Dynactin is a large multisubunit protein complex that enhances the processivity of cytoplasmic dynein and acts as an adapter between dynein and the cargo. It is composed of eleven different polypeptides of which eight are unique to this complex, namely dynactin1 (p150(Glued)), dynactin2 (p50 or dynamitin), dynactin3 (p24), dynactin4 (p62), dynactin5 (p25), dynactin6 (p27), and the actin-related proteins Arp1 and Arp10 (Arp11).

Dynein is the primary minus-end-directed microtubule motor protein. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the "activated dynein complex." The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and the adaptor. Ndel1 and its paralog Nde1 are dynein- and Lis1-binding proteins that help control dynein localization within the cell. Cell-based assays suggest that Ndel1-Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear. Using purified proteins and quantitative binding assays, here we found that the C-terminal region of Ndel1 contributes to dynein binding and negatively regulates binding to Lis1. Using single-molecule imaging and protein biochemistry, we observed that Ndel1 inhibits dynein activation in two distinct ways. First, Ndel1 disfavors the formation of the activated dynein complex. We found that phosphomimetic mutations in the C-terminal domain of Ndel1 increase its ability to inhibit dynein-dynactin-adaptor complex formation. Second, we observed that Ndel1 interacts with dynein and Lis1 simultaneously and sequesters Lis1 away from its dynein-binding site. In doing this, Ndel1 prevents Lis1-mediated dynein activation. Together, our work suggests that in vitro, Ndel1 is a negative regulator of dynein activation, which contrasts with cellular studies where Ndel1 promotes dynein activity. To reconcile our findings with previous work, we posit that Ndel1 functions to scaffold dynein and Lis1 together while keeping dynein in an inhibited state. We speculate that Ndel1 release can be triggered in cellular settings to allow for timed dynein activation.

Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.

Mutations in DCTN1, a component of the dynactin complex, are linked to neurodegenerative diseases characterized by a broad collection of neuropathologies. Because of the pleiotropic nature of dynactin complex function within the neuron, defining the causes of neuropathology in DCTN1 mutants has been difficult. We combined a genetic screen with cellular assays of dynactin complex function to identify genes that are critical for dynactin complex function in the nervous system. This approach identified the Drosophila homologue of Arfaptin, a multifunctional protein that has been implicated in membrane trafficking. We find that Arfaptin and the Drosophila DCTN1 homologue, Glued, function in the same pathway during synapse growth but not during axonal transport or synapse stabilization. Arfaptin physically associates with Glued and other dynactin complex components in the nervous system of both flies and mice and colocalizes with Glued at the Golgi in motor neurons. Mechanistically, membrane binding by Arfaptin mediates membrane association of the dynactin complex in motor neurons and is required for normal synapse growth. Arfaptin represents a novel dynactin complex-binding protein that specifies dynactin complex function during synapse growth.

During metazoan development, cell diversity arises primarily from asymmetric cell divisions which are executed in two phases: segregation of cytoplasmic factors and positioning of the mitotic spindle - and hence the cleavage plane -relative to the axis of segregation. When polarized cells divide, spindle alignment probably occurs through the capture and subsequent shortening of astral microtubules by a site in the cortex.

Kinesin and dynein motors transport intracellular cargos bidirectionally by pulling them in opposite directions along microtubules, through a process frequently described as a 'tug of war'. While kinesin produces 6 pN of force, mammalian dynein was found to be a surprisingly weak motor (0.5-1.5 pN) in vitro, suggesting that many dyneins are required to counteract the pull of a single kinesin. Mammalian dynein's association with dynactin and Bicaudal-D2 (BICD2) activates its processive motility, but it was unknown how this affects dynein's force output. Here, we show that formation of the dynein-dynactin-BICD2 (DDB) complex increases human dynein's force production to 4.3 pN. An in vitro tug-of-war assay revealed that a single DDB successfully resists a single kinesin. Contrary to previous reports, the clustering of many dyneins is not required to win the tug of war. Our work reveals the key role of dynactin and a cargo adaptor protein in shifting the balance of forces between dynein and kinesin motors during intracellular transport.

Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132:617-634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti-lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end-directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor-cargo binding.

Long-term changes in synaptic plasticity require gene transcription, indicating that signals generated at the synapse must be transported to the nucleus. Synaptic activation of hippocampal neurons is known to trigger retrograde transport of transcription factor NF-kappaB. Transcription factors of the NF-kappaB family are widely expressed in the nervous system and regulate expression of several genes involved in neuroplasticity, cell survival, learning and memory.

