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. 2017 Jul 31;114(33):E6857–E6866. doi: 10.1073/pnas.1705623114

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Fig. 3. — Analysis of average x and z profiles of SC proteins. (A) Image analysis workflow. Segments were traced of either flat SC with two clearly observable C(3)G-C tracks (type 1 SC) or turned SC where two C(3)G-C tracks were no longer distinguishable (type 2 SC). In type 1 SC, the microscope axes (XYZ) match the SC axes (xyz); however, in type 2 SC, the SC is turned on its side, placing the SC x-axis along the Z-axis of the microscope and the SC z-axis into the XY microscope plane. Using ImageJ, segments were straightened in 3D along the y-axis of the SC and each slice was then projected along the y-axis to create the average z profile. For type 2 SC, images were rotated to position the x-axis of the SC on the bottom for ease of viewing. Line profiles were drawn on the averaged xz images along either the x-axis (type 1 SC) or the z-axis (type 2 SC), as shown. Then line profiles were averaged together to plot the average distribution of fluorescence intensity. (Scale bars: expanded distances, 250 nm.) (B and C) Representative averaged xz images for type 1 SC (B) and type 2 SC (C) labeled for C(3)G-C (blue), Corolla (pink), CONA (yellow), N-C(3)G (red), and C(2)M (green). The variation observed in these images reflects the distortion from the microscope Z-axis; for a perfect image, the SC must lie completely flat with its side to the microscope, but type 2 SC frequently turns and twists and thus shows more variability than type 1 images. (Scale bars: expanded distances, 250 nm.) (D and E) Multiple line profiles along the x-axis [D: N-C(3)G, n = 21 SC fragments from 8 nuclei; C(3)G-C, n = 21 SC fragments from 8 nuclei; Corolla, n = 7 SC fragments from 6 nuclei; CONA, n = 9 SC fragments from 7 nuclei; C(2)M, n = 9 SC fragments from 4 nuclei] or z-axis [E: N-C(3)G, n = 15 SC fragments from 8 nuclei; C(3)G-C, n = 10 SC fragments from 8 nuclei; Corolla, n = 12 SC fragments from 6 nuclei; CONA, n = 12 SC fragments from 7 nuclei; C(2)M, n = 15 SC fragments from 4 nuclei] were averaged together and then mirrored to generate the distribution of the SC components along the axes. Error bars indicate SE. For both distributions, an expansion factor correction was applied (Materials and Methods) to determine the approximate unexpanded distances in nm. (F) Modeled positions of C(3)G-C (blue), Corolla (pink), CONA (yellow), N-C(3)G (red), and C(2)M (green) based on the line profiles in D and E. (Scale bar: biological distance, 50 nm.)