Protein purification

Dynein and dynactin were purified from stable HEK293 cell lines expressing IC2-SNAPf-3xFLAG or p62-HALO-3xFLAG, respectively. Cell lines were constructed using the FLP/FRT system (Thermo Fisher Scientific) as outlined above. Between 60–100 80% confluent, 15 cm plates were harvested per purification. Cells were collected by pipetting with ice-cold PBS and centrifuged at 1000 x g for 2 min to pellet. Cells were washed once more with ice-cold PBS. Cell pellets were either snap-frozen in liquid nitrogen in 50 mL conical tubes or immediately lysed for protein purification. To lyse, cell pellets were resuspended in dynein lysis buffer (25 mM HEPES pH 7.4, 50 mM KOAc, 2 mM MgOAc, 1 mM EGTA, 10% glycerol (v/v), and 1 mM DTT) supplemented with 0.2% Triton X-100, 0.5 mM Mg-ATP, and 1X protease inhibitors (cOmplete Protease Inhibitor Cocktail, Roche). To ensure complete lysis, resuspended cells were slowly rotated lengthwise at 4°C for 15 min. The lysate was clarified via centrifugation at 66,000 x g for 30 min in a Type 70 Ti rotor (Beckman Coulter) at 4°C. The clarified supernatant was mixed with 0.75–1 mL of Anti-FLAG M2 Affinity Gel (Sigma-Aldrich) overnight at 4°C. During incubation, the slurry was rotated about its long axis in a full 50 mL Falcon tube. Beads were collected by gravity flow and washed with 50 mL wash buffer (dynein lysis buffer with 0.02% Triton X-100 and 0.5 mM Mg-ATP) supplemented with protease inhibitors (cOmplete Protease Inhibitor Cocktail, Roche). Beads were then washed with 50 mL high salt wash buffer (25 mM HEPES, pH 7.4, 300 mM KOAc, 2 mM MgOAc, 10% glycerol, 1 mM DTT, 0.02% Triton X-100, 0.5 mM Mg-ATP, and 1X protease inhibitor (cOmplete Protease Inhibitor Cocktail, Roche) and then with 100 mL wash buffer.

To label with a fluorophore the beads were resuspended in 1 mL wash buffer and incubated with either 5 μM SNAP-Cell TMR Star (New England BioLabs; Ipswich, MA) (to label IC2) or 5 μM Halo-Atto647N (Promega) (to label p62) for 10 min at room temperature. Unreacted dye was removed from beads with 50–80 mL of wash buffer. Protein complexes were eluted with 0.5–1 mL of elution buffer (wash buffer with 2 mg/mL 3xFLAG peptide). Elution was collected, diluted to 2 mL in Buffer A (50 mM Tris pH 8, 2 mM MgOAc, 1 mM EGTA, and 1 mM DTT) and injected onto a MonoQ 5/50 GL column (GE Healthcare Life Sciences) at 0.5 mL/min. The column was washed with 20 CV of Buffer A at 1 mL/min. To elute, a linear gradient was run over 40 CV into Buffer B (50 mM Tris pH 8, 2 mM MgOAc, 1 mM EGTA, 1 mM DTT, 1 M KOAc). Pure dynein complex elutes from ~60–70% Buffer B, while pure dynactin complex elutes around ~70–80% Buffer B. Peak fractions were pooled and concentrated, Mg-ATP was added to 0.1 mM and glycerol was added to 10%. Samples were then snap frozen in 2 µL aliquots.

Activators and potential activators were cloned into pET-28a vectors with an N-terminal StrepII-sfGFP tag. Mouse BICD2 (mBICD2) (aa 25–400) was a gift from Rick McKenney (University of California, Davis), while NIN (aa 1–693) and NINL (aa 1–702) were sub-cloned from ORFs outlined above. All constructs were transformed into BL21-CodonPlus (DE3)-RIPL cells (Agilent Technologies; Santa Clara, CA). 2 L of cells were grown at 37°C in LB media to a 600 nm optical density of 0.4–0.8 before the temperature was reduced to 18°C and expression was induced with 0.5 mM IPTG. After 16–18 hr, cells were harvested via centrifugation for 6 min at 4°C at 6000 rpm in a JLA 8.1000 fixed angle rotor (Beckman Coulter). Pellets were resuspended in 30–40 mL of dynein lysis buffer with 0.5 mM PefaBloc SC (Sigma-Aldrich) and 1 mg/mL lysozyme and incubated at 4°C for 30 min. Cells were lysed via sonication (Branson Digital Sonifier, Emerson; Saint Louis, MA) and clarified via centrifugation at 66,000 x g for 30 min in a Type 70 Ti rotor (Beckman) at 4°C. Supernatant was loaded onto a 5 mL StrepTrap column (GE Healthcare Life Sciences) and washed with 50–100 mL of lysis buffer. Activators were then eluted with 25–50 mL of elution buffer (dynein lysis buffer with 3 mM d-Desthiobiotin). Finally, all activators were purified via size exclusion chromatography on either a Superdex 200 Increase 10/300 GL or a Superose 6 Increase 10/300 GL column (GE Healthcare Life Sciences) that had been equilibrated with degassed dynein lysis buffer. Peak fractions were collected and used for single molecule motility experiments immediately or snap-frozen in 2–20 µL aliquots. Care was taken not to concentrate the activators as we observed that this led to aggregation and inactivity.