Unlike transport vesicles or organelles, human adenovirus (HAdV) directly binds to the microtubule minus end-directed motor dynein for transport to the nucleus. The dynein cofactor dynactin enhances nuclear transport of HAdV and boosts infection. To determine if dynactin has a specific role in cytoplasmic trafficking of incoming HAdV on microtubules, we used live cell spinning disc confocal microscopy at 25 Hz acquisition frequency and automated tracking of single virus particles at 20-50 nm spatial resolution. Computational dissection by machine-learning algorithms extracted specific motion patterns of viral trajectories. We found that unperturbed cells supported two kinds of microtubule-dependent motions, directed motions (DM) and fast drifts (FD). DM had speeds of 0.2 to 2 μm/s and run lengths of 0.4 up to 7 μm, while FD were slower and less extensive at 0.02 to 0.4 μm/s and 0.05 to 2.5 μm. Dynactin interference by overexpression of p50/dynamitin or a coiled-coil domain of p150/Glued reduced the speeds and amounts of both center- and periphery-directed DM but not FD, and inhibited infection. These results indicate that dynactin enhances adenovirus infection by increasing the speed and efficiency of dynein-mediated virus motion to the nucleus, and, surprisingly, also supports a hereto unknown motor activity for virus transport to the cell periphery.

The transmembrane prostate androgen-induced protein (TMEPAI) has been reported to be elevated in various tumor cells, is localized to the lysosome and promotes lysosome stability. The molecular mechanism of TMEPAI trafficking however to the lysosome is unknown. Here we report that clathrin and CI-M6PR mediate TMEPAI transport from the Golgi directly into the endo-lysosomal pathway. TMEPAI is ubiquitinated at its C-terminal region and ubiquitination modification of TMEPAI is a signal for its lysosomal trafficking. Moreover, TMEPAI binds the ubiquitin binding proteins Hrs and STAM which is required for its lysosomal transport. In addition, TMEPAI interacts with the dynactin pointed-end complex subunits dynactin 5 and dynactin 6. The aa 132-155 domain is essential for specific TMEPAI binding and deletion of this binding site leads to mis-trafficking of TMEPAI to the plasma membrane. These results reveal the pathway and mechanism regulating transport of TMEPAI to the lysosome, which helps to further understand the role of TMEPAI in tumorigenesis.

Dynactin is a multisubunit protein complex necessary for dynein function. Here, we investigated the function of dynactin in budding yeast. Loss of dynactin impaired movement and positioning of the mitotic spindle, similar to loss of dynein. Dynactin subunits required for function included p150(Glued), dynamitin, actin-related protein (Arp) 1 and p24. Arp10 and capping protein were dispensable, even in combination. All dynactin subunits tested localized to dynamic plus ends of cytoplasmic microtubules, to stationary foci on the cell cortex and to spindle pole bodies. The number of molecules of dynactin in those locations was small, less than five. In the absence of dynactin, dynein accumulated at plus ends and did not appear at the cell cortex, consistent with a role for dynactin in offloading dynein from the plus end to the cortex. Dynein at the plus end was necessary for dynactin plus-end targeting. p150(Glued) was the only dynactin subunit sufficient for plus-end targeting. Interactions among the subunits support a molecular model that resembles the current model for brain dynactin in many respects; however, three subunits at the pointed end of brain dynactin appear to be absent from yeast.

We present evidence that synapse retraction occurs during normal synaptic growth at the Drosophila neuromuscular junction (NMJ). An RNAi-based screen to identify the molecular mechanisms that regulate synapse retraction identified Arp-1/centractin, a subunit of the dynactin complex. Arp-1 dsRNA enhances synapse retraction, and this effect is phenocopied by a mutation in P150/Glued, also a dynactin component. The Glued protein is enriched within the presynaptic nerve terminal, and presynaptic expression of a dominant-negative Glued transgene enhances retraction. Retraction is associated with a local disruption of the synaptic microtubule cytoskeleton. Electrophysiological, ultrastructural, and immunohistochemical data support a model in which presynaptic retraction precedes disassembly of the postsynaptic apparatus. Our data suggests that dynactin functions locally within the presynaptic arbor to promote synapse stability.

Dynactin is a multi-subunit complex that serves as a critical cofactor of the microtubule motor cytoplasmic dynein. We previously identified dynactin in the nerve growth cone. However, the function of dynactin in the growth cone is still unclear. Here we show that dynactin in the growth cone is required for constant forward movement of the growth cone. Chromophore-assisted laser inactivation (CALI) of dynamitin, a dynactin subunit, within the growth cone markedly decreases the rate of growth cone advance. CALI of dynamitin in vitro dissociates another dynactin subunit, p150(Glued), from dynamitin. These results indicate that dynactin, especially the interaction between dynamitin and p150(Glued), plays an essential role in growth cone advance.

