Dynein Motor Research Articles

LFA-1 and kindlin-3 enable the collaborative transport of SLP-76 microclusters by myosin and dynein motors.

Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB1) and SKAP55 (SKAP1) are recruited into microclusters and activate integrins via the effectors talin-1 and kindlin-3 (FERMT3). We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity and dynein motor function. Although immobilized VLA-4 (α4β1 integrin) and LFA-1 (αLβ2 integrin) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin β2, kindlin-3 and zyxin are required for complete centripetal transport, while integrin β1 and talin-1 are not. CD69 upregulation is similarly dependent on integrin β2, kindlin-3 and zyxin, but not talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and kindlin-3. This article has an associated First Person interview with the first author of the paper.

The Bardet-Biedl syndrome complex component BBS1 controls T cell polarity during immune synapse assembly.

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are co-opted by the non-ciliated T cell to orchestrate polarized endosome recycling and to sustain signaling during immune synapse formation. Here, we investigated the potential role of Bardet-Biedl syndrome 1 protein (BBS1), an essential core component of the BBS complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We demonstrated that BBS1 allows for centrosome polarization towards the immune synapse. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH1 (also known as WASHC1), a process that we demonstrated to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that functions upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling. This article has an associated First Person interview with the first author of the paper.

Dynein intermediate chain 2c (DNCI2c) complex is essential for exiting Mad2-dependent spindle assembly checkpoint

The Mad2 protein plays a key role in the spindle assembly checkpoint (SAC) function. The SAC pathway delays mitotic progression into anaphase until all kinetochores attach to the spindle during mitosis. The formation of the Mad2–p31comet complex correlates with the completion of spindle attachment and the entry into anaphase during mitosis.Herein, we showed that dynein intermediate chain 2c (DNCI2c)—a subunit of dynein motor protein—forms an immunocomplex with p31comet during mitosis. DNCI2c-knockdown resulted in prolonged mitotic arrest in a Mad2-dependent manner. Furthermore, DNCI2c-knockdown-induced mitotic arrest was not rescued by p31comet overexpression. However, the combination of p31comet overexpression with the mitotic drug treatment reversed the mitotic arrest in DNCI2c-knockdown. Together, these results indicate that the DNCI2c–p31comet complex plays an important role in exiting Mad2-dependent SAC.

Effects of Reactive Oxygen and Nitrogen Species on TrkA Expression and Signalling: Implications for proNGF in Aging and Alzheimer's Disease.

Nerve growth factor (NGF) and its precursor form, proNGF, are critical for neuronal survival and cognitive function. In the brain, proNGF is the only detectable form of NGF. Dysregulation of proNGF in the brain is implicated in age-related memory loss and Alzheimer’s disease (AD). AD is characterized by early and progressive degeneration of the basal forebrain, an area critical for learning, memory, and attention. Learning and memory deficits in AD are associated with loss of proNGF survival signalling and impaired retrograde transport of proNGF to the basal forebrain. ProNGF transport and signalling may be impaired by the increased reactive oxygen and nitrogen species (ROS/RNS) observed in the aged and AD brain. The current literature suggests that ROS/RNS nitrate proNGF and reduce the expression of the proNGF receptor tropomyosin-related kinase A (TrkA), disrupting its downstream survival signalling. ROS/RNS-induced reductions in TrkA expression reduce cell viability, as proNGF loses its neurotrophic function in the absence of TrkA and instead generates apoptotic signalling via the pan-neurotrophin receptor p75NTR. ROS/RNS also interfere with kinesin and dynein motor functions, causing transport deficits. ROS/RNS-induced deficits in microtubule motor function and TrkA expression and signalling may contribute to the vulnerability of the basal forebrain in AD. Antioxidant treatments may be beneficial in restoring proNGF signalling and axonal transport and reducing basal forebrain neurodegeneration and related deficits in cognitive function.

