Fig. 5

Multiple HSET motors conjugated to quantum dots drive self-assembly of GMPCPP-MTs into asters. a EGFP-HSET or EGFP-HSETΔTail was conjugated to streptavidin-QDots via the N-terminal 6× His-tag and a biotin anti-His antibody at a 3:1 ratio and visualized via TIRF. Representative kymographs of EGFP-HSET-QDots (left, 1 nM EGFP-HSET: 0.33 nM QDot) and EGFP-HSETΔTail-QDots (right, 0.5 nM EGFP-HSETΔTail: 0.17 nM QDot) are shown (x-scale, distance, 5 µm; y-scale, time, 10 s). b–d Velocities (b), run lengths (c), and end dwell times (d) for the indicated constructed conjugated to QDots at a 3:1 ratio (EGFP-HSET, black, EGFP-HSETΔTail, red) were determined by kymograph analysis and plotted as histograms for the population. Data are reported as the mean values (insets) from CDF fitting ± the 95% CI from bootstrapping for the indicated n particles from 2 independent experiments, where N ≥ 4 movies for each condition. Populations for EGFP-HSET-QDots (black, upper) and EGFP-HSETΔTail-QDots (red, lower) are shown. For run length/end dwell times, particles reaching the end of MTs/dissociating immediately (<1 frame) are color-coded on the histograms. e Self-organization of GMPCPP-stabilized MTs by EGFP-HSET-QDots and EGFP-HSETΔTail-QDots. Alexa594-labeled GMPCPP-MTs (10% labeled, 1 µM tubulin in polymeric form, magenta) were mixed in assay buffer with the indicated motor-QDot complexes (21:7 nM motor :QDots, green) and monitored by time-lapse microscopy at 37 °C. The yellow box indicates the field of view depicted in f. Technical replicates were repeated n ≥ 3 times for each condition, and representative images are shown. Scale bar, 50 µm. f Zoomed-in view of the indicated field. The yellow arrow indicates EGFP-HSET-QDots that have accumulated on the minus end of an MT bundle. Time is indicated in min:s. Scale bar, 10 µm