Fig. 1

Full-length HSET organizes growing MTs into asters. a Schematic of HSET truncations purified in this study. HSET contains two MT-binding domains: an ATP-independent globular tail domain located at the N terminus (amino acid 1–138, brown), and an ATP-dependent conserved kinesin motor domain located at the C terminus (aa 305–673, blue). HSET also contains a coiled-coil stalk domain necessary for dimerization (aa 139–304, black). All constructs contained an N-terminal 6× His tag used for affinity purification. b Aster formation of growing MTs by HSET. 20 µM tubulin (10% Alexa594-labeled, magenta) was mixed in assay buffer with the indicated EGFP-HSET truncation (green) and monitored by time-lapse microscopy at 37 °C. With the exception of EGFP-HSETΔTail (20 nM), all HSET constructs were present at 100 nM. c Bundle formation of nongrowing, GMPCPP-stabilized MTs by HSET. Alexa594-labeled GMPCPP-MTs (10% labeled, 1 µM tubulin in polymer form, magenta) were mixed in assay buffer with the indicated EGFP-HSET truncation (green) and monitored by time-lapse microscopy at 37 °C. HSET concentrations are identical to b. For contrast measurements over time, see Supplementary Figure 1d, e. For movies, see Supplementary Movies 1–2. For additional EGFP-HSET images on GMPCPP-MTs, see Fig. 4a. Technical replicates of experiments in b, c were repeated n ≥ 3 times for each condition, and representative images are shown. Scale bars, 50 µm