Microtubules (Mts) are dynamic cytoskeleton structures that play a key role in vesicular transport. The Mts-mediated transport depends on motor proteins named kinesins and the dynein/dynactin motor complex. The Rab7 adapter protein FYCO1 controls the anterograde transport of the endocytic compartments through the interaction with the kinesin KIF5. Rab7 and its partner RILP induce the recruitment of dynein/dynactin to late endosomes regulating its retrograde transport to the perinuclear area to fuse with lysosomes. The late endosomal-lysosomal fusion is regulated by the HOPS complex through its interaction with RILP and the GTPase Arl8. Coxiella burnetii (Cb), the causative agent of Q fever, is an obligate intracellular pathogen, which generates a large compartment with autophagolysosomal characteristics named Cb-containing vacuole (CCV). The CCV forms through homotypic fusion between small non-replicative CCVs (nrCCV) and through heterotypic fusion with other compartments, such as endosomes and lysosomes. In this work, we characterise the role of Mts, motor proteins, RILP/Rab7 and Arl8 on the CCV biogenesis. The formation of the CCV was affected when either the dynamics and/or the acetylation state of Mts were modified. Similarly, the overexpression of the dynactin subunit non-functional mutants p150Glued and RILP led to the formation of small nrCCVs. This phenomenon is not observed in cells overexpressing WT proteins, the motor KIF5 or its interacting protein FYCO1. The formation of the CCV was normal in infected cells that overexpressed Arl8 alone or together with hVps41 (a HOPS subunit) or in cells co-overexpressing hVps41 and RILP. The dominant negative mutant of Arl8 and the non-functional hVps41 inhibited the formation of the CCV. When the formation of CCV was affected, the bacterial multiplication diminished. Our results suggest that nrCCVs recruit the molecular machinery that regulate the Mts-dependent retrograde transport, Rab7/RILP and the dynein/dynactin system, as well as the tethering processes such as HOPS complex and Arl8 to finally originate the CCV where C. burnetii multiplies.

Mitotic spindle orientation in polarized cells determines whether they divide symmetrically or asymmetrically. Moreover, regulated spindle orientation may be important for embryonic development, stem cell biology, and tumor growth. Drosophila neuroblasts align their spindle along an apical/basal cortical polarity axis to self-renew an apical neuroblast and generate a basal differentiating cell. It is unknown whether spindle alignment requires both apical and basal cues, nor have molecular motors been identified that regulate spindle movement. Using live imaging of neuroblasts within intact larval brains, we detect independent movement of both apical and basal spindle poles, suggesting that forces act on both poles. We show that reducing astral microtubules decreases the frequency of spindle movement, but not its maximum velocity, suggesting that one or few microtubules can move the spindle. Mutants in the Lis1/dynactin complex strongly decrease maximum and average spindle velocity, consistent with this motor complex mediating spindle/cortex forces. Loss of either astral microtubules or Lis1/dynactin leads to spindle/cortical polarity alignment defects at metaphase, but these are rescued by telophase. We propose that an early Lis1/dynactin-dependent pathway and a late Lis1/dynactin-independent pathway regulate neuroblast spindle orientation.

Cells must divide strictly along a plane to form an epithelial layer parallel to the basal lamina. The axis of cell division is primarily governed by the orientation of the mitotic spindle and spindle misorientation pathways have been implicated in cancer initiation. While β1-Integrin and the Dynein/Dynactin complex are known to be involved, the pathways linking these complexes in positioning mitotic spindles relative to the basal cortex and extracellular matrix remain to be elucidated. Here, we show that Integrin-Linked Kinase (ILK) and α-Parvin regulate mitotic spindle orientation by linking Dynactin-1 and Dynactin-2 subunits of the Dynein/Dynactin complex to Integrin receptors at the basal cortex of mitotic cells. ILK and α-Parvin are required for spindle orientation. ILK interacts with Dynactin-1 and Dynactin-2 and ILK siRNA attenuates Dynactin-2 localization to the basal cortex. Furthermore we show that Dynactin-2 can no longer colocalize or interact with Integrins when ILK is absent, suggesting mechanistically that ILK is acting as a linking protein. Finally we demonstrate that spindle orientation and cell proliferation are disrupted in intestinal epithelial cells in vivo using tissue-specific ILK knockout mice. These data demonstrate that ILK is a linker between Integrin receptors and the Dynactin complex to regulate mitotic spindle orientation.

