Figure 2. BICD2FL mutants show increased binding to dynein/dynactin and single-molecule motility in vitro. (A) Porcine brain lysate pull-down using BICD2FL as bait. Rab6aGTP or Rab6aGDP was added to the lysate at a concentration of 1 µM. The amount of endogenous dynein (DIC) and dynactin (p150 subunit) bound was analyzed by immunoblot. The amount of BICD2 on beads was visualized using Coomassie stain. (B) The ratio of dynein or dynactin band intensity for mutants compared with WT BICD2 for the lysate only condition; mean and SEM from n = 3 independent experiments. Mean, SEM, and p-values for the mutants shown for DIC are as follows: N188T: 2.24 ± 0.42, P = 0.10; F739I: 1.95 ± 0.34, P = 0.11; I189F: 1.61 ± 0.27, P = 0.15; and S107L: 2.44 ± 0.41, P = 0.07. Mean, SEM, and p-values for p135/p150 are as follows: N188T: 3.86 ± 1.09, P = 0.12; F739I: 3.65 ± 1.5, P = 0.22; I189F: 1.88 ± 0.14, P = 0.027; and S107L: 2.88 ± 1.18, P = 0.25. Quantitation of the other conditions are shown in Fig. S2 (A and B). (C) Dynein and dynactin (purified from RPE-1 cells; see Materials and methods) were incubated with BICD2 and flowed into a motility chamber. The number of moving DDB motors, as visualized by sfGFP fluorescence on BICD2, was quantified per minute per micrometer length of microtubules in the assay; mean and SEM from n = 3 independent experiments (each experiment measuring a minimum of 30 microtubules); *, P ≤ 0.05. (D) Velocities of the moving motors from C is shown; mean and SEM from n = 3 independent protein preparations and experiments (each experiment measuring a minimum of 100 DDB complexes). (E) The run lengths of WT and mutant DDBFL; mean and SEM from n = 3 independent experiments (each experiment measuring a minimum of 130 DDB complexes). For processivity measurements, the NaCl concentration was increased from 50–65 mM to reduce the run length so that it could be more reliably measured (see Materials and methods). (F) Purified dynein and dynactin were incubated with truncated BICD225–400 (WT and mutant) and flowed into a motility chamber. The number of moving DDB complexes, as visualized by SNAP-TMR fluorescence on BICD2, was quantified per minute per micrometer of microtubule; mean and SEM from n = 3 independent protein preparations and experiments (each experiment measuring a minimum of 30 microtubules).
Figure 2.

BICD2FL mutants show increased binding to dynein/dynactin and single-molecule motility in vitro. (A) Porcine brain lysate pull-down using BICD2FL as bait. Rab6aGTP or Rab6aGDP was added to the lysate at a concentration of 1 µM. The amount of endogenous dynein (DIC) and dynactin (p150 subunit) bound was analyzed by immunoblot. The amount of BICD2 on beads was visualized using Coomassie stain. (B) The ratio of dynein or dynactin band intensity for mutants compared with WT BICD2 for the lysate only condition; mean and SEM from n = 3 independent experiments. Mean, SEM, and p-values for the mutants shown for DIC are as follows: N188T: 2.24 ± 0.42, P = 0.10; F739I: 1.95 ± 0.34, P = 0.11; I189F: 1.61 ± 0.27, P = 0.15; and S107L: 2.44 ± 0.41, P = 0.07. Mean, SEM, and p-values for p135/p150 are as follows: N188T: 3.86 ± 1.09, P = 0.12; F739I: 3.65 ± 1.5, P = 0.22; I189F: 1.88 ± 0.14, P = 0.027; and S107L: 2.88 ± 1.18, P = 0.25. Quantitation of the other conditions are shown in Fig. S2 (A and B). (C) Dynein and dynactin (purified from RPE-1 cells; see Materials and methods) were incubated with BICD2 and flowed into a motility chamber. The number of moving DDB motors, as visualized by sfGFP fluorescence on BICD2, was quantified per minute per micrometer length of microtubules in the assay; mean and SEM from n = 3 independent experiments (each experiment measuring a minimum of 30 microtubules); *, P ≤ 0.05. (D) Velocities of the moving motors from C is shown; mean and SEM from n = 3 independent protein preparations and experiments (each experiment measuring a minimum of 100 DDB complexes). (E) The run lengths of WT and mutant DDBFL; mean and SEM from n = 3 independent experiments (each experiment measuring a minimum of 130 DDB complexes). For processivity measurements, the NaCl concentration was increased from 50–65 mM to reduce the run length so that it could be more reliably measured (see Materials and methods). (F) Purified dynein and dynactin were incubated with truncated BICD225–400 (WT and mutant) and flowed into a motility chamber. The number of moving DDB complexes, as visualized by SNAP-TMR fluorescence on BICD2, was quantified per minute per micrometer of microtubule; mean and SEM from n = 3 independent protein preparations and experiments (each experiment measuring a minimum of 30 microtubules).

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