Mitochondrial adaptor TRAK2 activates and functionally links opposing kinesin and dynein motors

Mitochondria are transported along microtubules by opposing kinesin and dynein motors. Kinesin-1 and dynein-dynactin are linked to mitochondria by TRAK proteins, but it is unclear how TRAKs coordinate these motors. We used single-molecule imaging of cell lysates to show that TRAK2 robustly activates kinesin-1 for transport toward the microtubule plus-end. TRAK2 is also a novel dynein activating adaptor that utilizes a conserved coiled-coil motif to interact with dynein to promote motility toward the microtubule minus-end. However, dynein-mediated TRAK2 transport is minimal unless the dynein-binding protein LIS1 is present at a sufficient level. Using co-immunoprecipitation and co-localization experiments, we demonstrate that TRAK2 forms a complex containing both kinesin-1 and dynein-dynactin. These motors are functionally linked by TRAK2 as knockdown of either kinesin-1 or dynein-dynactin reduces the initiation of TRAK2 transport toward either microtubule end. We propose that TRAK2 coordinates kinesin-1 and dynein-dynactin as an interdependent motor complex, providing integrated control of opposing motors for the proper transport of mitochondria.

Ciliary Dyneins and Dynein Related Ciliopathies

Although ubiquitously present, the relevance of cilia for vertebrate development and health has long been underrated. However, the aberration or dysfunction of ciliary structures or components results in a large heterogeneous group of disorders in mammals, termed ciliopathies. The majority of human ciliopathy cases are caused by malfunction of the ciliary dynein motor activity, powering retrograde intraflagellar transport (enabled by the cytoplasmic dynein-2 complex) or axonemal movement (axonemal dynein complexes). Despite a partially shared evolutionary developmental path and shared ciliary localization, the cytoplasmic dynein-2 and axonemal dynein functions are markedly different: while cytoplasmic dynein-2 complex dysfunction results in an ultra-rare syndromal skeleto-renal phenotype with a high lethality, axonemal dynein dysfunction is associated with a motile cilia dysfunction disorder, primary ciliary dyskinesia (PCD) or Kartagener syndrome, causing recurrent airway infection, degenerative lung disease, laterality defects, and infertility. In this review, we provide an overview of ciliary dynein complex compositions, their functions, clinical disease hallmarks of ciliary dynein disorders, presumed underlying pathomechanisms, and novel developments in the field.

N4BP2L1 interacts with dynactin and contributes to GLUT4 trafficking and glucose uptake in adipocytes.

ABSTRACTAims/IntroductionIt was reported previously that N4bp2l1 expression increases in 3T3‐L1 cells in a differentiation‐dependent manner and N4bp2l1 knockdown suppresses adipocyte differentiation. However, the physiological function of N4BP2L1 in adipocytes remains unknown. This study aimed to elucidate the physiological mechanism of N4bp2l1 expression and the role of N4BP2L1 in the physiological function of adipocytes.Materials and MethodsAnalysis of gene expression levels of N4bp2l1 in adipose tissue during feeding in mice was conducted. Identification of transcription factors that regulate N4bp2l1 expression was conducted using a reporter assay. Investigation of N4BP2L1‐interacting proteins was carried out using immunoprecipitation. A GLUT4 translocation assay and a glucose uptake assay in 3T3‐L1 adipocytes were performed using N4bp2l1 overexpression and knockdown adenovirus.ResultsThe results indicated that N4bp2l1 is a novel FoxO1 target gene and its expression is controlled by the insulin‐mediated regulation of FoxO1. N4BP2L1 interacts with dynactin, which binds to the microtubule motor dynein, indicating that N4BP2L1 is involved in GLUT4 trafficking and glucose uptake in 3T3‐L1 adipocytes.ConclusionsOur results suggest that N4BP2L1 is involved in adipocyte homeostasis by interacting with dynein–dynactin and affecting GLUT4‐mediated glucose uptake and the insulin signaling pathway.

Transient Tertiary Structures of Disordered Dynein Intermediate Chain Regulate its Interactions with Multiple Partners

The N-terminal domain of dynein intermediate chain (N–IC) is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and regulation of motor activity. It is a prototypical intrinsically disordered protein (IDP), serving as a primarily disordered polybivalent molecular scaffold for numerous binding partners, including three dimeric dynein light chains and coiled coil domains of dynein partners dynactin p150Glued and NudE. At the very N-terminus, a 40 amino acid single alpha helix (SAH) forms the major binding site for both p150Glued and NudE, while a shorter nascent helix (H2) separated from SAH by a disordered linker, is necessary for tight binding to dynactin p150Glued but not to NudE. Here we demonstrate that transient tertiary interactions in this highly dynamic protein underlie the differences in its interactions with p150Glued and NudE. NMR paramagnetic relaxation enhancement experiments and restrained molecular dynamics simulations identify interactions between the two non-contiguous SAH and H2 helical regions, the extent of which correlates with the length and stability of H2, showing clearly that tertiary and secondary structure formation are coupled in IDPs. These interactions are significantly attenuated when N–IC is bound to NudE, suggesting that NudE binding shifts the conformational ensemble to one that is more extended and with less structure in H2. While the intrinsic disorder and flexibility in N–IC modulate its ability to serve as a binding platform for numerous partners, deviations of this protein from random-coil behavior provide a process for regulating these binding interactions and potentially the dynein motor.