The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells, perturbation of the dynein-dynactin interaction profoundly blocks mitotic spindle assembly, and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein-dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome-lysosome system, but the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components, gamma tubulin and dynactin, are lost from centrosomes, but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin-cargo interactions.

The multi-component cytoplasmic dynein transports cellular cargoes with the help of another multi-component complex dynactin, but we do not know enough about factors that may affect the assembly and functions of these proteins. From a genetic screen for mutations affecting early-endosome distribution in Aspergillus nidulans, we identified the prp40AL438* mutation in Prp40A, a homologue of Prp40, an essential RNA-splicing factor in the budding yeast. Prp40A is not essential for splicing, although it associates with the nuclear splicing machinery. The prp40AL438* mutant is much healthier than the ∆prp40A mutant, but both mutants exhibit similar defects in dynein-mediated early-endosome transport and nuclear distribution. In the prp40AL438* mutant, the frequency but not the speed of dynein-mediated early-endosome transport is decreased, which correlates with a decrease in the microtubule plus-end accumulations of dynein and dynactin. Within the dynactin complex, the actin-related protein Arp1 forms a mini-filament. In a pull-down assay, the amount of Arp1 pulled down with its pointed-end protein Arp11 is lowered in the prp40AL438* mutant. In addition, we found from published interactome data that a mammalian Prp40 homologue PRPF40A interacts with Arp1. Thus, Prp40 homologues may regulate the assembly or function of dynein-dynactin and their mechanisms deserve to be further studied.

The multisubunit protein complex, dynactin, is an essential component of the cytoplasmic dynein motor. High-resolution structural work on dynactin and the dynein/dynactin supercomplex has been limited to small subunits and recombinant fragments that do not report fully on either ≈1MDa assembly. In the present study, we used negative-stain electron microscopy and image analysis based on random conical tilt reconstruction to obtain a three-dimensional (3D) structure of native vertebrate dynactin. The 35-nm-long dynactin molecule has a V-shaped shoulder at one end and a flattened tip at the other end, both offset relative to the long axis of the actin-related protein (Arp) backbone. The shoulder projects dramatically away from the Arp filament core in a way that cannot be appreciated in two-dimensional images, which has implications for the mechanism of dynein binding. The 3D structure allows the helical parameters of the entire Arp filament core, which includes the actin capping protein, CP, to be determined for the first time. This structure exhibits near identity to F-actin and can be well fitted into the dynactin envelope. Molecular fitting of modeled CP-Arp polymers into the envelope shows that the filament contains between 7 and 9 Arp protomers and is capped at both ends. In the 7 Arp model, which agrees best with measured Arp stoichiometry and other structural information, actin capping protein (CP) is not present at the distal tip of the structure, unlike what is seen in the other models. The 3D structure suggests a mechanism for dynactin assembly and length specification.

BACKGROUND Dynactin (DCTN) is a multi-subunit protein encoded by DCTN genes for 6 subunits. In different diseases the DCTN genes may have different roles; therefore, we investigated the prognostic potential of DCTN mRNA expression in cutaneous melanoma (CM). MATERIAL AND METHODS Data for DCTN mRNA expression in CM patients were obtained from the OncoLnc database, which contains updated gene expression data for 459 CM patients based on the Cancer Genome Atlas. Kaplan-Meier analysis and a Cox regression model were used to determine overall survival (OS) with calculation of hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS The multivariate survival analysis showed that individually low expression of DCTN1, DCTN2, and DCTN5 and high expression of DCTN6 were associated with favorable OS (adjusted P=0.008, HR=0.676, 95% CI=0.506-0.903; adjusted P=0.004, HR=0.648, 95% CI=0.485-0.867; adjusted P=0.011, HR=0.686, 95% CI=0.514-0.916; and adjusted P=0.018, HR=0.706, 95% CI=0.530-0.942, respectively). In a joint-effects analysis, combinations of low expression of DCTN1, DCTN2, and DCTN5 and high expression of DCTN6 were found to be more highly correlated with favorable OS (all P<0.05). CONCLUSIONS Our findings suggest that downregulated DCTN1, DCTN2, and DCTN5 and upregulated DCTN6 mRNA expression in CM are associated with favorable prognosis and may represent potential prognostic biomarkers. Moreover, use of the 4 genes in combination can improve the sensitivity for predicting OS in CM patients.