Different modes of dynein/dynactin/NuMA ^FL -mediated microtubule transport. (A–E) Representative time course TIRF microscopy images with schematics illustrating the type of transport (A, C, and E) and related kymographs (B and D) of microtubule transport experiments performed in the presence of 14 nM mEGFP-dynein (prebound to surface-immobilized Atto647N-labeled GMPCPP-microtubules), 28 nM dynactin, 1,000 nM Lis1, and “lollipop” microtubule minus-end bound mScarlet-NuMA^FL. (A and B) A lollipop gets aligned to the immobilized microtubule, apparently through some crosslinking dynein bound at a distance from the lollipop minus end (red arrowhead, strongest NuMA signal). The orientation is parallel, as the lollipop minus end is facing the immobilized microtubule minus end (recognized by the direction of dynein-driven transport, and accumulated dynein). (C and D) Similarly to A and B, a lollipop is aligned to the immobilized microtubule, however, in antiparallel orientation, which results in the plus end of the lollipop being slid toward the minus end of the immobilized microtubule. (E) A lollipop-bound NuMA (red arrowhead) in solution lands directly on the minus end of the immobilized microtubule, where dynein has accumulated, resulting in transport-independent gathering of minus ends. The timestamps refer to mm:ss. Schematics as described in Fig. 8.