Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements.

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Abstract

Cilia are multifunctional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multimotor assemblies with kinesin-II. Combining these data with insights from single-particle EM, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2's mechanical 'linker' and 'stalk' domains within a novel motor-motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2's recycling activity during IFT.

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Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes.

Richard McKenney, Walter Huynh, Marvin Tanenbaum … (2014)

Cytoplasmic dynein is a molecular motor that transports a large variety of cargoes (e.g., organelles, messenger RNAs, and viruses) along microtubules over long intracellular distances. The dynactin protein complex is important for dynein activity in vivo, but its precise role has been unclear. Here, we found that purified mammalian dynein did not move processively on microtubules in vitro. However, when dynein formed a complex with dynactin and one of four different cargo-specific adapter proteins, the motor became ultraprocessive, moving for distances similar to those of native cargoes in living cells. Thus, we propose that dynein is largely inactive in the cytoplasm and that a variety of adapter proteins activate processive motility by linking dynactin to dynein only when the motor is bound to its proper cargo. Copyright © 2014, American Association for the Advancement of Science.

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Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer.

Mirco Castoldi, Andrei V. Popov (2003)

Microtubules can be assembled in vitro from purified alpha/beta tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymerization-depolymerization, followed by ion-exchange chromatography to remove any contaminating microtubule-associated proteins and motors. Here, we show that only two cycles of polymerization-depolymerization of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and production of a high-concentration tubulin solution. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatography-based procedures.

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Functions and mechanics of dynein motor proteins.

Anthony Roberts, Takahide Kon, Peter Knight … (2013)

Fuelled by ATP hydrolysis, dyneins generate force and movement on microtubules in a wealth of biological processes, including ciliary beating, cell division and intracellular transport. The large mass and complexity of dynein motors have made elucidating their mechanisms a sizable task. Yet, through a combination of approaches, including X-ray crystallography, cryo-electron microscopy, single-molecule assays and biochemical experiments, important progress has been made towards understanding how these giant motor proteins work. From these studies, a model for the mechanochemical cycle of dynein is emerging, in which nucleotide-driven flexing motions within the AAA+ ring of dynein alter the affinity of its microtubule-binding stalk and reshape its mechanical element to generate movement.