(A) Schematic overview of the dynein constructs used in this study. The N-terminal tail is shown in gray, the linker in purple, the six numbered AAA+ domains are in light blue and the stalk and MT binding domain are depicted in orange. GFP and GST tags are shown in green and blue, respectively. The Halo tag (DHA, Promega) is shown in red. (B) Coomassie brilliant blue stained gels showing purified dynein constructs used in this study. The associated subunits of the brain cytoplasmic dynein complex are labeled; HC–heavy chain, IC–intermediate chain, LIC–light intermediate chain, LC–light chain. Recombinant yeast dynein constructs do not contain associated subunits. Molecular weight markers are indicated. (C) MTs incubated in the absence or presence of dynein are visualized by attachment to a streptavidin-coated coverslip via a biotin tag. Brain dynein and GST-Dyn1331kDa crosslink MTs into large bundles, while the dynein monomer, Dyn1331kDa does not. Scale bar, 10 µm. (D) Cartoon depicting two different mechanisms by which dynein could crosslink MTs, either using its two motor domains or through the tail domain. Alexa-568 and Alexa-488 labeled MTs are crosslinked by dynein. The green MTs are attached to the coverslip through a biotin-streptavidin linkage and perfusion of 1 mM ATP induces sliding between the MTs. (E and F) Example of rat (E) and GST-Dyn1331kDa (F) dynein-driven sliding of red-labeled MTs within the bundle after 1 mM ATP addition. Arrowhead tracks the sliding MT within the bundle. The time relative to the start is noted in min:s at the bottom of each image. Scale bar, 5 µm.