Fig. 4: Plasmodium EB1 shows MT lattice affinity.

a Protein domain organization of H. sapiens EB1 (HsEB1) and P. yoelii (PyEB1). Full-length proteins and fragments of HsEB1 and PyEB1 were tested for MT-localization and MT-binding activity. CH: calponin homology domain, CC: coil-coiled domain, linker: the region between CH and CC. The numbers indicate the position of amino acids. b IFA of GFP-tagged EB1 proteins (green) and MTs (red) in the human cell line MRC5. Full-length proteins and N-terminus fragments of HsEB1 and PyEB1 were fused with GFP at the C-terminus, M or C-ternimus fragments of HsEB1 and PyEB1 were fused with GFP at the N-terminus and transiently expressed in the MRC5. Insets show enlargements of the boxed areas. Representative for three independent experiments. Scale bar = 5 μm. c IFA of GFP-tagged PyEB1 (green) and endogenous HsEB1 (red) in the MRC5 cells. Full-length PyEB1 were fused with GFP at the C-terminal and transiently expressed in the MRC5. Representative for three independent experiments. Scale bar = 5 μm. d in vitro MT binding of proteins detected by total internal reflection fluorescence (TIRF) microscopy. The GMPCPP-stabilized MT was used as nucleation seed for MT (red) growth in the presence of GFP-tagged HsEB1 or PyEB1 (green). Representative images and kymographs showed MT growth-end tracking of HsEB1 (left panel) and full-length MT binding of PyEB1 (right panel). Note that MT growth in vitro occurs in both MT-plus and -minus ends. Representative for three independent experiments. Horizontal scale bars: 2 μm; vertical scale bars: 1 min. e Quantification of GFP fluorescence signal for HsEB1 and PyEB1 along MT in d. Relative intensities of GFP signal on the MT lattice and MT plus-end segments were measured. n is the number of MTs measured in each group. Mean ± SD from three independent experiments, two-sided Mann–Whitney U test. f in vitro MT binding of HsEB1 and PyEB1 detected by TIRF. The GMPCPP-stabilized MT (red) was used as nucleation seed for MT growth in the presence of both mCherry-tagged HsEB1 and GFP-tagged PyEB1 (60 nM used). Note that MT growth in vitro occurs in both MT-plus and -minus ends. Representative images and kymographs showed MT growth-end tracking of HsEB1 and full-length MT binding of PyEB1. Representative for three independent experiments. Horizontal scale bars: 2 μm; vertical scale bars: 1 min. g Representative images and kymographs showing MT-stabilizing activity of PyEB1. Horizontal scale bars: 2 μm; vertical scale bars: 1 min. h Quantification of MT dynamic events in g, including MT shrinkage, catastrophe, and rescue in the presence of a titration of PyEB1. Mean ± SD from three independent experiments, two-tailed t-test.