Nexin

Abstract: Cilia and flagella are highly conserved microtubule (MT)-based organelles with motile and sensory functions, and ciliary defects have been linked to several human diseases. The 9 + 2 structure of motile axonemes contains nine MT doublets interconnected by nexin links, which surround a central pair of singlet MTs. Motility is generated by the orchestrated activity of thousands of dynein motors, which drive interdoublet sliding. A key regulator of motor activity is the dynein regulatory complex (DRC), but detailed structural information is lacking. Using cryoelectron tomography of wild-type and mutant axonemes from Chlamydomonas reinhardtii, we visualized the DRC in situ at molecular resolution. We present the three-dimensional structure of the DRC, including a model for its subunit organization and intermolecular connections that establish the DRC as a major regulatory node. We further demonstrate that the DRC is the nexin link, which is thought to be critical for the generation of axonemal bending.

Abstract: Interactions between microtubule and actin networks are thought to be crucial for mechanical and signalling events at the cell cortex. Cytoplasmic dynein has been proposed to mediate many of these interactions. Here, we report that dynein is localized to the cortex at adherens junctions in cultured epithelial cells and that this localization is sensitive to drugs that disrupt the actin cytoskeleton. Dynein is recruited to developing contacts between cells, where it localizes with the junctional proteins beta-catenin and E-cadherin. Microtubules project towards these early contacts and we hypothesize that dynein captures and tethers microtubules at these sites. Dynein immunoprecipitates with beta-catenin, and biochemical analysis shows that dynein binds directly to beta-catenin. Overexpression of beta-catenin disrupts the cellular localization of dynein and also dramatically perturbs the organization of the cellular microtubule array. In cells overexpressing beta-catenin, the centrosome becomes disorganized and microtubules no longer appear to be anchored at the cortex. These results identify a novel role for cytoplasmic dynein in capturing and tethering microtubules at adherens junctions, thus mediating cross-talk between actin and microtubule networks at the cell cortex.

Abstract: Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1–3 Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4–7 How retromer is assembled and how it is recruited to form coated tubules is not known Here we describe the structure of the retromer complex (Vps26–Vps29–Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface Vps35 forms the ‘legs’ of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions

Abstract: EM analyses reveal the architecture of cytoplasmic dynein in complex with dynactin and the BicD2 cargo adaptor on microtubules, showing the quaternary complex positioned for unidirectional movement and cargo recruitment.