Figure 2. The N-terminal coiled-coil of Nde1 tethers Lis1 to dynein and stimulates dynein motility.

a. AlphaFold2 prediction of Lis1 (purple) binding to Nde1 (orange: N-terminal, pink: C-terminal). b. Mass photometry profiles for Lis1 binding to Nde1. c. Schematics of single molecule colocalization assay. wtDyn was labeled with both biotin and Alexa488 and immobilized on a glass surface. 0.5 nM LD555-labeled Lis1 and different concentrations of unlabeled Nde1 were flown into the chamber for observation of Lis1 colocalization to surface-immobilized dynein. d. Percent colocalization of surface-immobilized dyneins with LD555-Lis1 under different concentrations of Nde1 (Left) and with LD655-Nde1 under different concentrations of Lis1 (Right). From left to right, N = 8, 10, 10, 11, 10, 5, 5, 5, and 5 imaging areas (40 µm x 40 µm) with at least 100 immobilized dynein spots. e. (Left) The inverse cumulative distribution function (1 - CDF) of Lis1 binding to dynein in the presence and absence of Nde1. (Right) Nde1 binding to dynein in the presence or absence of Lis1. Dashed curves represent a fit to an exponential decay to estimate the average residence time (±s.e.). f. Mass photometry profiles for Lis1 binding to Nde1^1–190. g. Representative kymographs show wtDDR motility with Nde1 or Nde1^1–190. h. The run frequency distribution of Nde1 and Nde1^1–190 under different Lis1 and Nde1 concentrations. Results were normalized to the 0 nM Lis1 and 0 nM Nde1 condition. From left to right, N = 14, 14, 10, 10, 10, 14, 10, and 10 microtubules. In b and f, solid curves represent a fit to multiple Gaussians to predict the average mass (Supplementary Table 2) and percentage of each population. In d and h, the center line and whiskers represent the mean and s.d., respectively. P values are calculated from a two-tailed t-test.