Dynein light chain-dependent dimerization of Egalitarian is essential for maintaining oocyte fate in Drosophila

Egalitarian (Egl) is an RNA adaptor for the Dynein motor and is thought to link numerous, perhaps hundreds, of mRNAs with Dynein. Dynein, in turn, is responsible for the transport and localization of these mRNAs. Studies have shown that efficient mRNA binding by Egl requires the protein to dimerize. We recently demonstrated that Dynein light chain (Dlc) is responsible for facilitating the dimerization of Egl. Mutations in Egl that fail to interact with Dlc do not dimerize, and as such, are defective for mRNA binding. Consequently, this mutant does not efficiently associate with BicaudalD (BicD), the factor responsible for linking the Egl/mRNA complex with Dynein. In this report, we tested whether artificially dimerizing this Dlc-binding mutant using a leucine zipper would restore mRNA binding and rescue mutant phenotypes in vivo. Interestingly, we found that although artificial dimerization of Egl restored BicD binding, it only partially restored mRNA binding. As a result, Egl-dependent phenotypes, such as oocyte specification and mRNA localization, were only partially rescued. We hypothesize that Dlc-mediated dimerization of Egl results in a three-dimensional conformation of the Egl dimer that is best suited for mRNA binding. Although the leucine zipper restores Egl dimerization, it likely does not enable Egl to assemble into the conformation required for maximal mRNA binding activity.

Spindle positioning and its impact on vertebrate tissue architecture and cell fate.

In multicellular systems, oriented cell divisions are essential for morphogenesis and homeostasis as they determine the position of daughter cells within the tissue and also, in many cases, their fate. Early studies in invertebrates led to the identification of conserved core mechanisms of mitotic spindle positioning centred on the Gαi-LGN-NuMA-dynein complex. In recent years, much has been learnt about the way this complex functions in vertebrate cells. In particular, studies addressed how the Gαi-LGN-NuMA-dynein complex dynamically crosstalks with astral microtubules and the actin cytoskeleton, and how it is regulated to orient the spindle according to cellular and tissue-wide cues. We have also begun to understand how dynein motors and actin regulators interact with mechanosensitive adhesion molecules sensing extracellular mechanical stimuli, such as cadherins and integrins, and with signalling pathways so as to respond to extracellular cues instructing the orientation of the division axis in vivo. In this Review, with the focus on epithelial tissues, we discuss the molecular mechanisms of mitotic spindle orientation in vertebrate cells, and how this machinery is regulated by epithelial cues and extracellular signals to maintain tissue cohesiveness during mitosis. We also outline recent knowledge of how spindle orientation impacts tissue architecture in epithelia and its emerging links to the regulation of cell fate decisions. Finally, we describe how defective spindle orientation can be corrected or its effects eliminated in tissues under physiological conditions, and the pathological implications associated with spindle misorientation.

Polarized endosome dynamics engage cytoplasmic Par-3 that recruits dynein during asymmetric cell division.

In the developing embryos, the cortical polarity regulator Par-3 is critical for establishing Notch signaling asymmetry between daughter cells during asymmetric cell division (ACD). How cortically localized Par-3 establishes asymmetric Notch activity in the nucleus is not understood. Here, using in vivo time-lapse imaging of mitotic radial glia progenitors in the developing zebrafish forebrain, we uncover that during horizontal ACD along the anteroposterior embryonic axis, endosomes containing the Notch ligand DeltaD (Dld) move toward the cleavage plane and preferentially segregate into the posterior (subsequently basal) Notchhi daughter. This asymmetric segregation requires the activity of Par-3 and dynein motor complex. Using label retention expansion microscopy, we further detect Par-3 in the cytosol colocalizing the dynein light intermediate chain 1 (Dlic1) onto Dld endosomes. Par-3, Dlic1, and Dld are associated in protein complexes in vivo. Our data reveal an unanticipated mechanism by which cytoplasmic Par-3 directly polarizes Notch signaling components during ACD.

