alexa594-labeled gmpcpp-mts  (Quantum Dot Inc)

Journal: Nature Communications

Article Title: Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

doi: 10.1038/s41467-018-04991-2

Figure Lengend Snippet: 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 . For movies, see Supplementary Movies – . For additional EGFP-HSET images on GMPCPP-MTs, see Fig. . Technical replicates of experiments in b , c were repeated n ≥ 3 times for each condition, and representative images are shown. Scale bars, 50 µm

Article Snippet: 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.

Techniques: Purification, Binding Assay, Construct, Affinity Purification, Labeling, Time-lapse Microscopy

Journal: Nature Communications

Article Title: Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

doi: 10.1038/s41467-018-04991-2

Figure Lengend Snippet: Soluble (non-MT) tubulin promotes the ability of HSET to drive aster self-organization of GMPCPP-MTs independent of MT polymerization. a EGFP-HSET-driven self-organization of GMPCPP-stabilized MTs with increasing tubulin concentration. Alexa594-labeled GMPCPP-MTs (10% labeled, 1 µM tubulin in polymer form, magenta) were mixed in assay buffer with EGFP-HSET (100 nM, green) and monitored by time-lapse microscopy at 37 °C. Unlabeled tubulin was added to the reaction at the indicated concentration. Technical replicates were repeated N ≥ 3 times for each condition, and representative images are shown. Scale bar, 50 µm. b EGFP-HSET-driven self-organization of GMPCPP-stabilized MTs in the absence of MT polymerization. Experiments were performed identically to a but in the absence of taxol and the presence of saturating colchicine and GDP to prevent polymerization. Technical replicates were repeated N ≥ 2 times for each condition, and representative images are shown. Scale bar, 50 µm

Article Snippet: 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.

Techniques: Concentration Assay, Labeling, Time-lapse Microscopy

Journal: Nature Communications

Article Title: Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

doi: 10.1038/s41467-018-04991-2

Figure Lengend Snippet: Soluble (non-MT) tubulin activates processive motility of HSET on single MTs. a Schematic. EGFP-HSET truncations were diluted in P12 buffer and monitored on GMPCPP-stabilized MTs by time-lapse TIRF. b Representative kymographs for time-lapse TIRF images for the indicated constructs at the following concentrations: EGFP-HSET and EGFP-HSETΔMotor, 50 pM. EGFP-HSETΔTail, 250 pM. Distance is on the x -axis (scale bar, 10 µm), and time is on the y -axis (scale bar, 10 s). c Mean-squared displacement (MSD) analysis of particle motion. The reported diffusion constant D is determined from a linear fit over the first 5 s, with the units nm 2 /s: EGFP-HSET: D = 6.3 × 10 4 , n = 206; EGFP-HSETΔMotor: D = 9.4 × 10 4 , n = 197; EGFP-HSETΔTail: D = 0.1 × 10 4 , n = 200. Data are presented as the calculated mean MSD ( y -axis) from two independent experiments over the indicated time intervals ( x -axis) for the indicated n particles ± SEM. d EGFP-HSET in BRB80 + 50 mM KCl was observed in the absence (left) or presence (right) of soluble tubulin and visualized by kymograph ( x -scale bar, distance, 10 µm; y -scale bar, time, 1 min). e Quantification of processive (≥5 s) event frequency as a function of [EGFP-HSET] in the presence (dark green) or absence (light green) of 2 µM tubulin. Data are presented as the number of processive events divided by the total observed MT length multiplied by the observation time for two independent experiments ± SD calculated from N ≥ 10 movies for each condition. Boxes represent first and third quartiles, whiskers represent detection limits, and lines represent median (mean overlaid). f Unlabeled HSET was mixed with 10 nM Cy5-tubulin in BRB80 + 50 mM KCl and observed. Velocities and run lengths of moving Cy5-tubulin particles were determined by kymograph and plotted as histograms. Data are reported as the mean velocity and run length values of n particles from CDF fitting ± the 95% CI from bootstrapping from two independent experiments. g 100 nM Cy5-tubulin (magenta) and 1 nM EGFP-HSET (green) were observed near-simultaneously by high-speed TIRF in BRB80 + 50 mM KCl, and visualized by kymograph ( x -scale bar, distance, 5 µm; y -scale bar, time, 10 s)

Article Snippet: 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.

Techniques: Construct, Diffusion-based Assay

Journal: Nature Communications

Article Title: Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

doi: 10.1038/s41467-018-04991-2

Figure Lengend Snippet: 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

Article Snippet: 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.

Techniques: Construct, Labeling, Time-lapse Microscopy