A family of small molecules called ‘ciliobrevins’ are described that can rapidly and reversibly modulate the AAA+ ATPase motor dynein, which transports cargo molecules along microtubule tracks. Enzymes of the AAA+ ATPase family convert chemical potential energy into the mechanical forces required for cellular processes, such as cargo transport and cytoskeleton disassembly. Here, Firestone et al. describe a class of small molecules called ciliobrevins. These molecules can rapidly and reversibly modulate the AAA+ ATPase motor, dynein, which transports cargo molecules along microtubule tracks. The ciliobrevins will be useful as molecular probes of dynein function. The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion1. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking2, mitotic spindle formation3 and organelle transport4, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore–microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.

The microtubules that attach kinetochores to chromosomes (K-fibres) are stabilized in prometaphase to allow for accurate chromosome segregation. Kapoor and colleagues find that the B56-PP2A phosphatase stabilizes K-fibres potentially by counteracting the phosphorylation of kinetochore substrates that is mediated by Aurora B and Plk1. Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore–microtubule attachments (K-fibres) on sister chromatids that have attached to opposite spindle poles (bi-oriented)1. In many instances, phosphorylation correlates with K-fibre destabilization2,3,4,5,6,7. Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes when compared with bi-oriented chromosomes, which have stable attachments2,8. Paradoxically, however, these kinases also target to prometaphase chromosomes that have not yet established spindle attachments and it is therefore unclear how kinetochore–microtubule interactions can be stabilized when kinase levels are high. Here we show that the generation of stable K-fibres depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibres are destabilized and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the level of phosphorylation of Aurora B and Plk1 kinetochore substrates as well as Plk1 recruitment to kinetochores. Consistent with increased substrate phosphorylation, we find that chemical inhibition of Aurora or Plk1 restores K-fibres in B56-PP2A-depleted cells. Our findings reveal that PP2A, an essential tumour suppressor9, tunes the balance of phosphorylation to promote chromosome–spindle interactions during cell division.