Nesprin-2 coordinates opposing microtubule motors during nuclear migration in neurons.

Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.

Quantitatively Dissecting Triple Roles of Dynactin in Dynein-Driven Transport of Influenza Virus by Quantum Dot-Based Single-Virus Tracking.

After entering host cells by endocytosis, influenza A virus (IAV) is transported along microfilaments and then transported by dynein along microtubules (MTs) to the perinuclear region for genome release. Understanding the mechanisms of dynein-driven transport is significant for a comprehensive understanding of IAV infection. In this work, the roles of dynactin in dynein-driven transport of IAV were quantitatively dissected in situ using quantum dot-based single-virus tracking. It was revealed that dynactin was essential for dynein to transport IAV toward the nucleus. After virus entry, virus-carrying vesicles bound to dynein and dynactin before being delivered to MTs. The attachment of dynein to the vesicles was dependent on dynactin and its subunits, p150Glued and Arp1. Once viruses reached MTs, dynactin-assisted dynein initiates retrograde transport of IAV. Importantly, the retrograde transport of viruses could be initiated at both plus ends (32%) and other regions on MTs (68%). Subsequently, dynactin accompanied and assisted dynein to persistently transport the virus along MTs in the retrograde direction. This study revealed the dynactin-dependent dynein-driven transport process of IAV, enhancing our understanding of IAV infection and providing important insights into the cell's endocytic transport mechanism.

Cholesterol-rich lysosomes induced by respiratory syncytial virus promote viral replication by blocking autophagy flux.

Respiratory syncytial virus (RSV) hijacks cholesterol or autophagy pathways to facilitate optimal replication. However, our understanding of the associated molecular mechanisms remains limited. Here, we show that RSV infection blocks cholesterol transport from lysosomes to the endoplasmic reticulum by downregulating the activity of lysosomal acid lipase, activates the SREBP2-LDLR axis, and promotes uptake and accumulation of exogenous cholesterol in lysosomes. High cholesterol levels impair the VAP-A-binding activity of ORP1L and promote the recruitment of dynein-dynactin, PLEKHM1, or HOPS VPS39 to Rab7-RILP, thereby facilitating minus-end transport of autophagosomes and autolysosome formation. Acidification inhibition and dysfunction of cholesterol-rich lysosomes impair autophagy flux by inhibiting autolysosome degradation, which promotes the accumulation of RSV fusion protein. RSV-F storage is nearly abolished after cholesterol depletion or knockdown of LDLR. Most importantly, the knockout of LDLR effectively inhibits RSV infection in vivo. These findings elucidate the molecular mechanism of how RSV co-regulates lysosomal cholesterol reprogramming and autophagy and reveal LDLR as a novel target for anti-RSV drug development.

Pan-cancer analysis of DCTN2 and its tumour-promoting role in HCC by modulating the AKT pathway.

Dynactin subunit 2 (DCTN2) has been reported to play a role in progression of several tumours; however, the involvement of DCTN2 in potential mechanism or the tumour immune microenvironment among various cancers still remains largely unknown. Therefore, the objective of this study was to comprehensively investigate the expression status and potential function of DCTN2 in various malignancies through different database, such as The Cancer Genome Atlas, the Genotype-Tissue Expression and Gene Expression Omnimus databases. We discovered that DCTN2 expression was high in many type of tumours tissues compared to adjacent non-tumour ones. High DCTN2 signified poor prognosis for patients with tumours. Additionally, Gene Set Enrichment Analysis (GSEA) analysis revealed that DCTN2 was positively correlated with oncogenic pathways, including cell cycle, tumour metastasis-related pathway, while it was negatively with anti-tumour immune signalling pathway, such as INF-γ response. More importantly, we elucidated the functional impact of DCTN2 on hepatocellular carcinoma (HCC) progression and its underlying mechanisms. DCTN2 expression was much higher in HCC tissues than in adjacent non-tumour tissues. Silencing DCTN2 dramatically suppressed the proliferative and metastasis capacities of tumour cell in vitro. Mechanistically, DCTN2 exerted tumour-promoting effects by modulating the AKT signalling pathway. DCTN2 knockdown in HCC cells inhibited AKT phosphorylation and its downstream targets as well. Rescue experiments revealed that the anti-tumour effects of DCTN2 knockdown were partially reversed upon AKT pathway activation. Overall, DCTN2 may be a potent biomarker signifying tumour prognosis and a promising therapeutic target for tumour treatment, particularly in HCC.

Herpes simplex virus type-1 cVAC formation in neuronal cells is mediated by dynein motor function and glycoprotein retrieval from the plasma membrane.

