Figure 4.
CSPP1 binds to MT lumen. (A) Denoised tomograms of dynamic MTs polymerized from GMPCPP-stabilized seeds in the presence or absence of 10 nM GFP-CSPP-L, with or without 250 nM vinblastine vitrified on EM grids. Scale bar, 50 nm. (B) Quantification of the percentage of MTs containing luminal densities from total MTs (from tomograms as shown in A). Orange and gray dots (single data points, tomograms), black circle (mean), SD (error bars). Number of MTs and tomograms are displayed in the graph. ***, P < 0.001, Mann-Whitney test. Analysis from two independent experiments. (C) Reconstituted images from automated segmentation of denoised tomograms as in A. Scale bar, 50 nm. (D and E) Single color 2D-MINFLUX measurements of in vitro reconstituted MTs polymerized in the presence of SNAP-CSPP-L (D) or SNAP-MTB-LZ (E). Images were rendered with 1 nm voxel size for visualization. White boxes in confocal image indicate the region shown in the rendered 2D-MINFLUX image, yellow boxes in the 2D-MINFLUX image indicate the region of the zoom, red dashed lines represent the MT outline from the confocal image. Scale bars, 5 μm (confocal image); 500 nm (2D-MINFLUX image and zoom). (F) Quantification of the fitted, FWHM values per MT (from 2D-MINFLUX images as shown in D and E; see Fig. S3 C analysis details). Single data points are shown. Error bars represent SD. Number of measured MTs; CSPP-L, n = 23; MTB+LZ, n = 50. Analysis from three independent experiments. (G and H) Two examples of dual color 3D-MINFLUX measurements of COS-7 cells overexpressing GFP-MAP7 plus SNAP-MTB-H5-H6. Images were rendered with a 4-nm voxel size for visualization. Black boxes in the confocal image indicate the region shown in the rendered 3D-MINFLUX image, yellow boxes in the 3D-MINFLUX image indicate the region of the zoom, red dashed lines represent the MT outline from the confocal image. Top right image shows a maximum intensity projection of the cross-section of the MT over 800 nm. The red dashed line there indicates the line scan related to the bottom right graph. Scale bars, 10 μm (confocal image); 500 nm (3D-MINFLUX image and zoom), 50 nm (maximum intensity projection image). See also Fig. S3 and Videos 3 and 4.

CSPP1 binds to MT lumen. (A) Denoised tomograms of dynamic MTs polymerized from GMPCPP-stabilized seeds in the presence or absence of 10 nM GFP-CSPP-L, with or without 250 nM vinblastine vitrified on EM grids. Scale bar, 50 nm. (B) Quantification of the percentage of MTs containing luminal densities from total MTs (from tomograms as shown in A). Orange and gray dots (single data points, tomograms), black circle (mean), SD (error bars). Number of MTs and tomograms are displayed in the graph. ***, P < 0.001, Mann-Whitney test. Analysis from two independent experiments. (C) Reconstituted images from automated segmentation of denoised tomograms as in A. Scale bar, 50 nm. (D and E) Single color 2D-MINFLUX measurements of in vitro reconstituted MTs polymerized in the presence of SNAP-CSPP-L (D) or SNAP-MTB-LZ (E). Images were rendered with 1 nm voxel size for visualization. White boxes in confocal image indicate the region shown in the rendered 2D-MINFLUX image, yellow boxes in the 2D-MINFLUX image indicate the region of the zoom, red dashed lines represent the MT outline from the confocal image. Scale bars, 5 μm (confocal image); 500 nm (2D-MINFLUX image and zoom). (F) Quantification of the fitted, FWHM values per MT (from 2D-MINFLUX images as shown in D and E; see Fig. S3 C analysis details). Single data points are shown. Error bars represent SD. Number of measured MTs; CSPP-L, n = 23; MTB+LZ, n = 50. Analysis from three independent experiments. (G and H) Two examples of dual color 3D-MINFLUX measurements of COS-7 cells overexpressing GFP-MAP7 plus SNAP-MTB-H5-H6. Images were rendered with a 4-nm voxel size for visualization. Black boxes in the confocal image indicate the region shown in the rendered 3D-MINFLUX image, yellow boxes in the 3D-MINFLUX image indicate the region of the zoom, red dashed lines represent the MT outline from the confocal image. Top right image shows a maximum intensity projection of the cross-section of the MT over 800 nm. The red dashed line there indicates the line scan related to the bottom right graph. Scale bars, 10 μm (confocal image); 500 nm (3D-MINFLUX image and zoom), 50 nm (maximum intensity projection image). See also Fig. S3 and Videos 3 and 4.

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