Dynein Regulation Research Articles

CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon.

Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.

Role of Disorder in the Regulation of Dynein's Intermediate Chain

Cytoplasmic dynein is a large, eukaryotic motor protein complex composed of heavy, intermediate, light intermediate, and light chain subunits. The motor domain of dynein has been well characterized, however in the tail domain there are almost 300 amino acids of disorder that remain missing from resolved dynein structures. This disorder lies in dynein's intermediate chain (IC), the key protein in dynein's cargo attachment subcomplex, involved in binding dynein's light chain subunits as well as various non-dynein regulatory proteins.

Cell scientist to watch – Anthony Roberts

ABSTRACT Anthony Roberts studied biochemistry at Imperial College London, UK. He then pursued a PhD in molecular and cellular biology with Peter Knight and Stan Burgess at the University of Leeds, UK, where he studied the mechanism of the dynein motor protein. After this, Anthony moved to Boston, USA, for his postdoctoral work with Samara Reck-Peterson, at the Harvard Medical School, focussing on cytoplasmic dynein regulation. In late 2014, he returned to London, UK to start his own lab at the Institute of Structural and Molecular Biology at Birkbeck, University of London, and University College London (UCL), where he is now a Wellcome Trust Senior Research Fellow and Proleptic Senior Lecturer. Anthony received the Biochemical Society Early Career Research Award in 2016, a Biotechnology and Biological Sciences Research Council New Investigator Award in 2017, and was elected to the EMBO Young Investigator Programme in 2018. His lab is focussing on the mechanisms of microtubule-based transport within cilia and flagella.

Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins.

Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly-acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use in vivo imaging in Xenopus to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity- purification mass-spectrometry of in vivo interaction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.

Cargo-Mediated Activation of Cytoplasmic Dynein in vivo.

Cytoplasmic dynein-1 is a minus-end-directed microtubule motor that transports a variety of cargoes including early endosomes, late endosomes and other organelles. In many cell types, dynein accumulates at the microtubule plus end, where it interacts with its cargo to be moved toward the minus end. Dynein binds to its various cargoes via the dynactin complex and specific cargo adapters. Dynactin and some of the coiled-coil-domain-containing cargo adapters not only link dynein to cargo but also activate dynein motility, which implies that dynein is activated by its cellular cargo. Structural studies indicate that a dynein dimer switches between the autoinhibited phi state and an open state; and the binding of dynactin and a cargo adapter to the dynein tails causes the dynein motor domains to have a parallel configuration, allowing dynein to walk processively along a microtubule. Recently, the dynein regulator LIS1 has been shown to be required for dynein activation in vivo, and its mechanism of action involves preventing dynein from switching back to the autoinhibited state. In this review, we will discuss our current understanding of dynein activation and point out the gaps of knowledge on the spatial regulation of dynein in live cells. In addition, we will emphasize the importance of studying a complete set of dynein regulators for a better understanding of dynein regulation in vivo.

New insights into the mechanism of dynein motor regulation by lissencephaly-1.

Lissencephaly (‘smooth brain’) is a severe brain disease associated with numerous symptoms, including cognitive impairment, and shortened lifespan. The main causative gene of this disease – lissencephaly-1 (LIS1) – has been a focus of intense scrutiny since its first identification almost 30 years ago. LIS1 is a critical regulator of the microtubule motor cytoplasmic dynein, which transports numerous cargoes throughout the cell, and is a key effector of nuclear and neuronal transport during brain development. Here, we review the role of LIS1 in cellular dynein function and discuss recent key findings that have revealed a new mechanism by which this molecule influences dynein-mediated transport. In addition to reconciling prior observations with this new model for LIS1 function, we also discuss phylogenetic data that suggest that LIS1 may have coevolved with an autoinhibitory mode of cytoplasmic dynein regulation.

Lis1 co-localizes with actin in the phagocytic cup and regulates phagocytosis.

Phagocytosis, the ingestion of solid particles by cells is essential for nutrient uptake, innate immune response, antigen presentation and organelle homeostasis. Here we show that Lissencephaly-1 (Lis1), a well-known regulator of the microtubule motor dynein, co-localizes with actin at the phagocytic cup in the early stages of phagocytosis. Both knockdown and overexpression of Lis1 perturb phagocytosis, suggesting that Lis1 levels may be regulated during particle engulfment to facilitate remodeling of actin filaments within the phagocytic cup. This requirement of Lis1 is replicated in mouse macrophage cells as well as in the amoeba Dictyostelium, indicating an evolutionarily conserved role for Lis1 in phagocytosis. In support of these findings, Dictyostelium cells overexpressing Lis1 show defects in migration possibly caused by dysregulated actin. Taken together, Lis1 localizes to the phagocytic cup and influences the actin cytoskeleton in a manner that appears important for the uptake of solid particles into cells.

