Microtubule- and dynein-dependent nuclear trafficking of rhesus rhadinovirus in rhesus fibroblasts

We investigated the role of microtubules in rhesus rhadinovirus (RRV) nuclear trafficking in rhesus fibroblasts. Intact microtubules and microtubule dynamics are required for RRV trafficking to perinuclear regions. RRV trafficking was reduced by an inhibitor of the dynein motor and overexpression of dynamitin. Furthermore, RRV particles are colocalized with microtubules and dynein proteins. These results highlight the important roles of microtubules and dynein-dynactin complexes in the transport of RRV particles to nuclei during primary infection.     

Dynein Light Chain 1 (LC8) Association Enhances Microtubule Stability and Promotes Microtubule Bundling

Dynein light chain 1 (LC8), a highly conserved protein, is known to bind to a variety of different polypeptides. It functions as a dimer, which is inactivated through phosphorylation at the Ser-88 residue. A loss of LC8 function causes apoptosis in Drosophila embryos, and its overexpression induces malignant transformation of breast cancer cells. Here we show that LC8 binds to tubulin, promotes microtubule assembly, and induces the bundling of reconstituted microtubules in vitro. Furthermore, LC8 decorates microtubules both in Drosophila embryos and in HeLa cells, increases the microtubule stability, and promotes microtubule bundling in these cells. Microtubule stability influences a number of different cellular functions including mitosis and cell differentiation. The LC8 overexpression reduces the susceptibility of microtubules to cold and nocodazole-induced depolymerization in tissue-cultured cells and increases microtubule acetylation, suggesting that LC8 stabilizes microtubules. We also show that LC8 knockdown or transfection with inhibitory peptides destabilizes microtubules and inhibits bipolar spindle assembly in HeLa cells. In addition, LC8 knockdown leads to the mitotic block in HeLa cells. Furthermore, molecular docking analysis using the crystal structures of tubulin and LC8 dimer indicated that the latter may bind at α-β tubulin junction in a protofilament at sites distinct from the kinesin and dynein binding sites. Together, we provide the first evidence of a novel microtubule-associated protein-like function of LC8 that could explain its reported roles in cellular metastasis and differentiation.     

Cytoplasmic dynein mediates adenovirus binding to microtubules

During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% +/- 3.5% to 80.7% +/- 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding.     

Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.     

Tau directs intracellular trafficking by regulating the forces exerted by kinesin and dynein teams

Organelles, proteins, and mRNA are transported bidirectionally along microtubules by plus‐end directed kinesin and minus‐end directed dynein motors. Microtubules are decorated by microtubule‐associated proteins (MAPs) that organize the cytoskeleton, regulate microtubule dynamics and modulate the interaction between motor proteins and microtubules to direct intracellular transport. Tau is a neuronal MAP that stabilizes axonal microtubules and crosslinks them into bundles. Dysregulation of tau leads to a range of neurodegenerative diseases known as tauopathies including Alzheimer's disease (AD). Tau reduces the processivity of kinesin and dynein by acting as an obstacle on the microtubule. Single‐molecule assays indicate that kinesin‐1 is more strongly inhibited than kinesin‐2 or dynein, suggesting tau might act to spatially modulate the activity of specific motors. To investigate the role of tau in regulating bidirectional transport, we isolated phagosomes driven by kinesin‐1, kinesin‐2, and dynein and reconstituted their motility along microtubules. We find that tau biases bidirectional motility towards the microtubule minus‐end in a dose‐dependent manner. Optical trapping measurements show that tau increases the magnitude and frequency of forces exerted by dynein through inhibiting opposing kinesin motors. Mathematical modeling indicates that tau controls the directional bias of intracellular cargoes through differentially tuning the processivity of kinesin‐1, kinesin‐2, and dynein. Taken together, these results demonstrate that tau modulates motility in a motor‐specific manner to direct intracellular transport, and suggests that dysregulation of tau might contribute to neurodegeneration by disrupting the balance of plus‐ and minus‐end directed transport.      

