Metazoan dynein moves processively with the aid of dynactin and the endosomal cargo adaptor Hook3. A structure–function study of Hook3 reveals how it assembles dynein with dynactin and suggests that an additional step of allosteric activation is required beyond complex assembly.

Cytoplasmic dynein, a member of the AAA family of ATPases, drives the processive movement of numerous intracellular cargos towards the minus end of microtubules. Here, we summarize the structural and motile properties of dynein and highlight features that distinguish this motor from kinesin-1 and myosin V, two well-studied transport motors. Integrating information from recent crystal and cryo-EM structures as well as high-resolution single molecule studies, we also discuss models for how dynein biases its movement in one direction along a microtubule track, and present a movie that illustrates these principles.

Cytoplasmic dynein, a microtubule-based motor protein, transports many intracellular cargos by means of its light intermediate chain (LIC). In this study, we have determined the crystal structure of the conserved LIC domain, which binds the motor heavy chain, from a thermophilic fungus. We show that the LIC has a Ras-like fold with insertions that distinguish it from Ras and other previously described G proteins. Despite having a G protein fold, the fungal LIC has lost its ability to bind nucleotide, while the human LIC1 binds GDP preferentially over GTP. We show that the LIC G domain binds the dynein heavy chain using a conserved patch of aromatic residues, whereas the less conserved C-terminal domain binds several Rab effectors involved in membrane transport. These studies provide the first structural information and insight into the evolutionary origin of the LIC as well as revealing how this critical subunit connects the dynein motor to cargo.DOI: http://dx.doi.org/10.7554/eLife.03351.001

Metazoan cytoplasmic dynein moves processively along microtubules with the aid of dynactin and an adaptor protein that joins dynein and dynactin into a stable ternary complex. Here, we have examined how Hook3, a cargo adaptor involved in Golgi and endosome transport, forms a motile dynein-dynactin complex. We show that the conserved Hook domain interacts directly with the dynein light intermediate chain 1 (LIC1). By solving the crystal structure of the Hook domain and using structure-based mutagenesis, we identify two conserved surface residues that are each critical for LIC1 binding. Hook proteins with mutations in these residues fail to form a stable dynein-dynactin complex, revealing a crucial role for LIC1 in this interaction. We also identify a region of Hook3 specifically required for an allosteric activation of processive motility.Our work reveals the structural details of Hook3's interaction with dynein and offers insight into how cargo adaptors form processive dynein-dynactin motor complexes. (Maldonado-Baez et al., 2013;Szebenyi et al., 2006;Moynihan et al., 2009;Sano et al., 2007;Walenta et al., 2001). All mammalian Hook isoforms promote endosomal trafficking by forming a complex with Fused Toes (FTS) and Hook Interacting Protein (FHIP) (Xu et al., 2008).Here, we sought to understand the mechanism by which Hook3 interacts with dynein and dynactin and activates processive motility. We report the crystal structure of the Hook domain and show that this domain binds directly to the C-terminal region of LIC1. Structure-based mutagenesis studies revealed two conserved surface residues that are essential for this interaction. Abrogation of the LIC interaction renders Hook3 unable to join dynein and dynactin into a stable complex. Interestingly, while the N-terminal 239 residues of Hook3 are sufficient for forming a stable complex with dynein-dynactin, this tripartite complex is immotile; activation of motility requires a more distal coiled coil region of Hook3. This result reveals that complex assembly and the activation of motility are separable activities. Our data suggest a model for how Hook3 joins dynein and dynactin into a motile complex.

Many cytoskeletal proteins perform fundamental biological processes and are evolutionarily ancient. For example, the superfamily of actin-related proteins (Arps) specialized early in eukaryotic evolution for diverse cellular roles in the cytoplasm and the nucleus. Despite its strict conservation across eukaryotes, we find that the Arp superfamily has undergone dramatic lineage-specific diversification in Drosophila. Our phylogenomic analyses reveal four independent Arp gene duplications that occurred in the common ancestor of the obscura group of Drosophila and have been mostly preserved in this lineage. All four obscura-specific Arp paralogs are predominantly expressed in the male germline and have evolved under positive selection. We focus our analyses on the divergent Arp2D paralog, which arose via a retroduplication event from Arp2, a component of the Arp2/3 complex that polymerizes branched actin networks. Computational modeling analyses suggest that Arp2D can replace Arp2 in the Arp2/3 complex and bind actin monomers. Together with the signature of positive selection, our findings suggest that Arp2D may augment Arp2’s functions in the male germline. Indeed, we find that Arp2D is expressed during and following male meiosis, where it localizes to distinct locations such as actin cones—specialized cytoskeletal structures that separate bundled spermatids into individual mature sperm. We hypothesize that this unprecedented burst of genetic innovation in cytoskeletal proteins may have been driven by the evolution of sperm heteromorphism in the obscura group of Drosophila.

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The tyrosine kinase ACK is an oncogene associated with poor prognosis in human cancers. ACK promotes proliferation, in part, by contributing to the activation of Akt, the major PI3-Kinase effector. We show that ACK also regulates PI3-Kinase directly, via interactions with the PI3-Kinase regulatory subunits. ACK interacts with all five regulatory subunit isoforms and directly phosphorylates p85α, p85β, p55α and p50α on Tyr607 (or equivalent). Phosphorylation of p85β at this residue promotes cell proliferation but, counterintuitively, ACK does not stimulate PI3-Kinase catalytic activity. We show that ACK stabilizes p85α levels by promoting an interaction between the p85 nSH2 domain and pTyr607, protecting p85 from ubiquitination. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions where the increased levels of the regulatory subunits, together with the nSH2-pTyr607 interaction, promote formation of dimeric p85. We postulate that these novel dimers undertake nuclear functions that contribute to Cdc42-ACK driven oncogenesis. We propose that ACK shapes PI3-Kinase signalling by dampening the PIP 3 response, whilst continuing to drive cell proliferation through Akt activation and hereto unexplored but crucial functions of nuclear dimeric p85. These new regulatory subunit dimers represent a previously undescribed mode of regulation for PI3-Kinase and potentially reveal additional avenues for therapeutic intervention.

The discovery of neocentromere activity by maize knobs heralded the field of meiotic drive, in which selfish genetic elements exploit meiotic asymmetry to enhance their propagation. A new study reveals the long-awaited basis of this meiotic drive: cytoskeletal motors enable neocentromeric knobs to achieve favorable meiotic positioning and preferential inheritance.