Supplemental Movie S1: DDB dwell and accumulate as stable clusters at
microtubule minus ends. Corresponds to Figure 1A. Taxol-stabilized microtubules
(blue) were fixed to coverglass. The microtubules were then continuously imaged by
TIRF-M while ~30 nM DDB (Red) was added to the system. Time and scalebar as
labeled in video.
Supplemental Movie S2: Single molecules of DDB dwell at microtubule minus
ends. Corresponds to Figure 2ABC. Left: Individual DDB (red) process and dwell at
microtubule (blue) minus ends. Middle: When differentially labeled DDB (Snap-Alexa488, green) was added to the system dwell times increased for individual DDB. Right:
Detyrosinated microtubules (blue) were generated by treating taxol-stabilized
microtubules with carboxypeptidase-A. Dwell times of DDB (red) were not affected.
Timescale as labeled in seconds, scalebar: 2 µm.
Supplemental Movie S3: DDB clusters drive microtubule sliding of all angles.
Corresponds to Figure 3C. Taxol-stabilized microtubules (blue) were fixed to the TIRFM field by methyl cellulose are free to move in the xy-plane. Under these conditions, we
observe DDB clusters (red) driving parallel, antiparallel, and perpendicular sliding. Time
as labeled in seconds, Scalebar: 2 µm.
Supplemental Movie S4: DDB clusters drive drastic reorganizations of
microtubule bundles. Taxol-stabilized microtubules (blue) were fixed to the TIRF-M
field by methyl cellulose are free to move in the xy-plane. Under these conditions,
multiple DDB-driven (red) sliding events lead to a stable topologically organized miniaster. Time as labeled in seconds, scalebar: 2 µm.
Supplemental Movie S5: DDB acculate and form MT-MT Foci on growing
microtubules. Corresponds to Figure 4B. 20 µM soluble tubulin (green) was added to
immobilized GMP-CPP stabilized MT seeds (blue) in the presence of 2 µM GTP/ATP.
DDB (red) form clusters on and slide growing microtubule minus ends. Sliding events
result in MT-MT foci (yellow arrows). Time and scalebar as labeled.
Supplemental Movie S6: DDB create asters from polymerizing MTs. Corresponds
to Figure 4C. 4 µM DDB (red) was added to 40 µM free tubulin (black) in the presence
of 2 uM ATP/GTP. Asters are seen coalescing and merging. Time is in seconds and
scalebar as labeled.
Supplemental Movie S7: Large-scale networks of stabilized microtubules undergo
bulk contractions in the presence of DDB but not GST-Dyn1. Upper: Corresponds
to Figure 5A. 1 µM DDB (red) was added to a microfluidic channel filled with 1 µM
taxol-stabilized microtubules (blue). The contraction kinetics could be described as an
active fluid, previously seen in cell extract. Lower: Corresponds to Figure 5E. 1 µM of
weakly processive GST-dynein (red) is unable to contract a microfluidic channel filled
with 1 µM taxol-stabilized microtubules (blue). Time and scalebar as labeled.
Supplemental Movie S8: DDB localize to the center of MT densities in bulk
contractions. Corresponds to Figure 5B. High magnification imaging of a contracting
network of 1 µM taxol-stabilized microtubules (blue) by 1 µM DDB (red) shows that DDB
localize to the centers of areas enriched in MT signal, reminiscent of asters.