On and off controls within dynein–dynactin on native cargoes

The dynein-dynactin nanomachine transports cargoes along microtubules in cells. Why dynactin interacts separately with the dynein motor and also with microtubules is hotly debated. Here we disrupted these interactions in a targeted manner on phagosomes extracted from cells, followed by optical trapping to interrogate native dynein-dynactin teams on single phagosomes. Perturbing the dynactin-dynein interaction reduced dynein's on rate to microtubules. In contrast, perturbing the dynactin-microtubule interaction increased dynein's off rate markedly when dynein was generating force against the optical trap. The dynactin-microtubule link is therefore required for persistence against load, a finding of importance because disease-relevant mutations in dynein-dynactin are known to interfere with "high-load" functions of dynein in cells. Our findings call attention to a less studied property of dynein-dynactin, namely, its detachment against load, in understanding dynein dysfunction.

Targeting allostery in the Dynein motor domain with small molecule inhibitors

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, four distinct nucleotide-binding sites, and conformational dynamics pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S.cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with functional assays of dynein motility and mutagenesis studies, suggest that the compounds inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein's main catalytic site in the AAA1 domain. A double Walker B mutation of the AAA3 and AAA4 sites substantially reduces enzyme activity, suggesting that targeting these regulatory domains is sufficient to inhibit dynein. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein's AAA domains.

Dynein light intermediate chains as pivotal determinants of dynein multifunctionality.

In animal cells, a single cytoplasmic dynein motor mediates microtubule minus-end-directed transport, counterbalancing dozens of plus-end-directed kinesins. The remarkable ability of dynein to interact with a diverse cargo spectrum stems from its tightly regulated recruitment of cargo-specific adaptor proteins, which engage the dynactin complex to make a tripartite processive motor. Adaptor binding is governed by the homologous dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which exist in mutually exclusive dynein complexes that can perform both unique and overlapping functions. The intrinsically disordered and variable C-terminal domains of the LICs are indispensable for engaging a variety of structurally divergent adaptors. Here, we hypothesize that numerous spatiotemporally regulated permutations of posttranslational modifications of the LICs, as well as of the adaptors and cargoes, exponentially expand the spectrum of dynein-adaptor-cargo complexes. We thematically illustrate the possibilities that could generate a vast set of biochemical variations required to support the wide range of dynein functions.

Dynein Light-Chain Dynlrb2 Is Essential for Murine Leukemia Virus Traffic and Nuclear Entry.

Murine leukemia virus (MLV) requires the infected cell to divide to access the nucleus to integrate into the host genome. It has been determined that MLV uses the microtubule and actin network to reach the nucleus at the early stages of infection. Several studies have shown that viruses use the dynein motor protein associated with microtubules for their displacement. We have previously reported that dynein light-chain roadblock type 2 (Dynlrb2) knockdown significantly decreases MLV infection compared to nonsilenced cells, suggesting a functional association between this dynein light chain and MLV preintegration complex (PIC). In this study, we aimed to determine if the dynein complex Dynlrb2 subunit plays an essential role in the retrograde transport of MLV. For this, an MLV mutant containing the green fluorescent protein (GFP) fused to the viral protein p12 was used to assay the PIC localization and speed in cells in which the expression of Dynlrb2 was modulated. We found a significant decrease in the arrival of MLV PIC to the nucleus and a reduced net speed of MLV PICs when Dynlrb2 was knocked down. In contrast, an increase in nuclear localization was observed when Dynlrb2 was overexpressed. Our results suggest that Dynlrb2 plays an essential role in MLV retrograde transport. IMPORTANCE Different viruses use different components of cytoplasmic dynein complex to traffic to their replication site. We have found that murine leukemia virus (MLV) depends on dynein light-chain Dynlrb2 for infection, retrograde traffic, and nuclear entry. Our study provides new information regarding the molecular requirements for retrograde transport of MLV preintegration complex and demonstrates the essential role of Dynlrb2 in MLV infection.

Structure and Mechanics of Dynein Motors.