Herpesvirus assembly requires the cytoplasmic association of large macromolecular and membrane structures that derive from both the nucleus and cytoplasmic membrane systems. Results from the study of human cytomegalovirus (HCMV) in cells where it organizes a perinuclear cytoplasmic virus assembly compartment (cVAC) show a clear requirement for the minus-end-directed microtubule motor, dynein, for virus assembly. In contrast, the assembly of herpes simplex virus -1 (HSV-1) in epithelial cells where it forms multiple dispersed, peripheral assembly sites is only mildly inhibited by the microtubule-depolymerizing agent, nocodazole. Here, we make use of a neuronal cell line system in which HSV-1 forms a single cVAC and show that dynein and its co-factor dynactin localize to the cVAC, and dynactin is associated with membranes that contain the virion tegument protein pUL11. We also show that the virus membrane-associated structural proteins pUL51 and the viral envelope glycoprotein gE arrive at the cVAC by different routes. Specifically, gE arrives at the cVAC after retrieval from the plasma membrane, suggesting the need for an intact retrograde transport system. Finally, we demonstrate that inhibition of dynactin function profoundly inhibits cVAC formation and virus production during the cytoplasmic assembly phase of infection.IMPORTANCEMany viruses reorganize cytoplasmic membrane systems and macromolecular transport systems to promote the production of progeny virions. Clarifying the mechanisms by which they accomplish this may reveal novel therapeutic strategies and illustrate mechanisms that are critical for normal cellular organization. Here, we explore the mechanism by which HSV-1 moves macromolecular and membrane cargo to generate a virus assembly compartment in the infected cell. We find that the virus makes use of a well-characterized, microtubule-based transport system that is stabilized against drugs that disrupt microtubules.

The CCTδ subunit of the molecular chaperone CCT is required for correct localisation of p150Glued to spindle poles during mitosis.

Chaperonin Containing Tailless complex polypeptide 1 (CCT) is a molecular chaperone composed of eight distinct subunits that can exist as individual monomers or as components of a double oligomeric ring, which is essential for the folding of actin and tubulin and other substrates. Here we assess the role of CCT subunits in the context of cell cycle progression by individual subunit depletions upon siRNA treatment in mammalian cells. The depletion of individual CCT subunits leads to variation in the distribution of cell cycle phases and changes in mitotic index. Mitotic defects, such as unaligned chromosomes occur when CCTδ is depleted, concurrent with a reduction in spindle pole-localised p150Glued, a component of the dynactin complex and a binding partner of monomeric CCTδ. In CCTδ-depleted cells, changes in the elution profile of p150Glued are observed consistent with altered conformations and or assembly states with the dynactin complex. Addition of monomeric CCTδ, in the form of GFP-CCTδ, restores correct p150Glued localisation to the spindle poles and rescues the mitotic segregation defects that occur when CCTδ is depleted. This study demonstrates a requirement for CCTδ in its monomeric form for correct chromosome segregation via a mechanism that promotes the correct localisation of p150Glued, thus revealing further complexities to the interplay between CCT, tubulin folding and microtubule dynamics.

Autophagy initiation triggers p150Glued-AP-2ß interaction on the lysosomes and facilitates their transport.

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

Molecular mechanism of dynein-dynactin complex assembly by LIS1.

Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.

Swedish Alzheimer's disease variant perturbs activity of retrograde molecular motors and causes widespread derangement of axonal transport pathways.

Experimental studies in flies, mice, and humans suggest a significant role of impaired axonal transport in the pathogenesis of Alzheimer's disease (AD). The mechanisms underlying these impairments in axonal transport, however, remain poorly understood. Here we report that the Swedish familial AD mutation causes a standstill of the amyloid precursor protein (APP) in the axons at the expense of its reduced anterograde transport. The standstill reflects the perturbed directionality of the axonal transport of APP, which spends significantly more time traveling in the retrograde direction. This ineffective movement is accompanied by an enhanced association of dynactin-1 with APP, which suggests that reduced anterograde transport of APP is the result of enhanced activation of the retrograde molecular motor dynein by dynactin-1. The impact of the Swedish mutation on axonal transport is not limited to the APP vesicles since it also reverses the directionality of a subset of early endosomes, which become enlarged and aberrantly accumulate in distal locations. In addition, it also reduces the trafficking of lysosomes due to their less effective retrograde movement. Altogether, our experiments suggest a pivotal involvement of retrograde molecular motors and transport in the mechanisms underlying impaired axonal transport in AD and reveal significantly more widespread derangement of axonal transport pathways in the pathogenesis of AD.

Rab32 family proteins regulate autophagosomal components recycling.

