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 due to 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 signaling 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.

Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore-microtubule attachments (K-fibers) on sister chromatids that have attached to opposite spindle poles (bi-oriented)1. In many instances, phosphorylation correlates with K-fiber destabilization2–7. Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes compared to 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 generation of stable K-fibers depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibers are destabilized and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the 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-fibers in B56-PP2A depleted cells. Our findings reveal that PP2A, an essential tumor suppressor9, tunes the balance of phosphorylation to promote chromosome-spindle interactions during cell division.

Accurate chromosome segregation depends on biorientation, whereby sister chromatids attach to microtubules emanating from opposite spindle poles. The spindle assembly checkpoint is a conserved surveillance mechanism in eukaryotes that inhibits anaphase onset until all chromosomes are bioriented1, 2, 3. In current models, the recruitment of Mad2, via Mad1, to improperly attached kinetochores is a key step needed to stop cell cycle progression3, 4, 5, 6. However, it is not known if the localization of Mad1-Mad2 to kinetochores is sufficient to block anaphase. Furthermore, it is unclear if other signalling proteins (e.g. Aurora kinases7) that regulate chromosome biorientation have checkpoint functions downstream of Mad1-Mad2 recruitment to kinetochores or if they act upstream to merely quench the primary error signal8. Here, to address both these issues, we engineered a Mad1 construct which, unlike endogenous Mad1, localizes to kinetochores that are bioriented. We show that Mad1’s constitutive localization at kinetochores is sufficient for a metaphase arrest that depends on Mad1-Mad2 binding. By uncoupling the checkpoint from its primary error signal, we show that Aurora kinase, Mps1 and BubR1, but not Polo-like kinase, are needed to maintain the checkpoint arrest even when Mad1 is present on bi-oriented kinetochores. Together, our data suggest a model in which the biorientation errors, which recruit Mad1-Mad2 to kinetochores, may be signalled not only through Mad2’s templated activation dynamics, but also through the activity of widely-conserved kinases, to ensure the fidelity of cell division.

Respiration is a core biological energy-converting process whose last steps are carried out by a chain of multi-subunit complexes in the inner mitochondrial membrane. To probe the functional and structural diversity of eukaryotic respiration, we examined the respiratory chain of the ciliate Tetrahymena thermophila (Tt). Using cryo-electron microscopy on a mixed sample, we solved structures of a supercomplex between Tt-complex I (CI) and Tt-CIII 2 (Tt-SC I+III 2 ) and a structure of Tt-CIV 2 . Tt-SC I+III 2 (~2.3 MDa) is a curved assembly with structural and functional symmetry breaking. Tt-CIV 2 is a ~2.7 MDa dimer with over 52 subunits per protomer, including mitochondrial carriers and a TIM8 3 -TIM13 3 -like domain. Our structural and functional study of the T. thermophila respiratory chain reveals divergence in key components of eukaryotic respiration, expanding our understanding of core metabolism.

Respiration, an essential metabolic process, provides cells with chemical energy. In eukaryotes, respiration occurs via the mitochondrial electron transport chain (mETC) composed of several large membrane-protein complexes. Complex I (CI) is the main entry point for electrons into the mETC. For plants, limited availability of mitochondrial material has curbed detailed biochemical and structural studies of their mETC. Here, we present the cryoEM structure of the known CI assembly intermediate CI* from Vigna radiata at 3.9 Å resolution. CI* contains CI’s NADH-binding and CoQ-binding modules, the proximal-pumping module and the plant-specific γ-carbonic-anhydrase domain (γCA). Our structure reveals significant differences in core and accessory subunits of the plant complex compared to yeast, mammals and bacteria, as well as the details of the γCA domain subunit composition and membrane anchoring. The structure sheds light on differences in CI assembly across lineages and suggests potential physiological roles for CI* beyond assembly.