Extended Data Fig. 2: Characterization of the dynein binding interface and dimerization domain of Lis1.
From: LIS1 promotes the formation of activated cytoplasmic dynein-1 complexes
a, Example SEC-MALS traces with Lis1 dimer (orange), dynein monomer (grey), and dynein monomer with Lis1 dimer (black). The intensity of the UV signal (solid line) and the molecular weight fit (dashed line) are shown. Dimeric Halo-tagged-Lis1 is expected to be 161.4 kDa and monomeric dynein is expected to be 380.4 kDa. In this experiment we observe Halo-tagged-Lis1 to be 157.6 kDa, monomeric dynein to be 489.5 kDa and the Lis1-dynein complex to be 700.1 kDa. The high apparent molecular weight of monomeric dynein may be due to a self-association species that appears as a shoulder in the UV trace. The experiment was repeated in triplicate yielding similar results, giving a stoichiometry of 1.2 ± 0.3 Lis1 dimers per dynein monomer. Based on this data we cannot rule out that some dynein monomers are bound to two Lis1 dimers (which has been reported to occur34), but our data suggest that most dynein monomers bind a single Lis1 dimer, and that Lis1 does not tether two dynein monomers. b, Single-molecule velocity of dynein–dynactin–BicD2-S complexes with increasing concentrations of Lis1. The median and interquartile range are shown. c, Single-molecule velocity of dynein–dynactin–Hook3 complexes in the presence of a higher ionic strength buffer in the absence (white circles) or presence (black circles) of 300 nM Lis1 or Lis1-5A. The data in the presence and absence of WT Lis1 was also presented in Fig. 1g. The median and interquartile range are shown. d, Example kymographs of dynein–dynactin–Hook3 complexes in a higher ionic strength buffer in the absence or presence of 300 nM Lis1 or Lis1-5A. Scale bars are 10 µm (x) and 20 sec (y). Data is quantified in Extended Data Fig. 2c. e, Example SEC-MALS trace of Lis1ΔN (orange). The intensity of the UV signal (solid line) and the molecular weight fit (dashed line) are shown. Monomeric Halo-tagged-Lis1ΔN is expected to be 71.5kDa. In this experiment we observe Halo-tagged-Lis1ΔN to have a monomer peak at 72.0 kDa and a dimer peak at 141.2 kDa. The experiment was repeated in triplicate yielding similar results. Statistical data is available as source data for Extended Data Fig. 2.
