A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules

Microtubule-associated proteins (MAPs) use particular microtubule-binding domains that allow them to interact with microtubules in a manner specific to their individual cellular functions. Here, we have identified a highly basic microtubule-binding domain in the p150 subunit of dynactin that is only present in the dynactin members of the CAP-Gly family of proteins. Using single-particle microtubule-binding assays, we found that the basic domain of dynactin moves progressively along microtubules in the absence of molecular motors - a process we term 'skating'. In contrast, the previously described CAP-Gly domain of dynactin remains firmly attached to a single point on microtubules. Further analyses showed that microtubule skating is a form of one-dimensional diffusion along the microtubule. To determine the cellular function of the skating phenomenon, dynein and the dynactin microtubule-binding domains were examined in single-molecule motility assays. We found that the basic domain increased dynein processivity fourfold whereas the CAP-Gly domain inhibited dynein motility. Our data show that the ability of the basic domain of dynactin to skate along microtubules is used by dynein to maintain longer interactions for each encounter with microtubules.     

Effects of dynactin disruption and dynein depletion on axonal microtubules

We investigated potential roles of cytoplasmic dynein in organizing axonal microtubules either by depleting dynein heavy chain from cultured neurons or by experimentally disrupting dynactin. The former was accomplished by siRNA while the latter was accomplished by overexpressing P50-dynamitin. Both methods resulted in a persistent reduction in the frequency of transport of short microtubules. To determine if the long microtubules in the axon also undergo dynein-dependent transport, we ascertained the rates of EGFP-EB3 "comets" observed at the tips of microtubules during assembly. The rates of the comets, in theory, should reflect a combination of the assembly rate and any potential transport of the microtubule. Comets were initially slowed during P50-dynamitin overexpression, but this effect did not persist beyond the first day and was never observed in dynein-depleted axons. In fact, the rates of the comets were slightly faster in dynein-depleted axons. We conclude that the transient effect of P50-dynamitin overexpression reflects a reduction in microtubule polymerization rates. Interestingly, after prolonged dynein depletion, the long microtubules were noticeably misaligned in the distal regions of axons and failed to enter the filopodia of growth cones. These results suggest that the forces generated by cytoplasmic dynein do not transport long microtubules, but may serve to align them with one another and also permit them to invade filopodia.     

Bidirectional transport along microtubules

Active transport by microtubule motors has a plethora of crucial roles in eukaryotic cells. Organelles often move bidirectionally, employing both plus-end and minus-end directed motors. Bidirectional motion is widespread and may allow dynamic regulation, error correction and the establishment of polarized organelle distributions. Emerging evidence suggests that motors for both directions are simultaneously present on cellular 'cargo', but that their activity is coordinated so that when plus-end motors are active, minus-end motors are not, and vice versa. Both the dynein cofactor dynactin and the Klarsicht (Klar) protein appear to be important for such coordination. The direction of net transport depends on the balance between plus-end directed and minus-end directed motion. In several model systems, factors crucial for setting this balance have now been identified, setting the stage for a molecular dissection of the underlying regulatory mechanisms. These analyses will likely provide insight into motor cooperation in general.     

Role of cytoplasmic dynein in the axonal transport of microtubules and neurofilaments

Recent studies have shown that the transport of microtubules (MTs) and neurofilaments (NFs) within the axon is rapid, infrequent, asynchronous, and bidirectional. Here, we used RNA interference to investigate the role of cytoplasmic dynein in powering these transport events. To reveal transport of MTs and NFs, we expressed EGFP-tagged tubulin or NF proteins in cultured rat sympathetic neurons and performed live-cell imaging of the fluorescent cytoskeletal elements in photobleached regions of the axon. The occurrence of anterograde MT and retrograde NF movements was significantly diminished in neurons that had been depleted of dynein heavy chain, whereas the occurrence of retrograde MT and anterograde NF movements was unaffected. These results support a cargo model for NF transport and a sliding filament model for MT transport.     

