Fig. 8: CLASP2 mediates condensate attachment in other TBM-containing proteins.
From: CLASP-mediated competitive binding in protein condensates directs microtubule growth
a Sequence alignment of TBM-containing proteins in the human genome. To identify more TBM-containing proteins in silico, a motif search was performed on the Scansite website (https://scansite4.mit.edu/) with the consensus sequence pattern of TBM (Fig. 1k). The helical conformation of the potential TBMs in candidate proteins was checked using AlphaFold2-based predictions (https://www.alphafold.ebi.ac.uk). The identified TBM-containing proteins in this study are boxed. b, c Co-immunoprecipitation assays showing the TOG4-mediated interaction between CLASP2α and JAKMIP1 (b) or CENP-J (c). The experiment was repeated twice. d, e ITC-based analyses of the interaction between CLASP2_TOG4 and TBM-containing fragments in JAKMIP1 (d) or CENP-J (e). The boundaries used are residues 549–627 for JAKMIP1 and 571 – 589 for CENP-J. f–i Three-dimensional reconstruction (f, h) and quantification analysis (g, i) of attachment between the CLIP170 and JAKMIP1 (f, g) or BFP-CENP-J (h, i) condensates. Competitive binding to CLASP2 is required for the condensate interactions, as the L1467E mutation in CLASP2α abolished condensate attachment. Images were captured every 0.2 μm on the Z-axis, and the number of images depended on the distribution of condensates along the Z-axis. The 3D pictures for each cell were generated using NIS-Elements A1R Analysis software. The cell boundaries were generated using Imaris software. Data are mean ± s.d. The middle, bottom, and top of boxplots represent, respectively, the median, the 25th percentile, and the 75th percentile (n = 20 cells). j A schematic model illustrating TBM-containing proteins in MT organization in various cellular compartments. CLASP2 promotes condensate attachment by mediating mesoscale protein-protein interactions in the condensed phase.
