local curvature. D - An example of a convex HA surface (D35) on which only a mono-layer of tissue was formed.</p

Data_Sheet_1_Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation.docx

IntroductionNeurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release.MethodsWe employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system.Results and discussionElectrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels.</p

Quantitative results: curvature profile and growth rate.

<p>Quantitative analysis of tissue growth in circular pores (<i>CIR</i>) and on semi-circular channels (<i>SC</i>) of 1 mm diameter. A - The average curvature along the perimeter of the circular pore and on a given portion of the semi-circular surfaces is measured on experimental images at different culture times. As theoretically circular pores should be filled in about 432 steps or 25.4 days. B - The projected tissue area (PTA) is normalised by the area of the pore (PA) at D4 (reference) and reported as a function of culture time. In A and B, the full lines correspond to the prediction given by CCTG (; ). A lag time is used to overlap simulated and experimental data ( and ). C - Growth rates are calculated between D7 and D14 with the experimental and the simulated data as well as data simulated on ideal geometries with a radius derived from the experimental images. ANOVA analysis shows no significant differences between the methods used but a statistical difference in the tissue growth rates achieved in CIR and SC (). Dots and error bars represent mean values and standard errors, respectively ().</p

Experimental protocol.

<p>A - Moulds are produced by rapid prototyping. A build wax (blue) is used to print the mould in 3 D. A support wax (red) is added to reinforce the object while printing, and then removed by dissolution. B - Hydroxyapatite slurry is cast into the moulds, slowly dried and sintered. C - Pre-osteoblast cells are seeded (105 cells/cm²) on the scaffolds and cultured for 28 days. D - Tissue growth is quantified by phase contrast microscopy twice a week by measuring the projected tissue area (PTA) in each pore.</p

Importance of boundary conditions.

<p>Tissue growth (orange) is simulated on different artificial images using the CCTG description (A to F). The predicted PTA is reported as a function of iteration steps. Each initial interface (black) contains a semi-circle with a radius of 0.5 mm. The different boundary conditions show the influence expected on tissue growth rate and organisation. On A, B and C, the model predicts that the sharper the convex corners, the slower the growth. Tissue is eventually deposited on convex surfaces after the surroundings have been filled and the interface has locally become concave (red arrows). Comparing A, D and E reveals that shifting the convex corners upward prolongs the duration of a constant growth rate which is half of the one obtained in a full circle (F). Tissue deposition can expand on the walls until it reaches the convex corners. From this time point (inset), the surface joining the pinning points is minimised, which decreases the curvature and slows the growth.</p