Association of microtubules and neurofilaments in vitro is not mediated by ATP

Runge et al. [Runge, M.S., Laue, T.M., Yphantis, D.A., Lifsics, M.R., Saito, A., Altin, M., Reinke, K., & Williams, R.C., Jr. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1431-1435] found that mixtures of microtubules and neurofilaments formed a viscous, sedimentable complex when incubated at 37 degrees C for 20 min in the presence of ATP. They did not observe the high viscosities associated with the complex when the incubation was carried out in the absence of ATP. This paper reports an investigation of the roles of time and ATP in the formation of the complex. Microtubules assembled in a mixture containing GTP and neurofilaments prepared from bovine brain remained assembled for a shorter period of time than they did in similar solutions containing no neurofilaments. Adding ATP to the neurofilament-containing solutions, or doubling their GTP concentration, extended the time during which the microtubules remained assembled. These mixtures then became highly viscous. These phenomena resulted from the action of at least two enzymes present in the neurofilament preparation. A GTPase raised the GDP/GTP ratio, in the mixtures in which ATP was absent, to levels sufficient to cause disassembly of the microtubules. When ATP was present, a nucleotide diphosphokinase catalyzed regeneration of GTP from GDP while converting ATP to ADP. This process kept the GDP/GTP ratio low and delayed the disassembly of the microtubules. These results show that the apparent ATP dependence of formation of the microtubule-neurofilament complex observed by Runge et al. is attributable to a GDP-induced disassembly of microtubules rather than to a disruption of microtubule-neurofilament contacts. Those contacts can form in the absence of ATP.     

Cytoplasmic dynein/dynactin mediates the assembly of aggresomes

Aggresomes are pericentrosomal cytoplasmic structures into which aggregated, ubiquitinated, misfolded proteins are sequestered. Misfolded proteins accumulate in aggresomes when the capacity of the intracellular protein degradation machinery is exceeded. Previously, we demonstrated that an intact microtubule cytoskeleton is required for the aggresome formation [Johnston et al., 1998: J. Cell Biol. 143:1883-1898]. In this study, we have investigated the involvement of microtubules (MT) and MT motors in this process. Induction of aggresomes containing misfolded DeltaF508 CFTR is accompanied by a redistribution of the retrograde motor cytoplasmic dynein that colocalizes with aggresomal markers. Coexpression of the p50 (dynamitin) subunit of the dynein/dynactin complex prevents the formation of aggresomes, even in the presence of proteasome inhibitors. Using in vitro microtubule binding assays in conjunction with immunogold electron microscopy, our data demonstrate that misfolded DeltaF508 CFTR associate with microtubules. We conclude that cytoplasmic dynein/dynactin is responsible for the directed transport of misfolded protein into aggresomes. The implications of these findings with respect to the pathogenesis of neurodegenerative disease are discussed.     

Coordination of outer arm dynein activity along axonemal doublet microtubules

This study considers the mechanism by which ODA based sliding is produced and the relationship of that mechanism to the determination of beat frequency. Two models of activity have been examined: a stochastic model, where ODA activity is random and a metachronal model, where activity is sequentially triggered along a doublet. Inactivation of a few ODAs would have virtually no effect on stochastic activity, but would completely block metachronal activity. We (Seetharam and Satir [2005]: Cell Motil Cytoskeleton 60:96-103) previously demonstrated that ODAs produce high speed sliding of about 200 mum/s, followed by a pause. IDAs produce slow, 5 mum/s, continuous sliding. We have examined the effects of nM concentrations of vanadate on sliding, measuring velocity and extent of high speed sliding and pause distribution or sliding cessation. In 5 nM vanadate, where photocleavage experiments show about 16/270 ODAs per doublet are affected, no differences from control are seen, but at 10 and 25 nM vanadate, high speed velocity is greatly reduced and pause distribution changes. The results support a model, in which high speed sliding is produced by metachronal activity. Blockage of two or more heavy chains of one ODA or a small group of adjacent ODAs produces cessation of sliding, but cessation is only temporary, probably because IDA activity continues, allowing ODA activity re-initiation beyond the block. These conclusions are consistent with Sugino and Naitoh's [1982; Nature 295:609-611] proposal, whereby during each beat, every ODA along a doublet becomes activated in succession, with repetitive activation determining beat frequency.     

