Fig. 5: Disrupting the CRACR2a–LIC1 interaction impairs dynein motility.

a Mutating residues F58 (red) in the hydrophobic cleft of CRACR2a (orange) that bids the LIC1 helix (blue) to aspartic acid inhibits this interaction, as revealed by the ITC titration of CRACR2a_47–122 F58D into MBP-LIC_FL (experimental conditions given in the figure). The lack of change in the heat of injection precludes fitting to a binding isotherm. b Representative kymographs of the dynein–dynactin driven motility of single Halo-CRACR2a-positive particles (WT and mutant F58D CRACR2a) analyzed by TIRF microscopy with either 2 µM free Ca²⁺ (left) or 2 mM EGTA (right). Kymographs are oriented with the + end of the microtubule (MT) on the left (scale bars: horizontal, 3 µm; vertical, 5 s). c Quantitative analysis of the number of processive motile events, defined as runs of ≥1 µm, observed under the conditions shown in part b of this figure. Each point represents a single microtubule (n = 123 per condition). The statistical significance of the measurements was determined using the Kruskal–Wallis test, followed by a Dunn’s multiple comparisons test. Bars represent the median and 95% confidence interval. d Velocities of WT or mutant F58D CRACR2a with 2 µM free Ca²⁺, statistically analyzed using an unpaired t test and two-tailed p values were calculated. Data is pooled from three replicates (WT n = 326; F58D n = 95). Bars represent the median and 95% confidence interval; p = 0.0579. e Histogram of run lengths for WT and mutant F58D Halo-CRACR2a-positive particles with 2 µM free Ca²⁺. The data were analyzed using a Mann–Whitney test and two-tailed p values were calculated (****p < 0.0001; n.s. nonsignificant or p > 0.05). The data were pooled from three replicates (WT n = 317; F58D n = 87). Source data for panels b–e are provided in the Source Data file.