Figure 8.
Full-length NuMA can mediate dynein/dynactin-driven microtubule transport. (A) Schematic (top) of the polymerization protocol to obtain microtubule “lollipops” with representative TIRF microscopy image (bottom) of lollipops obtained from a mixture of 0.8 µM Atto647N-tubulin (magenta), 40 nM mScarlet-NuMAFL (green), and 1 mM GMPCPP. Minus ends can be identified by the selective presence of mScarlet-NuMAFL. (B) Schematic of a two-flush TIRF microscopy dynein-driven microtubule transport assay: first, dynein, dynactin, and Lis1 are flowed into a channel containing long surface-immobilized GMPCPP-microtubules; dynein is allowed to accumulate on microtubules for ≈3 min; in a second step, lollipops are introduced into the channel, with dynactin and Lis1, leading to dynein/dynactin/lollipop-bound NuMA transporting lollipop microtubules. (C and D) Representative time course TIRF microscopy images (C) and related kymograph (D) of transport events performed in the presence of 14 nM mEGFP-dynein (pre-bound to immobilized GMPCPP-microtubules), 28 nM dynactin and 1,000 nM Lis1. For both the faster (red arrowheads) and slower (yellow arrowheads) transport events, the lollipop is bound to the immobilized microtubule through a single anchoring point, corresponding to co-localizing mEGFP-dynein and mScarlet-NuMAFL (arrowheads), resulting in it to dangle while being transported. Related to Video 4. (E) Left: frequency of lollipop microtubule behaviors on immobilized microtubules (mean ± SEM, three biological replicates); each distinct symbol represents a replicate; protein concentrations as in C and D; n = 82 landed lollipops. Right: cartoons representing the three categories of transported lollipops; dynein and NuMA are depicted in cyan and green, respectively, the lollipop microtubule is shorter and brighter; the polarity of both lollipop and immobilized microtubule are indicated; arrow points to the direction of dynein motility (blue) and microtubule transport (magenta). Experiments were performed in dynein microscopy buffer with the omission of methylcellulose.

Full-length NuMA can mediate dynein/dynactin-driven microtubule transport. (A) Schematic (top) of the polymerization protocol to obtain microtubule “lollipops” with representative TIRF microscopy image (bottom) of lollipops obtained from a mixture of 0.8 µM Atto647N-tubulin (magenta), 40 nM mScarlet-NuMAFL (green), and 1 mM GMPCPP. Minus ends can be identified by the selective presence of mScarlet-NuMAFL. (B) Schematic of a two-flush TIRF microscopy dynein-driven microtubule transport assay: first, dynein, dynactin, and Lis1 are flowed into a channel containing long surface-immobilized GMPCPP-microtubules; dynein is allowed to accumulate on microtubules for ≈3 min; in a second step, lollipops are introduced into the channel, with dynactin and Lis1, leading to dynein/dynactin/lollipop-bound NuMA transporting lollipop microtubules. (C and D) Representative time course TIRF microscopy images (C) and related kymograph (D) of transport events performed in the presence of 14 nM mEGFP-dynein (pre-bound to immobilized GMPCPP-microtubules), 28 nM dynactin and 1,000 nM Lis1. For both the faster (red arrowheads) and slower (yellow arrowheads) transport events, the lollipop is bound to the immobilized microtubule through a single anchoring point, corresponding to co-localizing mEGFP-dynein and mScarlet-NuMAFL (arrowheads), resulting in it to dangle while being transported. Related to Video 4. (E) Left: frequency of lollipop microtubule behaviors on immobilized microtubules (mean ± SEM, three biological replicates); each distinct symbol represents a replicate; protein concentrations as in C and D; n = 82 landed lollipops. Right: cartoons representing the three categories of transported lollipops; dynein and NuMA are depicted in cyan and green, respectively, the lollipop microtubule is shorter and brighter; the polarity of both lollipop and immobilized microtubule are indicated; arrow points to the direction of dynein motility (blue) and microtubule transport (magenta). Experiments were performed in dynein microscopy buffer with the omission of methylcellulose.

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