Richard J. McKenney

LIS1 and NudE Induce a Persistent Dynein Force-Producing State

Cytoplasmic dynein is responsible for many aspects of cellular and subcellular movement. LIS1, NudE, and NudEL are dynein interactors initially implicated in brain developmental disease but now known to be required in cell migration, nuclear, centrosomal, and microtubule transport, mitosis, and growth cone motility. Identification of a specific role for these proteins in cytoplasmic dynein motor regulation has remained elusive. We find that NudE stably recruits LIS1 to the dynein holoenzyme molecule, where LIS1 interacts with the motor domain during the prepowerstroke state of the dynein crossbridge cycle. NudE abrogates dynein force production, whereas LIS1 alone or with NudE induces a persistent-force dynein state that improves ensemble function of multiple dyneins for transport under high-load conditions. These results likely explain the requirement for LIS1 and NudE in the transport of nuclei, centrosomes, chromosomes, and the microtubule cytoskeleton as well as the particular sensitivity of migrating neurons to reduced LIS1 expression.

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.

Multiple modes of cytoplasmic dynein regulation

In performing its multiple cellular functions, the cytoplasmic dynein motor is subject to complex regulation involving allosteric mechanisms within the dynein complex, as well as numerous extramolecular interactions controlling subcellular targeting and motor activity. Recent work has distinguished high- and low-load regulatory modes for cytoplasmic dynein, which, combined with a diversity of targeting mechanisms, accounts for a very broad range of functions.

NudE and NudEL are required for mitotic progression and are involved in dynein recruitment to kinetochores

NudE and NudEL are related proteins that interact with cytoplasmic dynein and LIS1. Their functional relationship and involvement in LIS1 and dynein regulation are not completely understood. We find that NudE and NudEL each localize to mitotic kinetochores before dynein, dynactin, ZW10, and LIS1 and exhibit additional temporal and spatial differences in distribution from the motor protein. Inhibition of NudE and NudEL caused metaphase arrest with misoriented chromosomes and defective microtubule attachment. Dynein and dynactin were both displaced from kinetochores by the injection of an anti-NudE/NudEL antibody. Dynein but not dynactin interacted with NudE surprisingly through the dynein intermediate and light chains but not the motor domain. Together, these results identify a common function for NudE and NudEL in mitotic progression and identify an alternative mechanism for dynein recruitment to and regulation at kinetochores.

Mechanical stochastic tug-of-war models cannot explain bidirectional lipid-droplet transport

Intracellular transport via the microtubule motors kinesin and dynein plays an important role in maintaining cell structure and function. Often, multiple kinesin or dynein motors move the same cargo. Their collective function depends critically on the single motors’ detachment kinetics under load, which we experimentally measure here. This experimental constraint—combined with other experimentally determined parameters—is then incorporated into theoretical stochastic and mean-field models. Comparison of modeling results and in vitro data shows good agreement for the stochastic, but not mean-field, model. Many cargos in vivo move bidirectionally, frequently reversing course. Because both kinesin and dynein are present on the cargos, one popular hypothesis explaining the frequent reversals is that the opposite-polarity motors engage in unregulated stochastic tugs-of-war. Then, the cargos’ motion can be explained entirely by the outcome of these opposite-motor competitions. Here, we use fully calibrated stochastic and mean-field models to test the tug-of-war hypothesis. Neither model agrees well with our in vivo data, suggesting that, in addition to inevitable tugs-of-war between opposite motors, there is an additional level of regulation not included in the models.

Mutually exclusive cytoplasmic dynein regulation by nude-LIS1 and dynactin

Background: Cytoplasmic dynein performs a great variety of cellular functions using a diversity of regulators. Results: NudE and dynactin compete for a common site within the dynein complex. Conclusion: This mechanism prevents dual regulation by dynactin and LIS1 and suggests a major new mode of regulatory control. Significance: This is the first insight into coordination of cytoplasmic dynein regulators. Cytoplasmic dynein is responsible for a wide range of cellular roles. How this single motor protein performs so many functions has remained a major outstanding question for many years. Part of the answer is thought to lie in the diversity of dynein regulators, but how the effects of these factors are coordinated in vivo remains unexplored. We previously found NudE to bind dynein through its light chain 8 (LC8) and intermediate chain (IC) subunits (1), the latter of which also mediates the dynein-dynactin interaction (2). We report here that NudE and dynactin bind to a common region within the IC, and compete for this site. We find LC8 to bind to a novel sequence within NudE, without detectably affecting the dynein-NudE interaction. We further find that commonly used dynein inhibitory reagents have broad effects on the interaction of dynein with its regulatory factors. Together these results reveal an unanticipated mechanism for preventing dual regulation of individual dynein molecules, and identify the IC as a nexus for regulatory interactions within the dynein complex.

High-resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high-load axonal transport

High-resolution particle tracking shows a specific role for the dynein regulatory factor LIS1 in high-load axonal transport of large vesicles but no evidence for mechanical activation of opposite-directed motors.

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.

Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains

Cytoplasmic dynein is the predominant minus-end-directed microtubule (MT) motor in most eukaryotic cells. In addition to transporting vesicular cargos, dynein helps to organize MTs within MT networks such as mitotic spindles. How dynein performs such non-canonical functions is unknown. Here we demonstrate that dynein crosslinks and slides anti-parallel MTs in vitro. Surprisingly, a minimal dimeric motor lacking a tail domain and associated subunits can cause MT sliding. Single molecule imaging reveals that motors pause and frequently reverse direction when encountering an anti-parallel MT overlap, suggesting that the two motor domains can bind both MTs simultaneously. In the mitotic spindle, inward microtubule sliding by dynein counteracts outward sliding generated by kinesin-5, and we show that a tailless, dimeric motor is sufficient to drive this activity in mammalian cells. Our results identify an unexpected mechanism for dynein-driven microtubule sliding, which differs from filament sliding mechanisms described for other motor proteins. DOI: http://dx.doi.org/10.7554/eLife.00943.001

Expression patterns of LIS1, dynein and their interaction partners dynactin, NudE, NudEL and NudC in human gliomas suggest roles in invasion and proliferation.

Diffusely infiltrating gliomas are the most common type of primary intracranial neoplasm in humans. One of the major obstacles to the effective treatment of these tumors is their highly infiltrative growth. However, mechanisms controlling their migration and proliferation are poorly understood. Glioma cells resemble neural progenitors, and we hypothesize that gliomas recapitulate the capacity of migration and proliferation of progenitors that takes place during brain development. Based on recent evidence implicating cytoplasmic dynein and its regulatory proteins in neural progenitor migration and division, we conducted immunohistochemical evaluation of surgically resected human glioma samples for the presence and distribution of these proteins. We examined expression of LIS1, the gene responsible for type I lissencephaly, cytoplasmic dynein and the dynein- and LIS1-interacting factors dynactin, NudE/NudEL and NudC, which play significant roles in neural progenitor cell behavior. We found that each of these proteins is expressed in all histological types and grades of human neuroectodermal tumors examined. Immunohistochemical analysis revealed that the levels of expression varied from cell to cell within each tumor, ranging from very high to undetectable. This stands in contrast to the low levels of diffuse staining seen in non-neoplastic brain tissue. Of particular interest, we noted tumor cells infiltrating the white matter and tumor cells undergoing cell division amongst the cells with notably high expression levels. These findings are compatible with the idea that LIS1 and its interacting proteins play a role in glioma migration and proliferation analogous to their role during brain development.