Multiple mechanisms of chromosome movement in vertebrate cells mediated through the Ndc80 complex and dynein/dynactin

Kinetochores bind microtubules laterally in a transient fashion and stably, by insertion of plus ends. These pathways may exist to carry out distinct tasks during different stages of mitosis and likely depend on distinct molecular mechanisms. On isolated chromosomes, we found microtubule nucleation/binding depended additively on both dynein/dynactin and on the Ndc80/Hec1 complex. Studying chromosome movement in living Xenopus cells within the simplified geometry of monopolar spindles, we quantified the relative contributions of dynein/dynactin and the Ndc80/Hec1 complex. Inhibition of dynein/dynactin alone had minor effects but did suppress transient, rapid, poleward movements. In contrast, inhibition of the Ndc80 complex blocked normal end-on attachments of microtubules to kinetochores resulting in persistent rapid poleward movements that required dynein/dynactin. In normal cells with bipolar spindles, dynein/dynactin activity on its own allowed attachment and rapid movement of chromosomes on prometaphase spindles but failed to support metaphase alignment and chromatid movement in anaphase. Thus, in prometaphase, dynein/dynactin likely mediates early transient, lateral interactions of kinetochores and microtubules. However, mature attachment via the Ndc80 complex is essential for metaphase alignment and anaphase A. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mutually exclusive cytoplasmic dynein regulation by NudE-Lis1 and dynactin

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

Activation of the dynein adenosinetriphosphatase by microtubules

Previous work has indicated that following the rapid adenosine 5'-triphosphate (ATP) induced dissociation of the microtubule-dynein complex, the rate-limiting step in the ATPase cycle is product release [Johnson, K. A. (1983) J. Biol. Chem. 258, 13825-13832], which occurs at a rate of approximately 2-6 s-1. In this report we complete the analysis of the ATPase cycle by examining the effect of microtubules on the rate of product release. For these studies we used repolymerized Tetrahymena axonemal microtubules and microtubule-associated protein (MAP) free bovine brain microtubules which were shown to be free of any measureable ATPase activity. Tetrahymena 22S dynein bound to these microtubules predominantly by the ATP-sensitive site and at a rate giving an apparent second-order rate constant of (0.2-1) X 10(6) M-1 s-1, which is 50-fold greater than the rate observed with brain microtubules containing MAPs. ATP induced the rapid dissociation of the microtubule-dynein complex with an apparent second-order rate constant vs. ATP concentration equal to 1.6 X 10(6) M-1 s-1; this value is only slightly lower than that observed in the presence of MAPs. After the ATP-induced dissociation, the dynein reassociated with the microtubules following a lag period due to the time required to hydrolyze the ATP. The duration of the lag time for reassociation decreased with increasing microtubule concentration, suggesting that microtubules increased the rate of ATP turnover. Direct measurements at steady state showed that the specific activity of the dynein increased with increasing microtubule concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dephosphorylation-induced interactions of neurofilaments with microtubules