LAX28 is required for the stable assembly of the inner dynein arm f complex, and the tether and tether head complex in Leishmania flagella.

Motile eukaryotic flagella beat through coordinated activity of dynein motor proteins; however, the mechanisms of dynein coordination and regulation are incompletely understood. The inner dynein arm (IDA) f complex (also known as the I1 complex), and the tether and tether head (T/TH) complex are thought to be key regulators of dynein action but, unlike the IDA f complex, T/TH proteins remain poorly characterised. Here, we characterised T/TH-associated proteins in the protist Leishmania mexicana Proteome analysis of axonemes from null mutants for the CFAP44 T/TH protein showed that they lacked the IDA f protein IC140 and a novel 28-kDa axonemal protein, LAX28. Sequence analysis identified similarities between LAX28 and the uncharacterised human sperm tail protein TEX47, both sharing features with sensory BLUF-domain-containing proteins. Leishmania lacking LAX28, CFAP44 or IC140 retained some motility, albeit with reduced swimming speed and directionality and a propensity for flagellar curling. Expression of tagged proteins in different null mutant backgrounds showed that the axonemal localisation of LAX28 requires CFAP44 and IC140, and the axonemal localisations of CFAP44 and IC140 both depend on LAX28. These data demonstrate a role for LAX28 in motility and show mutual dependencies of IDA f and T/TH-associated proteins for axonemal assembly in Leishmania.

Mechanistic insights into the interactions of dynein regulator Ndel1 with neuronal ankyrins and implications in polarity maintenance

Ankyrin-G (AnkG), a highly enriched scaffold protein in the axon initial segment (AIS) of neurons, functions to maintain axonal polarity and the integrity of the AIS. At the AIS, AnkG regulates selective intracellular cargo trafficking between soma and axons via interaction with the dynein regulator protein Ndel1, but the molecular mechanism underlying this binding remains elusive. Here we report that Ndel1's C-terminal coiled-coil region (CT-CC) binds to giant neuron-specific insertion regions present in both AnkG and AnkB with 2:1 stoichiometry. The high-resolution crystal structure of AnkB in complex with Ndel1 CT-CC revealed the detailed molecular basis governing the AnkB/Ndel1 complex formation. Mechanistically, AnkB binds with Ndel1 by forming a stable 5-helix bundle dominated by hydrophobic interactions spread across 6 distinct interaction layers. Moreover, we found that AnkG is essential for Ndel1 accumulation at the AIS. Finally, we found that cargo sorting at the AIS can be disrupted by blocking the AnkG/Ndel1 complex formation using a peptide designed based on our structural data. Collectively, the atomic structure of the AnkB/Ndel1 complex together with studies of cargo sorting through the AIS establish the mechanistic basis for AnkG/Ndel1 complex formation and for the maintenance of axonal polarity. Our study will also be valuable for future studies of the interaction between AnkB and Ndel1 perhaps at distal axonal cargo transport.

Asymmetries in the cilia of Chlamydomonas.

The generation of ciliary waveforms requires the spatial and temporal regulation of dyneins. This review catalogues many of the asymmetric structures and proteins in the cilia of Chlamydomonas, a unicellular alga with two cilia that are used for motility in liquid medium. These asymmetries, which have been identified through mutant analysis, cryo-EM tomography and proteomics, provide a wealth of information to use for modelling how waveforms are generated and propagated. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

NuMA1 promotes axon initial segment assembly through inhibition of endocytosis.

Axon initial segments (AISs) initiate action potentials and regulate the trafficking of vesicles between somatodendritic and axonal compartments. However, the mechanisms controlling AIS assembly remain poorly defined. We performed differential proteomics and found nuclear mitotic apparatus protein 1 (NuMA1) is downregulated in AIS-deficient neonatal mouse brains and neurons. NuMA1 is transiently located at the AIS during development where it interacts with the scaffolding protein 4.1B and the dynein regulator lissencephaly 1 (Lis1). Silencing NuMA1 or protein 4.1B by shRNA disrupts AIS assembly, but not maintenance. Silencing Lis1 or overexpressing NuMA1 during AIS assembly increased the density of AIS proteins, including ankyrinG and neurofascin-186 (NF186). NuMA1 inhibits the endocytosis of AIS NF186 by impeding Lis1's interaction with doublecortin, a potent facilitator of NF186 endocytosis. Our results indicate the transient expression and AIS localization of NuMA1 stabilizes the developing AIS by inhibiting endocytosis and removal of AIS proteins.

Role of Dynactin in the Intracellular Localization and Activation of Cytoplasmic Dynein.