Construction of centrosomes and spindle poles by molecular motor-driven assembly of protein particles

Centrosomes and other microtubule organizing centers are the largest non-membranous organelles in most cells. This morphologically diverse class of organelles shares a common ability to nucleate and organize microtubules in interphase and participates in the formation of mitotic spindles during cell division. This review summarizes recent evidence suggesting that assembly of centrosomes and mitotic spindle poles require transport of large protein particles along microtubules by the molecular motor cytoplasmic dynein.     

Identification of novel cellular proteins that bind to the LC8 dynein light chain using a pepscan technique

Dynein is a minus end-directed microtubule motor that serves multiple cellular functions. We have performed a fine mapping of the 8 kDa dynein light chain (LC8) binding sites throughout the development of a library of consecutive synthetic dodecapeptides covering the amino acid sequences of the various proteins known to interact with this dynein member according to the yeast two hybrid system. Two different consensus sequences were identified: GIQVD present in nNOS, in DNA cytosine methyl transferase and also in GKAP, where it is present twice in the protein sequence. The other LC8 binding motif is KSTQT, present in Bim, dynein heavy chain, Kid-1, protein 4 and also in swallow. Interestingly, this KSTQT motif is also present in several viruses known to associate with microtubules during retrograde transport from the plasma membrane to the nucleus during viral infection.     

Cdc42 Regulates Microtubule‐Dependent Golgi Positioning

The molecular mechanisms underlying cytoskeleton‐dependent Golgi positioning are poorly understood. In mammalian cells, the Golgi apparatus is localized near the juxtanuclear centrosome via dynein‐mediated motility along microtubules. Previous studies implicate Cdc42 in regulating dynein‐dependent motility. Here we show that reduced expression of the Cdc42‐specific GTPase‐activating protein, ARHGAP21, inhibits the ability of dispersed Golgi membranes to reposition at the centrosome following nocodazole treatment and washout. Cdc42 regulation of Golgi positioning appears to involve ARF1 and a binding interaction with the vesicle‐coat protein coatomer. We tested whether Cdc42 directly affects motility, as opposed to the formation of a trafficking intermediate, using a Golgi capture and motility assay in permeabilized cells. Disrupting Cdc42 activation or the coatomer/Cdc42 binding interaction stimulated Golgi motility. The coatomer/Cdc42‐sensitive motility was blocked by the addition of an inhibitory dynein antibody. Together, our results reveal that dynein and microtubule‐dependent Golgi positioning is regulated by ARF1‐, coatomer‐, and ARHGAP21‐dependent Cdc42 signaling.     

Dynein,Lis1 and CLIP‐170 counteract Eg5‐dependent centrosome separation during bipolar spindle assembly

Bipolar spindle assembly critically depends on the microtubule plus‐end‐directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus‐end‐directed motor dynein, or the dynein‐binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5‐dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus‐end‐tracking protein CLIP‐170. This function of CLIP‐170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP‐115, a highly homologous protein that lacks the dynein–dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP‐170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.     

Constitutive dynein activity in She1 mutants reveals differences in microtubule attachment at the yeast spindle pole body

The organization of microtubules is determined in most cells by a microtubule-organizing center, which nucleates microtubule assembly and anchors their minus ends. In Saccharomyces cerevisiae cells lacking She1, cytoplasmic microtubules detach from the spindle pole body at high rates. Increased rates of detachment depend on dynein activity, supporting previous evidence that She1 inhibits dynein. Detachment rates are higher in G1 than in metaphase cells, and we show that this is primarily due to differences in the strengths of microtubule attachment to the spindle pole body during these stages of the cell cycle. The minus ends of detached microtubules are stabilized by the presence of γ-tubulin and Spc72, a protein that tethers the γ-tubulin complex to the spindle pole body. A Spc72-Kar1 fusion protein suppresses detachment in G1 cells, indicating that the interaction between these two proteins is critical to microtubule anchoring. Overexpression of She1 inhibits the loading of dynactin components, but not dynein, onto microtubule plus ends. In addition, She1 binds directly to microtubules in vitro, so it may compete with dynactin for access to microtubules. Overall, these results indicate that inhibition of dynein activity by She1 is important to prevent excessive detachment of cytoplasmic microtubules, particularly in G1 cells.     

Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein

Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3). The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses. The P protein-dynein LC8 interaction was confirmed by colocalization with laser confocal microscopy in infected cells and by coimmunoprecipitation. The dynein-interacting P protein domain was mapped to the 186 amino acid residues of the N-terminal half of the protein. Dynein LC8 is a component of both cytoplasmic dynein and myosin V, which are involved in a wide range of intracellular motile events, such as microtubule minus-end directed organelle transport in axon "retrograde transport" and actin-based vesicle transport, respectively. Our results provide support for a model of viral nucleocapsid axoplasmic transport. Furthermore, the role of LC8 in cellular mechanisms other than transport, e.g., inhibition of neuronal nitric oxide synthase, suggests that the P protein interactions could be involved in physiopathological mechanisms of rabies virus-induced pathogenesis.     

Protein friction exerted by motor enzymes through a weak-binding interaction.

Recently Vale et al. (1989, Cell 59, 915-925.) reported an observation of the one-dimensional Brownian movement of microtubules bound to flagellar dynein through a weak-binding interaction. In this study, we propose a theoretical model of this phenomenon. Our model consists of a rigid microtubule associated with a number of elastic dynein heads through a weak-binding interaction at equilibrium. The model implies that (1) the Brownian motion of the microtubule is not directly driven by the atomic collision of the solvent particles, but is driven by the thermally-generated structural fluctuations of the dynein heads which interact with the microtubule; (2) dynein heads through a weak-binding interaction exert a frictional drag force on the sliding motion of the microtubule and the drag force is proportional to the sliding velocity the same as in hydrodynamic viscous friction. This protein friction, with such viscous-like characteristics, may well play a role as a velocity-limiting factor in the normal ATP-induced sliding movement of motile proteins.     

Proteomic identification of brain proteins that interact with dynein light chain LC8

Cytoplasmic dynein is a large minus end-directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two-hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain-associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post-synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.     

The Sireviruses, a plant-specific lineage of the Ty1/copia retrotransposons, interact with a family of proteins related to dynein light chain 8

Plant genomes are rich in long terminal repeat retrotransposons, and here we describe a plant-specific lineage of Ty1/copia elements called the Sireviruses. The Sireviruses vary greatly in their genomic organization, and many have acquired additional coding information in the form of an envelope-like open reading frame and an extended gag gene. Two-hybrid screens were conducted with the novel domain of Gag (the Gag extension) encoded by a representative Sirevirus from maize (Zea mays) called Hopie. The Hopie Gag extension interacts with a protein related to dynein light chain 8 (LC8). LC8 also interacts with the Gag extension from a Hopie homolog from rice (Oryza sativa). Amino acid motifs were identified in both Hopie Gag and LC8 that are responsible for the interaction. Two amino acids critical for Gag recognition map within the predicted LC8-binding cleft. Two-hybrid screens were also conducted with the Gag extension encoded by the soybean (Glycine max) SIRE1 element, and an interaction was found with light chain 6 (LC6), a member of the LC8 protein family. LC8 and LC6 proteins are components of the dynein microtubule motor, with LC8 being a versatile adapter that can bind many unrelated cellular proteins and viruses. Plant LC8 and LC6 genes are abundant and divergent, yet flowering plants do not encode other components of the dynein motor. Although, to our knowledge, no cellular roles for plant LC8 family members have been proposed, we hypothesize that binding of LC8 proteins to Gag aids in the movement of retrotransposon virus-like particles within the plant cell or possibly induces important conformational changes in the Gag protein.     