Effects of dephosphorylation on interactions of neurofilaments (NFs) with microtubules (MTs) were studied by the cosedimentation method. Centrifugation conditions were chosen so that MTs pelleted but NFs did not. While NFs isolated from bovine spinal cords did not cosediment with MTs polymerized in the presence of taxol, NFs dephosphorylated with Escherichia coli alkaline phosphatase began to coprecipitate with MTs. The dephosphorylated NFs bound to MTs but not to the unpolymerized tubulin dimer. The binding was not observed in the presence of high salt or with MTs containing microtubule-associated proteins. The cosedimentation experiments using purified NF subunit proteins showed that the dephosphorylation-induced binding of NFs to MTs was mediated by the largest subunit of NF (NF-H). Negative staining electron microscopy confirmed bindings of the dephosphorylated NFs and NF-H to MTs. Densitometric measurement of the bound and unbound NF-H after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the binding of the dephosphorylated NF-H to MT was saturable and gave the following binding parameters. Approximately 1 mol of NF-H bound per 10 mol of tubulin dimer with a high affinity site (Kd = 3.8 x 10(-8) M) and per 16 mol of tubulin dimer with a low affinity site (Kd = 1.1 x 10(-7) M).     

Cell cycle-regulated cortical dynein/dynactin promotes symmetric cell division by differential pole motion in anaphase

In cultured mammalian cells, how dynein/dynactin contributes to spindle positioning is poorly understood. To assess the role of cortical dynein/dynactin in this process, we generated mammalian cell lines expressing localization and affinity purification (LAP)-tagged dynein/dynactin subunits from bacterial artificial chromosomes and observed asymmetric cortical localization of dynein and dynactin during mitosis. In cells with asymmetrically positioned spindles, dynein and dynactin were both enriched at the cortex distal to the spindle. NuMA, an upstream targeting factor, localized asymmetrically along the cell cortex in a manner similar to dynein and dynactin. During spindle motion toward the distal cortex, dynein and dynactin were locally diminished and subsequently enriched at the new distal cortex. At anaphase onset, we observed a transient increase in cortical dynein, followed by a reduction in telophase. Spindle motion frequently resulted in cells entering anaphase with an asymmetrically positioned spindle. These cells gave rise to symmetric daughter cells by dynein-dependent differential spindle pole motion in anaphase. Our results demonstrate that cortical dynein and dynactin dynamically associate with the cell cortex in a cell cycle-regulated manner and are required to correct spindle mispositioning in LLC-Pk1 epithelial cells.     

Cytoplasmic dynein and dynactin in cell division and intracellular transport

Since the initial discovery of cytoplasmic dynein, it has become apparent that this microtubule-based motor is involved in several cellular functions including cell division and intracellular transport. Another multisubunit complex, dynactin, may be required for most, if not all, cytoplasmic dynein-driven activities and may provide clues to dynein's functional diversity. Recent genetic and biochemical findings have illuminated the cellular roles of dynein and dynactin and provided insight into the functional mechanism of this complex motor.     

Centering of a radial microtubule array by translocation along microtubules spontaneously nucleated in the cytoplasm

Positioning of a radial array of microtubules (MTs) in the cell centre is crucial for cytoplasmic organization, but the mechanisms of such centering are difficult to study in intact cells that have pre-formed radial arrays. Here, we use cytoplasmic fragments of melanophores, and cytoplasts of BS-C-1 cells to study MT centering mechanisms. Using live imaging and computer modelling, we show that the MT aster finds a central location in the cytoplasm by moving along spontaneously nucleated non-astral MTs towards a point at which MT nucleation events occur equally on all sides. We hypothesize that similar mechanisms, in the presence of the centrosome, contribute to this centering mechanism and ensure the robustness of cytoplasmic organization.     

Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration

To test the hypothesis that inhibition of axonal transport is sufficient to cause motor neuron degeneration such as that observed in amyotrophic lateral sclerosis (ALS), we engineered a targeted disruption of the dynein-dynactin complex in postnatal motor neurons of transgenic mice. Dynamitin overexpression was found to disassemble dynactin, a required activator of cytoplasmic dynein, resulting in an inhibition of retrograde axonal transport. Mice overexpressing dynamitin demonstrate a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, and denervation of muscle. Previous transgenic mouse models of ALS have shown abnormalities in microtubule-based axonal transport. In this report, we describe a mouse model that confirms the critical role of disrupted axonal transport in the pathogenesis of motor neuron degenerative disease.     