Cytoplasmic dynein, the major minus end-directed motor protein in several cell types, transports a variety of intracellular cargo upon forming a processive tripartite complex with its activator dynactin and cargo adaptors such as Hook3 and BicD2. Our current understanding of dynein regulation stems from a combination of in vivo studies of cargo movement upon perturbation of dynein activity, in vitro single-molecule experiments, and cryo-electron microscopy studies of dynein structure and its interaction with dynactin and cargo adaptors. In this Perspective, we first consolidate data from recent publications to understand how perturbations to the dynein-dynactin interaction and dynactin's in vivo localization alter the behavior of dynein-driven cargo transport in a cell type- and experimental condition-specific manner. In addition, we touch upon results from in vivo and in vitro studies to elucidate how dynein's interaction with dynactin and cargo adaptors activates dynein and enhances its processivity. Finally, we propose questions that need to be addressed in the future with appropriate experimental designs so as to improve our understanding of the spatiotemporal regulation of dynein's function in the context of the distribution and dynamics of dynactin in living cells.

FIGNL1 associates with KIF1Bβ and BICD1 to restrict dynein transport velocity during axon navigation.

Neuronal connectivity relies on molecular motor-based axonal transport of diverse cargoes. Yet the precise players and regulatory mechanisms orchestrating such trafficking events remain largely unknown. We here report the ATPase Fignl1 as a novel regulator of bidirectional transport during axon navigation. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we showed that Fignl1 binds the kinesin Kif1bβ and the dynein/dynactin adaptor Bicaudal D-1 (Bicd1) in a molecular complex including the dynactin subunit dynactin 1. Fignl1 colocalized with Kif1bβ and showed bidirectional mobility in zebrafish axons. Notably, Kif1bβ and Fignl1 loss of function similarly altered zebrafish motor axon pathfinding and increased dynein-based transport velocity of Rab3 vesicles in these navigating axons, pinpointing Fignl1/Kif1bβ as a dynein speed limiter complex. Accordingly, disrupting dynein/dynactin activity or Bicd1/Fignl1 interaction induced motor axon pathfinding defects characteristic of Fignl1 gain or loss of function, respectively. Finally, pharmacological inhibition of dynein activity partially rescued the axon pathfinding defects of Fignl1-depleted larvae. Together, our results identify Fignl1 as a key dynein regulator required for motor circuit wiring.

Dynein tails: how to hitch a ride on an IFT train.

Researchers have sought to understand the function and regulation of the motor protein dynein since its discovery more than 50 years ago1. Dynein-2 is one of the motors that move the intraflagellar transport (IFT) trains ― large protein complexes that are needed for the assembly and function of eukaryotic cilia and flagella. Toropova et al. report the single-particle cryo-EM structure of the human dynein-2 complex2, which unexpectedly reveals two different conformations of the motor subunit tails. One tail forms a zigzag that matches the periodicity of the IFT trains, which reinforces the auto-inhibition of dynein motor activity and the binding of multiple dynein-2 complexes along the train during anterograde transport.

Molecular basis for dyneinopathies reveals insight into dynein regulation and dysfunction.

Cytoplasmic dynein plays critical roles within the developing and mature nervous systems, including effecting nuclear migration, and retrograde transport of various cargos. Unsurprisingly, mutations in dynein are causative of various developmental neuropathies and motor neuron diseases. These 'dyneinopathies' define a broad spectrum of diseases with no known correlation between mutation identity and disease state. To circumvent complications associated with dynein studies in human cells, we employed budding yeast as a screening platform to characterize the motility properties of seventeen disease-correlated dynein mutants. Using this system, we determined the molecular basis for several classes of etiologically related diseases. Moreover, by engineering compensatory mutations, we alleviated the mutant phenotypes in two of these cases, one of which we confirmed with recombinant human dynein. In addition to revealing molecular insight into dynein regulation, our data provide additional evidence that the type of disease may in fact be dictated by the degree of dynein dysfunction.

Distinct roles for dynein light intermediate chains in neurogenesis, migration, and terminal somal translocation.

Cytoplasmic dynein participates in multiple aspects of neocortical development. These include neural progenitor proliferation, morphogenesis, and neuronal migration. The cytoplasmic dynein light intermediate chains (LICs) 1 and 2 are cargo-binding subunits, though their relative roles are not well understood. Here, we used in utero electroporation of shRNAs or LIC functional domains to determine the relative contributions of the two LICs in the developing rat brain. We find that LIC1, through BicD2, is required for apical nuclear migration in neural progenitors. In newborn neurons, we observe specific roles for LIC1 in the multipolar to bipolar transition and glial-guided neuronal migration. In contrast, LIC2 contributes to a novel dynein role in the little-studied mode of migration, terminal somal translocation. Together, our results provide novel insight into the LICs' unique functions during brain development and dynein regulation overall.

