Abstract
Regulation of cytoplasmic dynein's motor activity is essential for diverse eukaryotic functions, including cell division, intracellular transport, and brain development. The dynein regulator Lis1 is known to keep dynein bound to microtubules; however, how this is accomplished mechanistically remains unknown. We have used three-dimensional electron microscopy, single-molecule imaging, biochemistry, and in vivo assays to help establish this mechanism. The three-dimensional structure of the dynein-Lis1 complex shows that binding of Lis1 to dynein's AAA+ ring sterically prevents dynein's main mechanical element, the 'linker', from completing its normal conformational cycle. Single-molecule experiments show that eliminating this block by shortening the linker to a point where it can physically bypass Lis1 renders single dynein motors insensitive to regulation by Lis1. Our data reveal that Lis1 keeps dynein in a persistent microtubule-bound state by directly blocking the progression of its mechanochemical cycle.
Highlights
Cytoplasmic dynein (‘dynein’ here), the largest and least understood of the cytoskeletal motors, uses the energy from ATP hydrolysis to move towards the minus ends of microtubules (Vale, 2003; Carter, 2013)
We previously showed that the propeller domain alone can regulate dynein in vitro and used negative stain electron microscopy (EM) and two-dimensional (2D) image processing to show that Lis1 binds to dynein's motor domain near AAA3/4 (Huang et al, 2012)
In order to visualize the spatial relationship between Lis1 and dynein's multiple structural elements and to better understand the mechanism by which Lis1 regulates dynein, we used cryo-negative stain EM and single-particle image processing to obtain the 3D structure of the dynein–Lis1 complex (Figure 1C)
Summary
Cytoplasmic dynein (‘dynein’ here), the largest and least understood of the cytoskeletal motors, uses the energy from ATP hydrolysis to move towards the minus ends of microtubules (Vale, 2003; Carter, 2013). Despite progress in understanding the architecture and mechanism of dynein's large motor domain, how this structure is acted upon by regulatory factors is not yet known. Two appendages to the ring are essential for dynein function; the ‘stalk’, an intramolecular anti-parallel coiled-coil at the end of which lies the microtubule-binding domain (Gee et al, 1997; Carter et al, 2008) and the ‘linker’, which is dynein's key mechanical element and is an elongated structure N-terminal to AAA1.
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