Dyneins make up a family of AAA+ motors that move toward the minus end of microtubules. Cytoplasmic dynein is responsible for transporting intracellular cargos in interphase cells and mediating spindle assembly and chromosome positioning during cell division. Other dynein isoforms transport cargos in cilia and power ciliary beating. Dyneins were the least studied of the cytoskeletal motors due to challenges in the reconstitution of active dynein complexes in vitro and the scarcity of high-resolution methods for in-depth structural and biophysical characterization of these motors. These challenges have been recently addressed, and there have been major advances in our understanding of the activation, mechanism, and regulation of dyneins. This review synthesizes the results of structural and biophysical studies for each class of dynein motors. We highlight several outstanding questions about the regulation of bidirectional transport along microtubules and the mechanisms that sustain self-coordinated oscillations within motile cilia.

PI(3,4)P2-mediated membrane tubulation promotes integrin trafficking and invasive cell migration

Invadopodia are integrin-mediated adhesions with abundant PI(3,4)P2 However, the functional role of PI(3,4)P2 in adhesion signaling remains unclear. Here, we find that the PI(3,4)P2 biogenesis regulates the integrin endocytosis at invadopodia. PI(3,4)P2 is locally produced by PIK3CA and SHIP2 and is concentrated at the trailing edge of the invadopodium arc. The PI(3,4)P2-rich compartment locally forms small puncta (membrane buds) in a SNX9-dependent manner, recruits dynein activator Hook1 through AKTIP, and rearranges into micrometer-long tubular invaginations (membrane tubes). The uncurving membrane tube extends rapidly, follows the retrograde movement of dynein along microtubule tracks, and disconnects from the plasma membrane. Activated integrin-beta3 is locally internalized through the pathway of PI(3,4)P2-mediated membrane invagination and is then actively recycled. Blockages of PI3K, SHIP2, and SNX9 suppress integrin-beta3 endocytosis, delay adhesion turnover, and impede transwell invasion of MEF-Src and MDA-MB-231 cells. Thus, the production of PI(3,4)P2 promotes invasive cell migration by stimulating the trafficking of integrin receptor at the invadopodium.

Flagellar energetics from high-resolution imaging of beating patterns in tethered mouse sperm

We demonstrate a technique for investigating the energetics of flagella or cilia. We record the planar beating of tethered mouse sperm at high resolution. Beating waveforms are reconstructed using proper orthogonal decomposition of the centerline tangent-angle profiles. Energy conservation is employed to obtain the mechanical power exerted by the dynein motors from the observed kinematics. A large proportion of the mechanical power exerted by the dynein motors is dissipated internally by the motors themselves. There could also be significant dissipation within the passive structures of the flagellum. The total internal dissipation is considerably greater than the hydrodynamic dissipation in the aqueous medium outside. The net power input from the dynein motors in sperm from Crisp2-knockout mice is significantly smaller than in wildtype samples, indicating that ion-channel regulation by cysteine-rich secretory proteins controls energy flows powering the axoneme.

Optimal RNA binding by Egalitarian, a Dynein cargo adaptor, is critical for maintaining oocyte fate in Drosophila

ABSTRACT The Dynein motor is responsible for the localization of numerous mRNAs within Drosophila oocytes and embryos. The RNA binding protein, Egalitarian (Egl), is thought to link these various RNA cargoes with Dynein. Although numerous studies have shown that Egl is able to specifically associate with these RNAs, the nature of these interactions has remained elusive. Egl contains a central RNA binding domain that shares limited homology with an exonuclease, yet Egl binds to RNA without degrading it. Mutations have been identified within Egl that disrupt its association with its protein interaction partners, BicaudalD (BicD) and Dynein light chain (Dlc), but no mutants have been described that are specifically defective for RNA binding. In this report, we identified a series of positively charged residues within Egl that are required for RNA binding. Using corresponding RNA binding mutants, we demonstrate that specific RNA cargoes are more reliant on maximal Egl RNA biding activity for their correct localization in comparison to others. We also demonstrate that specification and maintenance of oocyte fate requires maximal Egl RNA binding activity. Even a subtle reduction in Egl’s RNA binding activity completely disrupts this process. Our results show that efficient RNA localization at the earliest stages of oogenesis is required for specification of the oocyte and restriction of meiosis to a single cell.

C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility.

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we show using patient stem cell-derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.