Regulation of in vivo dynein force production by CDK5 and 14-3-3\u03b5 and KIAA0528

Single-molecule cytoplasmic dynein function is well understood, but there are major gaps in mechanistic understanding of cellular dynein regulation. We reported a mode of dynein regulation, force adaptation, where lipid droplets adapt to opposition to motion by increasing the duration and magnitude of force production, and found LIS1 and NudEL to be essential. Adaptation reflects increasing NudEL-LIS1 utilization; here, we hypothesize that such increasing utilization reflects CDK5-mediated NudEL phosphorylation, which increases the dynein-NudEL interaction, and makes force adaptation possible. We report that CDK5, 14-3-3ε, and CDK5 cofactor KIAA0528 together promote NudEL phosphorylation and are essential for force adaptation. By studying the process in COS-1 cells lacking Tau, we avoid confounding neuronal effects of CDK5 on microtubules. Finally, we extend this in vivo regulatory pathway to lysosomes and mitochondria. Ultimately, we show that dynein force adaptation can control the severity of lysosomal tug-of-wars among other intracellular transport functions involving high force.

Dynamic regulation of dynein localization revealed by small molecule inhibitors of ubiquitination enzymes.

Cytoplasmic dynein is a minus-end-directed microtubule-based motor that acts at diverse subcellular sites. During mitosis, dynein localizes simultaneously to the mitotic spindle, spindle poles, kinetochores and the cell cortex. However, it is unclear what controls the relative targeting of dynein to these locations. As dynein is heavily post-translationally modified, we sought to test a role for these modifications in regulating dynein localization. We find that dynein rapidly and strongly accumulates at mitotic spindle poles following treatment with NSC697923, a small molecule that inhibits the ubiquitin E2 enzyme, Ubc13, or treatment with PYR-41, a ubiquitin E1 inhibitor. Subsets of dynein regulators such as Lis1, ZW10 and Spindly accumulate at the spindle poles, whereas others do not, suggesting that NSC697923 differentially affects specific dynein populations. We additionally find that dynein relocalization induced by NSC697923 or PYR-41 can be suppressed by simultaneous treatment with the non-selective deubiquitinase inhibitor, PR-619. However, we did not observe altered dynein localization following treatment with the selective E1 inhibitor, TAK-243. Although it is possible that off-target effects of NSC697923 and PYR-41 are responsible for the observed changes in dynein localization, the rapid relocalization upon drug treatment highlights the highly dynamic nature of dynein regulation during mitosis.

Nde1 promotes diverse dynein functions through differential interactions and exhibits an isoform-specific proteasome association.

Nde1 is a key regulator of cytoplasmic dynein, binding directly to both dynein itself and the dynein adaptor, Lis1. Nde1 and Lis1 are thought to function together to promote dynein function, yet mutations in each result in distinct neurodevelopment phenotypes. To reconcile these phenotypic differences, we sought to dissect the contribution of Nde1 to dynein regulation and explore the cellular functions of Nde1. Here we show that an Nde1–Lis1 interaction is required for spindle pole focusing and Golgi organization but is largely dispensable for centrosome placement, despite Lis1 itself being required. Thus, diverse functions of dynein rely on distinct Nde1- and Lis1-mediated regulatory mechanisms. Additionally, we discovered a robust, isoform-specific interaction between human Nde1 and the 26S proteasome and identify precise mutations in Nde1 that disrupt the proteasome interaction. Together, our work suggests that Nde1 makes unique contributions to human neurodevelopment through its regulation of both dynein and proteasome function.

Cdk1 phosphorylation of the dynein adapter Nde1 controls cargo binding from G2 to anaphase.

Cytoplasmic dynein is involved in diverse cell cycle-dependent functions regulated by several accessory factors, including Nde1 and Ndel1. Little is known about the role of these proteins in dynein cargo binding, and less is known about their cell cycle--dependent dynein regulation. Using Nde1 RNAi, mutant cDNAs, and a phosphorylation site-specific antibody, we found a specific association of phospho-Nde1 with the late G2-M nuclear envelope and prophase to anaphase kinetochores, comparable to the pattern for the Nde1 interactor CENP-F. Phosphomutant-Nde1 associated only with prometaphase kinetochores and showed weaker CENP-F binding in in vitro assays. Nde1 RNAi caused severe delays in mitotic progression, which were substantially rescued by both phosphomimetic and phosphomutant Nde1. Expression of a dynein-binding-deficient Nde1 mutant reduced kinetochore dynein by half, indicating a major role for Nde1 in kinetochore dynein recruitment. These results establish CENP-F as the first well-characterized Nde1 cargo protein, and reveal phosphorylation control of Nde1 cargo binding throughout a substantial fraction of the cell cycle.