Additional file 1 of Nanoscale organization of Nicastrin, the substrate receptor of the γ-secretase complex, as independent molecular domains
Creators: name: Kedia, Shekhar
name: Mandal, Kousik
name: Netrakanti, Pallavi Rao
name: Jose, Mini
name: Sisodia, Sangram S.
name: Nair, Deepak
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Evolutionary Biology
subject: FOS: Biological sciences
subject: Cancer
subject: Computational Biology

description: Additional file 1: Table S1: Summary of the quantitative estimation of morphological and biophysical properties of the different nanodomains of Nicastrin obtained using STED microscopy. Figure S1. Distribution of Nicastrin within functional zones of an excitatory synapse using Airyscan super resolution microscopy. A, C Indicate the pseudocolour coded distribution of the post- (Shank2 and PSD95) and pre-synaptic markers (Bassoon and Piccolo). B, D Indicate the pseudocolour coded distribution of Nicastrin and overlay with corresponding markers for functional zones of the synapse. The pseudocolour overlay of Nicastrin (green) with the postsynaptic marker Shank2 in red and the presynaptic marker Bassoon in blue is shown in B. The pseudocolour overlay of Nicastrin (green) with the postsynaptic marker PSD95 in red and the presynaptic marker Piccolo in blue is shown in D. E Magnified view of the boxed regions from pseudocolour overlay in B and D. Scale bar in B, D indicate 15 μm and in E 2 μm. F Represents line scans connecting the centroids of pre- and post-synaptic reference molecules, indicating the distribution of Nicastrin with Shank2, Bassoon and PSD95, Piccolo. The X-and Y-axis represent the length (μm) and normalized intensity (a.u.) respectively. Figure S2. Quantification of the nanoscale architecture of Nicastrin clusters within different functional zones of a synapse and on neuronal processes using STED microscopy. A, B Diversity in Nicastrin (median/IQR 25–75% interval) clusters with respect to nanodomain length A and intensity B in pre/post/perisynapse. Significance was determined by Kruskal-Wallis test followed by Dunn’s multiple comparison test. Indications of significance correspond to P values *P ≤ 0.05, **P ≤ 0.01,and ***P ≤ 0.001, ns P &gt; 0.05. n = 5155 (pre), 3016 (post) and 3966 (peri) puncta from 3-4 biological repeats. C, D Indicate the nanoscale architecture of Nicastrin clusters on the neuronal processes. The distribution of the length of Nicastrin nanodomains is shown in C and the intensity in D. n = 3521nanodomains from 3-4 biological repeats. Figure S3. Distribution of Nicastrin in inhibitory neurons and within inhibitory synapses of pyramidal neurons using confocal microscopy. A Evaluation of the presence of Nicastrin in Parvalbumin expressing GABAergic inhibitory neurons. The pseudocolour overlay represents Nicastrin in green and parvalbuminin red. The white arrows indicate the presence of both Nicastrin and parvalbumin, while the red arrows indicate the presence of Nicastrin in the absence of parvalbumin. Scale bar in A indicate 28 μm (upper panel) and 11 μm (lowerpanel). B Evaluation of the presence of Nicastrin in Parvalbumin/Calbindin expressing GABAergic inhibitory neurons. The pseudocolour overlay represents Nicastrin in green, parvalbumin in red and Calbindin in blue. Scale bar in B indicate 18 μm. C Indicate the distribution of Nicastrin within inhibitory synapses of pyramidal neurons. The pseudocolour overlay indicates Nicastrin ingreen and Gephyrin, a marker for the inhibitory postsynapses in magenta. The blue arrows indicate the clusters of Nicastrin overlapping with Gephyrin, while the yellow arrows indicate their independent distribution. Scale bar in C indicate 11 μm (left) and 2 μm (right, inset). Figure S4. Discrete nanoscale association of Nicastrin with PS1 and APP on neuronal processes using STED microscopy. A STED image of Nicastrin (magenta) with pseudocolour overlay of PS1 (green). (1, 2, 3) are magnifiedinsets of regions indicated in A. B STED image of Nicastrin (magenta) withpseudocolour overlay of APP (green). (1, 2, 3) are magnified insets of regions indicated in B. Scale bar in A, B indicates 3 μm (left) and 750 nm (right).The black contours represent segmented regions marking the continuous stretc.hof neuronal processes, marking the presence of Nicastrin. Figure S5. Quantification of the nanoscale architecture of Nicastrin clusters associating with PS1 and APP on neuronal processes using STED microscopy. A, B Indicate the distribution of the length A and intensity B of Nicastrin nanodomains with PS1 and APP. C, D Diversity in nanodomain length C and intensity D of Nicastrin clusters (median/IQR25–75% interval) associating with PS1 and APP. Significance was determined by unpaired two-tailed Mann-Whitney test. E, F Comparison of RSP E and RSE F for quantifying colocalization of Nicastrin with respect to PS1 and APP. The data are represented as mean ± SEM. Significance was determined by two-tailed unpaired Student’s t-test with Welch’s correction. Indications of significance correspond to P values *P ≤0.05, **P ≤ 0.01, and ***P ≤ 0.001, ns P &gt; 0.05. n = 5417 (Nicastrin on PS1) and 7163 (Nicastrinon APP) puncta from 3-4 biological repeats. Figure S6. Identification of synapse associated endocytic zone. A, B The workflow to detect synapse associated Dynamin (endocytic zones) using a combination of both super resolution (STED) and conventional (confocal) imaging paradigms. A The confocal image of PSD marker and STED image of Dynamin were selected and thresholded to detect regions with high molecular content. They were then size filtered and obtained regions were converted into binary images. B The overlay of the masks confirms the overlap of segmented clusters of Dynamin with PSD. The automated evaluation of synaptic masks in the Dynamin positive domains was performed. Absence of Dynamin in PSD positive regions (green regions) were considered as negative, presence of both markers as positive (red regions) and dynamin alone as false positive (magenta regions). The paradigm involves sequential segmentation protocols to isolate Dynamin that is associated with the synaptic compartments and marking these segmented regions as synaptic endocytic zones. Scale bar in A, B indicate 5 μm. (Please refer to Supplementary information (Additional file 1: Materials and methods)) Figure S7. Identification of Dynamin associated with Clathrin. A Pseudocolour overlay of super resolved images of Clathrin (green) associated with Dynamin (Magenta) obtained using STED microscopy. B The workflow on super resolution images (STED) to detect Dynamin clusters colocalized with Clathrin, another marker for endocytic zone. The STED image of endocytic markers namely, Clathrin and Dynamin were selected and thresholded to detect regions with high molecular content. They were then size filtered and obtained regions were converted into binary images. The overlay of the masks confirms overlap of segmented clusters of Clathrin with Dynamin. Automated evaluation of Clathrin in the Dynamin positive domains was performed. For this purpose, the automatically detected Dynamin positive regions were transferred to the binary masks positive for Clathrin. Absence of Clathrin signal in Dynamin positive region was considered as negative, and those regions were not considered as endocytic regions. This segmentation protocol selectively evaluates domains enriched in both Clathrin and Dynamin, and therefore the presence of functional endocytic machinery. Scale bar in A indicate 5 μm and in B 2.25 μm. (Please Refer to Supplementary information (Additional file 1: Materials and methods)).