Related to Fig. 1 . Characterization of CSPP-L in vitro and in cells. (A) Analysis of purified GFP-CSPP-L by SDS-PAGE. Asterisk indicates the full-length protein band. Protein concentrations were determined from BSA standard. (B) Mass spectrometry analysis of purified GFP-CSPP-L. (C) Schematic representation of the in vitro reconstitution assays with dynamic MTs for imaging with TIRF microscopy. GMPCPP-stabilized MT seeds containing fluorescent tubulin, such as rhodamine tubulin (for visualization) and biotinylated tubulin (for surface attachment via NeutrAvidin), are immobilized on a plasma-cleaned coverslip coated with biotinylated poly(L-lysine)-[g]-poly(ethylene glycol) (PLL-PEG-biotin), which is coupled to NeutrAvidin. MT growth from GMPCPP-stabilized seeds is initiated and visualized by the addition of tubulin supplemented with fluorescently labeled tubulin or by the addition of unlabeled tubulin combined with fluorescently-tagged EB3. MT plus- and minus-ends are indicated. (D) Schematic representation of a kymograph visualizing the various transition events observed and quantified in this paper. (E) Kymographs illustrating MT growth with 20 nM GFP-EB3 together with 9.5 nM mCherry-CSPP-L and 0.5 nM GFP-CSPP-L. Scale bars, 2 μm (horizontal) and 60 s (vertical). (F) Maximum projections of z-stacks of interphase RPE-1 cells expressing low levels of mNG-CSPP-L transfected with control siRNA or siRNA against PCM1 and stained for PCM1 (blue) and α-tubulin (magenta). Scale bar, 5 μm. (G) Western blot analysis of RPE-1 cells treated with the indicated siRNAs. (H) FRAP analysis of ciliated RPE-1 cells stably expressing low levels of mNG-CSPP-L or mNG-ARL13B imaged in TIRF mode on DeltaVision OMX V4 Blaze 3D-SIM. FRAP areas are indicated by the red circles. Scale bar, 2 μm. (I) Average normalized fluorescence intensity recovery after photobleaching of axonemes and basal bodies as shown in H. Values were normalized to the fluorescence signal in the FRAP area of the first acquired frame. Number of analyzed FRAP areas, mNG-CSPP1 axoneme, n = 15; mNG-CSPP1 basal body, n = 10; mNG-ARL13B axoneme, n = 15. Thick lines represent pooled data from three independent experiments. Light thin lines represent SEM. (J) Images of COS-7 cells stained for CSPP1 and α-tubulin (widefield, left) or PCM1 (confocal, right). Left scale bars, 25 and 2 µm (zoom); right scale bars, 5 and 2 µm (zoom). (K) Widefield fluorescence images of COS-7 cells overexpressing GFP-CSPP-L and stained for α-tubulin and acetylated tubulin or EB1. Scale bar, 20 μm. (L) Quantification of the mean acetylated tubulin intensity in COS-7 cells (from images as in H). The average mean intensity of cells overexpressing GFP-CSPP-L was normalized to the average mean intensity in control cells. Total number of cells analyzed: control cells, n = 137; cells overexpressing GFP-CSPP-L, n = 77. Bars represent pooled data from two independent experiments. Error bars represent SEM. ***, P < 0.001; Mann-Whitney test. (M) Quantification of the number of EB1 comets per 100 µm² in COS-7 cells (from images as in H). Total number of cells analyzed: control cells, n = 111; cells overexpressing GFP-CSPP-L, n = 75. Bars represent pooled data from two independent experiments. Error bars represent SEM. ***, P < 0.001; Mann–Whitney test. Source data are available for this figure: SourceData FS1.