Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes1,2. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules3. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B4,5. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules6. Moreover, tension increases the lifetimes of the reconstituted attachments directly, via a catch bond-like mechanism that does not require Aurora B7-10. Based on these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. We describe a 1.9 Å resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic N-and C-termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We could therefore build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions. KeywordsAquaporin-0; lens; MIP; two-dimensional crystal; lipid-protein interaction; electron crystallography Members of the aquaporin (AQP) family form membrane pores that are either highly selective for water (aquaporins) or also permeable to other small neutral solutes such as glycerol and urea (aquaglyceroporins) (reviewed in 1 ). Structural studies have revealed that all AQPs share the same basic architecture, which consists of two tandem repeats, each containing a bundle of three transmembrane α-helices and a hydrophobic loop with the highly conserved asparagine-proline-alanine (NPA) motif 2 -8 . The two NPA-containing loops B and E fold back into the membrane and form short α-helices (HB and HE) that line the water pore. The ar/R constriction site, so named because it is formed by an aromatic and an arginine residue, confers water selectivity to AQP pores, while the NPA motifs play an important role in the proton exclusion mechanism (reviewed in 9 ).Correspondence to: Thomas Walz.Correspondence and requests for materials should be addressed to T.W. (twalz@hms.harvard.edu). Coordinates and structure factors for junctional and non-junctional AQP0 have been deposited in the Protein Data Bank (accession codes 2B6O and 2B6P, respectively).. Suplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests. AQP0 is the most abundant protein in lens fibre cell membranes, where it forms not only water pores but also the 11-13 nm "thin lens junctions" that assemble upon proteolytic cleavage of the cytoplasmic termini 10 , 11 . We recently presented the structure of the AQP0-mediated membrane junction at 3 Å resolution as determined by electron crystallography of doublelayered two-dimensional (2D) crystals 7 . The structure showed that AQP0 junctions are stabilised by specific interactions between tetramers in adjoining membranes involving almost exclusively proline residues. Calculated pore profiles also showed that the pore in junctional AQP0 is highly constricted due to a substantially ...

Subcellular membrane-less assemblies are a reinvigorated area study in biology with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces we determined atomic structures of five segments of protein low-complexity domains associated with membrane-less assemblies. Their common structural feature is the stacking of segments into kinked β-sheets which pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic sidechains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, known to form networks and localize to membrane-less assemblies.