Walter Huynh

Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes

How dynein makes the right moves The molecular motor cytoplasmic dynein moves a wide range of different intracellular cargoes. Dyneins activity in vivo requires another protein, dynactin, but exactly why that should be has been very unclear. Although in vitro experiments have provided some evidence that dynactin increases dyneins processivity, the resulting dynein motility has never come close to matching dyneins cargo-transporting activity in living cells. Now, McKenney et al. show that tripartite complexes of dynein, dynactin, and an adaptor molecule are highly processive in vitro, moving the sort of distances that dynein transports cargo in vivo (see the Perspective by Allan). Science, this issue p. 337; see also p. 271 Single-molecule studies reveal a mechanism to activate the molecular motor cytoplasmic dynein in a cargo-specific manner. [Also see Perspective by Allan] Cytoplasmic dynein is a molecular motor that transports a large variety of cargoes (e.g., organelles, messenger RNAs, and viruses) along microtubules over long intracellular distances. The dynactin protein complex is important for dynein activity in vivo, but its precise role has been unclear. Here, we found that purified mammalian dynein did not move processively on microtubules in vitro. However, when dynein formed a complex with dynactin and one of four different cargo-specific adapter proteins, the motor became ultraprocessive, moving for distances similar to those of native cargoes in living cells. Thus, we propose that dynein is largely inactive in the cytoplasm and that a variety of adapter proteins activate processive motility by linking dynactin to dynein only when the motor is bound to its proper cargo.

Electron cryo-microscopy structure of the mechanotransduction channel NOMPC

Mechanosensory transduction for senses such as proprioception, touch, balance, acceleration, hearing and pain relies on mechanotransduction channels, which convert mechanical stimuli into electrical signals in specialized sensory cells. How force gates mechanotransduction channels is a central question in the field, for which there are two major models. One is the membrane-tension model: force applied to the membrane generates a change in membrane tension that is sufficient to gate the channel, as in the bacterial MscL channel and certain eukaryotic potassium channels. The other is the tether model: force is transmitted via a tether to gate the channel. The transient receptor potential (TRP) channel NOMPC is important for mechanosensation-related behaviours such as locomotion, touch and sound sensation across different species including Caenorhabditis elegans, Drosophila and zebrafish. NOMPC is the founding member of the TRPN subfamily, and is thought to be gated by tethering of its ankyrin repeat domain to microtubules of the cytoskeleton. Thus, a goal of studying NOMPC is to reveal the underlying mechanism of force-induced gating, which could serve as a paradigm of the tether model. NOMPC fulfils all the criteria that apply to mechanotransduction channels and has 29 ankyrin repeats, the largest number among TRP channels. A key question is how the long ankyrin repeat domain is organized as a tether that can trigger channel gating. Here we present a de novo atomic structure of Drosophila NOMPC determined by single-particle electron cryo-microscopy. Structural analysis suggests that the ankyrin repeat domain of NOMPC resembles a helical spring, suggesting its role of linking mechanical displacement of the cytoskeleton to the opening of the channel. The NOMPC architecture underscores the basis of translating mechanical force into an electrical signal within a cell.

Tyrosination of α-tubulin controls the initiation of processive dynein–dynactin motility

Post‐translational modifications (PTMs) of α/β‐tubulin are believed to regulate interactions with microtubule‐binding proteins. A well‐characterized PTM involves in the removal and re‐ligation of the C‐terminal tyrosine on α‐tubulin, but the purpose of this tyrosination–detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactins microtubule‐binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the α‐tubulin tyrosine facilitates initial motor–tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C‐terminal α‐tubulin tyrosine on dynein–dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein‐driven motility in cells.

HkRP3 Is a Microtubule-Binding Protein Regulating Lytic Granule Clustering and NK Cell Killing

NK cells provide host defense by killing viral-infected and cancerous cells through the secretion of preformed lytic granules. Polarization of the lytic granules toward the target cell is dependent on an intact microtubule (MT) network as well as MT motors. We have recently shown that DOCK8, a gene mutated in a primary immunodeficiency syndrome, is involved in NK cell killing in part through its effects on MT organizing center (MTOC) polarization. In this study, we identified Hook-related protein 3 (HkRP3) as a novel DOCK8- and MT-binding protein. We further show that HkRP3 is present in lytic granule fractions and interacts with the dynein motor complex and MTs. Significantly, depletion of HkPR3 impaired NK cell cytotoxicity, which could be attributed to a defect in not only MTOC polarity, but also impaired clustering of lytic granules around the MTOC. Our results demonstrate an important role for HkRP3 in regulating the clustering of lytic granules and MTOC repositioning during the development of NK cell–mediated killing.

Disease-associated mutations in human BICD2 hyperactivate motility of dynein–dynactin

Bicaudal D2 (BICD2) joins dynein with dynactin into a ternary complex (termed DDB) capable of processive movement. Point mutations in the BICD2 gene have been identified in patients with a dominant form of spinal muscular atrophy, but how these mutations cause disease is unknown. To investigate this question, we have developed in vitro motility assays with purified DDB and BICD2’s membrane vesicle partner, the GTPase Rab6a. Rab6a–GTP, either in solution or bound to artificial liposomes, released BICD2 from an autoinhibited state and promoted robust dynein–dynactin transport. In these assays, BICD2 mutants showed an enhanced ability to form motile DDB complexes. Increased retrograde transport by BICD2 mutants also was observed in cells using an inducible organelle transport assay. When overexpressed in rat hippocampal neurons, the hyperactive BICD2 mutants decreased neurite growth. Our results reveal that dominant mutations in BICD2 hyperactivate DDB motility and suggest that an imbalance of minus versus plus end–directed microtubule motility in neurons may underlie spinal muscular atrophy.

A new calcium-activated dynein adaptor protein, CRACR2a, regulates clathrin-independent endocytic traffic in T cells

Cytoplasmic dynein is a microtubule minus-end-directed motor that transports numerous intracellular cargoes. Mammalian dynein transport is initiated by coiled-coil adaptor proteins that 1) join dynein and its co-factor dynactin into a complex capable of processive motility, and 2) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, which have a coiled-coil adaptor domain, a pair of EF hands, and a Rab GTPase domain fused into a single polypeptide. We find that CRACR2a-mediated dynein-dynactin motility is activated by calcium in vitro and in cells. In activated T cells, CRACR2a localizes to clathrin-independent endosomes that require microtubule-based transport to detach from the actin cortex and travel towards the microtubule organizing center. Together these results represent the first known examples of Rab GTPases that directly act as dynein adaptors and implicate CRACR2a-dynein in regulation of endocytic trafficking in T cells.

Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots

We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 μg protein, with yield of ∼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.