Activation of the dynein adenosinetriphosphatase by cross-linking to microtubules

The microtubule-dynein complex consisting of 22S dynein from Tetrahymena cilia and MAP-free microtubules was subjected to treatment with various concentrations of 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC), a zero-length cross-linker, at 28 degrees C for 1 h. Following cross-linking of the microtubule-dynein complex, nearly all of the ATPase activity cosedimented with the microtubules in the presence of ATP. Electron microscopic observation by negative staining revealed that, following treatment with 1 mM EDC, the complex did not dissociate in the presence of ATP, although the dynein decoration pattern was disordered. The complex treated with 3 mM EDC exhibited normal microtubule-dynein patterns even after the addition of ATP. The ATPase activity of the microtubule-dynein complex was enhanced about 30-fold by the treatment with 1-3 mM EDC. These results indicate that the ATPase activation was caused by the close proximity of the dynein ATPase sites to the microtubules and provide further support for the functional interaction of all three dynein heads with the microtubule. The maximal specific activity was 12 mumol min-1 (mg of dynein)-1, corresponding to a turnover rate of 150 s-1, which may be the rate-limiting step at infinite microtubule concentration and may represent the maximum rate of force production in the axoneme.     

LIS1, CLIP-170's key to the dynein/dynactin pathway

CLIP-170 is a plus-end tracking protein which may act as an anticatastrophe factor. It has been proposed to mediate the association of dynein/dynactin to microtubule (MT) plus ends, and it also binds to kinetochores in a dynein/dynactin-dependent fashion, both via its C-terminal domain. This domain contains two zinc finger motifs (proximal and distal), which are hypothesized to mediate protein-protein interactions. LIS1, a protein implicated in brain development, acts in several processes mediated by the dynein/dynactin pathway by interacting with dynein and other proteins. Here we demonstrate colocalization and direct interaction between CLIP-170 and LIS1. In mammalian cells, LIS1 recruitment to kinetochores is dynein/dynactin dependent, and recruitment there of CLIP-170 is dependent on its site of binding to LIS1, located in the distal zinc finger motif. Overexpression of CLIP-170 results in a zinc finger-dependent localization of a phospho-LIS1 isoform and dynactin to MT bundles, raising the possibility that CLIP-170 and LIS1 regulate dynein/dynactin binding to MTs. This work suggests that LIS1 is a regulated adapter between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in the control of MT dynamics.     

Evidence for glucocorticoid receptor transport on microtubules by dynein

Rapid, ligand-dependent movement of glucocorticoid receptors (GR) from cytoplasm to the nucleus is hsp90-dependent, and much of the movement system has been defined. GR.hsp90 heterocomplexes isolated from cells contain one of several hsp90-binding immunophilins that link the complex to cytoplasmic dynein, a molecular motor that processes along microtubular tracks to the nucleus. The immunophilins link to dynein indirectly via the dynamitin component of the dynein-associated dynactin complex (Galigniana, M. D., Harrell, J. M., O'Hagen, H. M., Ljungman, M., and Pratt, W. B. (2004) J. Biol. Chem. 279, 22483-22489). Although it is known that rapid, hsp90-dependent GR movement requires intact microtubules, it has not been shown that the movement is dynein-dependent. Here, we show that overexpression of dynamitin, which blocks movement by dissociating the dynein motor from its cargo, inhibits ligand-dependent movement of the GR to the nucleus. We show that native GR.hsp90.immnunophilin complexes contain dynamitin as well as dynein and that GR heterocomplexes isolated from cytosol containing paclitaxel and GTP to stabilize microtubules also contain tubulin. The complete movement system, including the dynein motor complex and tubulin, can be assembled under cell-free conditions by incubating GR immune pellets with paclitaxel/GTP-stabilized cytosol prepared from GR(-) L cells. This is the first evidence that the movement of a steroid receptor is dynein-dependent, and it is the first isolation of a steroid receptor bound to the entire system that determines its retrograde movement.