Journal: Nature Structural & Molecular Biology

Article Title: A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

doi: 10.1038/s41594-025-01483-y

Figure Lengend Snippet: a , Schematic representation of CTM members and summary table highlighting individual protein effects on MT dynamics. MTB, MT-binding domain; PD, pause domain. b , c , Fields of view (left; scale bar: 2 µm) and kymographs (right; scale bars: 2 µm and 60 s) illustrating MT dynamics from GMPCPP-stabilized seeds with either 15 µM tubulin supplemented with 3% HiLyte-488-labeled tubulin or 20 nM GFP–EB3. d – f , Parameters of MT plus-end dynamics in the presence of 15 µM tubulin alone or with 20 nM EB3 in combination with indicated concentrations of proteins (from kymographs shown in b , c , i – p and Extended Data Fig. ). d , Bars represent pooled data from three independent experiments (growth rate) or averaged means from three independent experiments (pause and block duration); total number of growth events, pauses/blocks: tubulin alone, n = 356, 0; 10 nM CCDC66, n = 411, 0; 10 nM CEP104, n = 306, 0; 100 nM CEP104, n = 0, 138; 10 nM CSPP1, n = 422, 97; 10 nM TOGARAM1, n = 347, 0; EB3 alone, n = 938, 0; EB3 with 10 nM CCDC66, n = 562, 0; EB3 with 10 nM CEP104, n = 213, 101; EB3 with CSPP1, n = 1715, 273; EB3 with TOGARAM1, n = 861, 0. **** P < 0.0001; NS, not significant (Kruskal–Wallis test followed by Dunn’s post hoc test; all conditions compared to their relevant control (either tubulin alone or tubulin with 20 nM EB3)). Videos were acquired for 10 min; therefore, this is the maximum time for pause duration. In e , f , bars represent averaged means from three independent experiments. Error bars represent the s.e.m. g – p , Fields of view (left; scale bar: 2 µm) and kymographs (right; scale bars: 2 µm and 60 s) illustrating MT dynamics from GMPCPP-stabilized seeds either with 15 µM tubulin supplemented with 3% HiLyte-488-labeled or rhodamine-labeled tubulin or with 20 nM GFP–EB3 or mCherry–EB3 and indicated concentrations and colors of CTM proteins. Orange arrowheads, blocked plus ends; orange arrows, CEP104-tracking minus ends; yellow arrowheads, pauses; blue arrowheads, rescues.

Article Snippet: A tubulin mix consisting of 70% unlabeled porcine brain tubulin, 18% biotin-labeled porcine tubulin and 12% HiLyte-488-labeled, rhodamine-labeled or HiLyte647-labeled porcine tubulin (all from Cytoskeleton) was incubated with 1 mM GMPCPP (Jena Biosciences) at 37 °C for 30 min. Polymerized MTs were then pelleted using an Airfuge for 5 min at 199,000 g and subsequently depolymerized on ice for 20 min. Next, 1 mM GMPCPP was added and MTs were let to polymerize again at 37 °C for 30 min. Polymerized MTs were again pelleted (as above) and diluted tenfold in MRB80 buffer containing 10% glycerol before snap-freezing to store them at −80 °C.

Techniques: Binding Assay, Labeling, Blocking Assay, Control

Journal: Nature Structural & Molecular Biology

Article Title: A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

doi: 10.1038/s41594-025-01483-y

Figure Lengend Snippet: a , Analysis of purified GFP-tagged CTM proteins by SDS-PAGE. Asterisks indicate the full-length protein bands. Protein concentrations were determined from a BSA standard. Assays were repeated independently at least two times. b , SEC-MALS analysis of purified GFP-tagged CTM proteins. The human sequence was used for all module members, except for TOGARAM1. Due to technical difficulties, the mouse sequence of TOGARAM1 was used, which is 84% identical to human TOGARAM1. c , Mass spectrometry analysis of purified GFP-tagged CTM proteins. d , Fields of view (top, scale bar 2 µm) and kymograph (bottom, scale bars 2 µm and 60 s) illustrating MT dynamics from GMPCPP-stabilized seed with HiLyte-488-tubulin and mCherry-CEP104. e , Kymographs illustrating mobility of DmKHC(1-421) on CEP104-blocked MT labelled with HiLyte-488-tubulin and mCherry-CEP104 (top) and DmKHC (bottom) proving that the blocked end of the MT is the plus end. Scale bars 2 µm and 60 s for both kymographs.

Article Snippet: A tubulin mix consisting of 70% unlabeled porcine brain tubulin, 18% biotin-labeled porcine tubulin and 12% HiLyte-488-labeled, rhodamine-labeled or HiLyte647-labeled porcine tubulin (all from Cytoskeleton) was incubated with 1 mM GMPCPP (Jena Biosciences) at 37 °C for 30 min. Polymerized MTs were then pelleted using an Airfuge for 5 min at 199,000 g and subsequently depolymerized on ice for 20 min. Next, 1 mM GMPCPP was added and MTs were let to polymerize again at 37 °C for 30 min. Polymerized MTs were again pelleted (as above) and diluted tenfold in MRB80 buffer containing 10% glycerol before snap-freezing to store them at −80 °C.

Techniques: Purification, SDS Page, Sequencing, Mass Spectrometry

Journal: Nature Structural & Molecular Biology

Article Title: A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

doi: 10.1038/s41594-025-01483-y

Figure Lengend Snippet: a , Analysis of purified GFP-tagged CEP104 constructs by SDS-PAGE. Asterisks show protein bands. Protein concentrations were determined from a BSA standard. Assays were repeated independently at least two times. b , Fields of view (left, scale bar 2 µm) and kymograph (right, scale bars 2 µm and 60 s) illustrating MT dynamics from GMPCPP-stabilized seed with either HiLyte-488-tubulin or GFP-EB3 and indicated concentrations and colors of CEP104 constructs. c , Co-immunoprecipitation of either CSPP1 (left) or CCDC66 (right) with indicated CEP104 constructs, both CSPP1 and CCDC66 interact with the jelly-roll domain of CEP104. Assays were repeated independently at least two times. d , Analysis of purified GFP-tagged CEP104 ΔJR construct by SDS-PAGE. Asterisk indicates the full-length protein band. Protein concentration was determined from a BSA standard. Assays were repeated independently at least two times.

Article Snippet: A tubulin mix consisting of 70% unlabeled porcine brain tubulin, 18% biotin-labeled porcine tubulin and 12% HiLyte-488-labeled, rhodamine-labeled or HiLyte647-labeled porcine tubulin (all from Cytoskeleton) was incubated with 1 mM GMPCPP (Jena Biosciences) at 37 °C for 30 min. Polymerized MTs were then pelleted using an Airfuge for 5 min at 199,000 g and subsequently depolymerized on ice for 20 min. Next, 1 mM GMPCPP was added and MTs were let to polymerize again at 37 °C for 30 min. Polymerized MTs were again pelleted (as above) and diluted tenfold in MRB80 buffer containing 10% glycerol before snap-freezing to store them at −80 °C.

Techniques: Purification, Construct, SDS Page, Immunoprecipitation, Protein Concentration