In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane components were recycled via autophagosomal components recycling (ACR), which is mediated by the recycler complex. The recycler complex, composed of SNX4, SNX5, and SNX17, cooperate with the dynein-dynactin complex to mediate ACR. However, how ACR is regulated remains unknown. Here, we found that Rab32 family proteins localize to autolysosomes and are required for ACR, rather than other autophagosomal or lysosomal Rab proteins. The GTPase activity of Rab32 family proteins, governed by their guanine nucleotide exchange factor and GTPase-activating protein, plays a key role in regulating ACR. This regulation occurs through the control of recycler complex formation, as well as the connection between the recycler-cargo and dynactin complex. Together, our study reveals an unidentified Rab32 family-dependent regulatory mechanism for ACR.

Dysregulation of stress granule dynamics by DCTN1 deficiency exacerbates TDP-43 pathology in Drosophila models of ALS/FTD.

The abnormal aggregation of TDP-43 into cytoplasmic inclusions in affected neurons is a major pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although TDP-43 is aberrantly accumulated in the neurons of most patients with sporadic ALS/FTD and other TDP-43 proteinopathies, how TDP-43 forms cytoplasmic aggregates remains unknown. In this study, we show that a deficiency in DCTN1, a subunit of the microtubule-associated motor protein complex dynactin, perturbs the dynamics of stress granules and drives the formation of TDP-43 cytoplasmic aggregation in cultured cells, leading to the exacerbation of TDP-43 pathology and neurodegeneration in vivo. We demonstrated using a Drosophila model of ALS/FTD that genetic knockdown of DCTN1 accelerates the formation of ubiquitin-positive cytoplasmic inclusions of TDP-43. Knockdown of components of other microtubule-associated motor protein complexes, including dynein and kinesin, also increased the formation of TDP-43 inclusions, indicating that intracellular transport along microtubules plays a key role in TDP-43 pathology. Notably, DCTN1 knockdown delayed the disassembly of stress granules in stressed cells, leading to an increase in the formation of pathological cytoplasmic inclusions of TDP-43. Our results indicate that a deficiency in DCTN1, as well as disruption of intracellular transport along microtubules, is a modifier that drives the formation of TDP-43 pathology through the dysregulation of stress granule dynamics.

Dynein and dynactin move long-range but are delivered separately to the axon tip.

Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level, it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here, we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction, we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with the initiation of dynein transport. Intriguingly, in the anterograde direction, dynein/LIS1 moves faster than dynactin/NDEL1, consistent with transport on different cargos. Therefore, neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required.

Wild-Type DCTN1 Suppresses the Aggregation of DCTN1 Mutants Associated with Perry Disease.

Perry disease, a rare autosomal dominant neurodegenerative disorder, is characterized by parkinsonism, depression or apathy, unexpected weight loss, and central hypoventilation. Genetic analyses have revealed a strong association between point mutations in the dynactin I gene (DCTN1) coding p150glued and Perry disease. Although previous reports have suggested a critical role of p150glued aggregation in Perry disease pathology, whether and how p150glued mutations affect protein aggregation is not fully understood. In this study, we comprehensively investigated the intracellular distribution of the p150glued mutants in HEK293T cells. We further assessed the effect of co-overexpression of the wild-type p150glued protein with mutants on the formation of mutant aggregates. Notably, overexpression of p150glued mutants identified in healthy controls, which is also associated with amyotrophic lateral sclerosis, showed a thread-like cytoplasmic distribution, similar to the wild-type p150glued. In contrast, p150glued mutants in Perry disease and motor neuron disease caused aggregation. In addition, the co-overexpression of the wild-type protein with p150glued mutants in Perry disease suppressed aggregate formation. In contrast, the p150glued aggregation of motor neuron disease mutants was less affected by the wild-type p150glued. Further investigation of the mechanism of aggregate formation, contents of the aggregates, and biological mechanisms of Perry disease could help develop novel therapeutics.

Role of BicDR in bristle shaft construction and support of BicD functions.

Cell polarization requires asymmetric localization of numerous mRNAs, proteins and organelles. The movement of cargo towards the minus end of microtubules mostly depends on cytoplasmic dynein motors. In the dynein-dynactin-Bicaudal-D transport machinery, Bicaudal-D (BicD) links the cargo to the motor. Here, we focus on the role of Drosophila BicD-related (BicDR, CG32137) in the development of the long bristles. Together with BicD, it contributes to the organization and stability of the actin cytoskeleton in the not-yet-chitinized bristle shaft. BicD and BicDR also support the stable expression and distribution of Rab6 and Spn-F in the bristle shaft, including the distal tip localization of Spn-F, pointing to the role of microtubule-dependent vesicle trafficking for bristle construction. BicDR supports the function of BicD, and we discuss the hypothesis whereby BicDR might transport cargo more locally, with BicD transporting cargo over long distances, such as to the distal tip. We also identified embryonic proteins that interact with BicDR and appear to be BicDR cargo. For one of them, EF1γ (also known as eEF1γ), we show that the encoding gene EF1γ interacts with BicD and BicDR in the construction of the bristles.

Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells.

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin, and a cargo adaptor. How this process of dynein activation occurs is unclear since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualize and track fluorescently tagged dynein. First, we observed that only ∼30% of dynein molecules that bound to the microtubule (MT) engaged in minus end-directed movement, and that too for a short duration of ∼0.6 s. Next, using high-resolution imaging in live and fixed cells and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-color imaging to visualize cargo movement effected by single motor binding. Finally, we performed long-term imaging to show that short movements are sufficient to drive cargo to the perinuclear region of the cell. Taken together, we discovered a search mechanism that is facilitated by dynein's frequent MT binding-unbinding kinetics: (i) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (ii) when dynein encounters cargo and dynactin upon MT binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end-directed transport in cellulo and that complex cargo behavior emerges from stochastic motor-cargo interactions.

Branched microtubule nucleation and dynein transport organize RanGTP asters in Xenopus laevis egg extract.

Chromosome segregation relies on the correct assembly of a bipolar spindle. Spindle pole self-organization requires dynein-dependent microtubule (MT) transport along other MTs. However, during M-phase RanGTP triggers MT nucleation and branching generating polarized arrays with nonastral organization in which MT minus ends are linked to the sides of other MTs. This raises the question of how branched-MT nucleation and dynein-mediated transport cooperate to organize the spindle poles. Here, we used RanGTP-dependent MT aster formation in Xenopus laevis (X. laevis) egg extract to study the interplay between these two seemingly conflicting organizing principles. Using temporally controlled perturbations of MT nucleation and dynein activity, we found that branched MTs are not static but instead dynamically redistribute over time as poles self-organize. Our experimental data together with computer simulations suggest a model where dynein together with dynactin and NuMA directly pulls and move branched MT minus ends toward other MT minus ends.

Vesicles driven by dynein and kinesin exhibit directional reversals without regulators.

Intracellular vesicular transport along cytoskeletal filaments ensures targeted cargo delivery. Such transport is rarely unidirectional but rather bidirectional, with frequent directional reversals owing to the simultaneous presence of opposite-polarity motors. So far, it has been unclear whether such complex motility pattern results from the sole mechanical interplay between opposite-polarity motors or requires regulators. Here, we demonstrate that a minimal system, comprising purified Dynein-Dynactin-BICD2 (DDB) and kinesin-3 (KIF16B) attached to large unilamellar vesicles, faithfully reproduces in vivo cargo motility, including runs, pauses, and reversals. Remarkably, opposing motors do not affect vesicle velocity during runs. Our computational model reveals that the engagement of a small number of motors is pivotal for transitioning between runs and pauses. Taken together, our results suggest that motors bound to vesicular cargo transiently engage in a tug-of-war during pauses. Subsequently, stochastic motor attachment and detachment events can lead to directional reversals without the need for regulators.

RZZ-Spindly and CENP-E form an integrated platform to recruit dynein to the kinetochore corona.

Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin-7) and dynein-dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The corona's building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we show that when corona assembly is prevented through MPS1 inhibition, CENP-E is absolutely required to retain RZZS at kinetochores. An RZZS phosphomimetic mutant bypasses this requirement, demonstrating the existence of a second receptor for polymeric RZZS. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.

Nde1 promotes Lis1-mediated activation of dynein.

Cytoplasmic dynein drives the motility and force generation functions towards the microtubule minus end. The assembly of dynein with dynactin and a cargo adaptor in an active transport complex is facilitated by Lis1 and Nde1/Ndel1. Recent studies proposed that Lis1 relieves dynein from its autoinhibited conformation, but the physiological function of Nde1/Ndel1 remains elusive. Here, we investigate how human Nde1 and Lis1 regulate the assembly and subsequent motility of mammalian dynein using in vitro reconstitution and single molecule imaging. We find that Nde1 recruits Lis1 to autoinhibited dynein and promotes Lis1-mediated assembly of dynein-dynactin adaptor complexes. Nde1 can compete with the α2 subunit of platelet activator factor acetylhydrolase 1B (PAF-AH1B) for the binding of Lis1, which suggests that Nde1 may disrupt PAF-AH1B recruitment of Lis1 as a noncatalytic subunit, thus promoting Lis1 binding to dynein. Before the initiation of motility, the association of dynactin with dynein triggers the dissociation of Nde1 from dynein by competing against Nde1 binding to the dynein intermediate chain. Our results provide a mechanistic explanation for how Nde1 and Lis1 synergistically activate the dynein transport machinery.