Figure S3.
Related toFig. 4. Characterization of CSPP1 inside the MT lumen. (A) Additional 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. See also Fig. 4 A. (B) Additional zooms of confocal images of in vitro MINFLUX regions shown in Fig. 4, D–E. Large confocal field of view (FOV) and 2D-MINFLUX images are identical to the images in Fig. 4, D–E. Panel representation as in Fig. 4, D–E. Scale bars, 5 μm (Large FOV confocal image); 500 nm (zoom FOV confocal image and 2D-MINFLUX image). (C) Analysis workflow of FWHM value determination in the plot shown in Fig. 4 F. An MT in from in the image in Fig. 4 E was chosen as an example. First, images of MTs were rendered from MINFLUX data as density map (step 1). Then, for a given MT filament, we extracted the central line along its longitudinal axis (step 2). The signal intensities of the nearby regions were then plotted against its distance to the central line and summed along the length of the filament (step 3). We then extracted the FWHM of the intensity profile plot, as the estimation of the width of the given filament (step 4). Imax is maximum intensity and Imax/2 is half-maximum intensity. Scale bar, 500 nm. (D) Standard deviation histograms (x- and y-axis) of groups of greater than four successive localizations from the same fluorophore depicting the localization precision of one example of a single color 2D-MINFLUX measurement of in vitro MTs polymerized in presence of SNAP-CSPP-L. The localization precision of all single-color 2D-MINFLUX measurements used for the FWHM analysis was in the range of 3.0 and 4.3 nm. (E) Dual color 3D-MINFLUX measurements of COS-7 cells overexpressing GFP-MAP7 together with SNAP-MTB-H5-H6. Panel representation as in Fig. 4, G–H. Scale bars, 10 μm (large FOV confocal image); 5 μm (medium FOV confocal image); 500 nm (small FOV confocal image, 3D-MINFLUX image and zoom); 50 nm (maximum intensity projection image).

Related to Fig. 4 . Characterization of CSPP1 inside the MT lumen. (A) Additional 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. See also Fig. 4 A. (B) Additional zooms of confocal images of in vitro MINFLUX regions shown in Fig. 4, D–E. Large confocal field of view (FOV) and 2D-MINFLUX images are identical to the images in Fig. 4, D–E. Panel representation as in Fig. 4, D–E. Scale bars, 5 μm (Large FOV confocal image); 500 nm (zoom FOV confocal image and 2D-MINFLUX image). (C) Analysis workflow of FWHM value determination in the plot shown in Fig. 4 F. An MT in from in the image in Fig. 4 E was chosen as an example. First, images of MTs were rendered from MINFLUX data as density map (step 1). Then, for a given MT filament, we extracted the central line along its longitudinal axis (step 2). The signal intensities of the nearby regions were then plotted against its distance to the central line and summed along the length of the filament (step 3). We then extracted the FWHM of the intensity profile plot, as the estimation of the width of the given filament (step 4). Imax is maximum intensity and Imax/2 is half-maximum intensity. Scale bar, 500 nm. (D) Standard deviation histograms (x- and y-axis) of groups of greater than four successive localizations from the same fluorophore depicting the localization precision of one example of a single color 2D-MINFLUX measurement of in vitro MTs polymerized in presence of SNAP-CSPP-L. The localization precision of all single-color 2D-MINFLUX measurements used for the FWHM analysis was in the range of 3.0 and 4.3 nm. (E) Dual color 3D-MINFLUX measurements of COS-7 cells overexpressing GFP-MAP7 together with SNAP-MTB-H5-H6. Panel representation as in Fig. 4, G–H. Scale bars, 10 μm (large FOV confocal image); 5 μm (medium FOV confocal image); 500 nm (small FOV confocal image, 3D-MINFLUX image and zoom); 50 nm (maximum intensity projection image).

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