The 8-kDa dynein light chain binds to p53-binding protein 1 and mediates DNA damage-induced p53 nuclear accumulation

The tumor suppressor protein p53 is known to undergo cytoplasmic dynein-dependent nuclear translocation in response to DNA damage. However, the molecular link between p53 and the minus end-directed microtubule motor dynein complex has not been described. We report here that the 8-kDa light chain (LC8) of dynein binds to p53-binding protein 1 (53BP1). The LC8-binding domain was mapped to a short peptide segment immediately N-terminal to the kinetochore localization region of 53BP1. The LC8-binding domain is completely separated from the p53-binding domain in 53BP1. Therefore, 53BP1 can potentially act as an adaptor to assemble p53 to the dynein complex. Unlike other known LC8-binding proteins, 53BP1 contains two distinct LC8-binding motifs that are arranged in tandem. We further showed that 53BP1 can directly associate with the dynein complex. Disruption of the interaction between LC8 and 53BP1 in vivo prevented DNA damage-induced nuclear accumulation of p53. These data illustrate that LC8 is able to function as a versatile acceptor to link a wide spectrum of molecular cargoes to the dynein motor.     

Molecular analysis of the klarsicht gene and its role in nuclear migration within differentiating cells of the Drosophila eye.

BACKGROUND: The temporally regulated, cell-type-specific transport of organelles has great biological significance, yet little is known about the regulation of organelle transport during development. The Drosophila gene klarsicht is required for temporally regulated lipid droplet transport in developing embryos and for the stereotypical nuclear migrations in differentiating cells of the developing eye. Klarsicht is thought to coordinate the function of several molecular motors bound to a single lipid droplet or to facilitate the attachment of dynein to the cargo, but it is not known whether Klarsicht affects motors directly or indirectly. RESULTS: Here, we have cloned the klarsicht gene and shown that it encodes a unique large protein. Drosophila klarsicht null mutants were viable, with obvious defects only in adult eye morphology. Epitope-tagged Klarsicht expressed in the eye from a transgene was perinuclear. In flies carrying transgenes that express markers for microtubule plus and minus ends, microtubules in differentiating cells of the eye were oriented with their plus ends apical and their minus ends at the nucleus. CONCLUSIONS: Drosophila klarsicht null mutants were viable and fertile, demonstrating that klarsicht is essential only for specific motor protein functions. Perinuclear localization of Klarsicht protein indicates that Klarsicht has a direct mechanical role in nuclear migration. Taken together with the finding that the minus ends of the microtubules are associated with the photoreceptor nuclei, the observation that Klarsicht is largely perinuclear supports the idea that Klarsicht associates with dynein, consistent with a model in which Klarsicht assists dynein in 'reeling in' the nucleus.     

Cytoplasmic Dynein and Dynactin Are Required for the Transport of Microtubules into the Axon

Previous work from our laboratory suggested that microtubules are released from the neuronal centrosome and then transported into the axon (Ahmad, F.J., and P.W. Baas. 1995. J. Cell Sci. 108: 2761–2769). In these studies, cultured sympathetic neurons were treated with nocodazole to depolymerize most of their microtubule polymer, rinsed free of the drug for a few minutes to permit a burst of microtubule assembly from the centrosome, and then exposed to nanomolar levels of vinblastine to suppress further microtubule assembly from occurring. Over time, the microtubules appeared first near the centrosome, then dispersed throughout the cytoplasm, and finally concentrated beneath the periphery of the cell body and within developing axons. In the present study, we microinjected fluorescent tubulin into the neurons at the time of the vinblastine treatment. Fluorescent tubulin was not detected in the microtubules over the time frame of the experiment, confirming that the redistribution of microtubules observed with the experimental regime reflects microtubule transport rather than microtubule assembly. To determine whether cytoplasmic dynein is the motor protein that drives this transport, we experimentally increased the levels of the dynamitin subunit of dynactin within the neurons. Dynactin, a complex of proteins that mediates the interaction of cytoplasmic dynein and its cargo, dissociates under these conditions, resulting in a cessation of all functions of the motor tested to date (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132: 617–633). In the presence of excess dynamitin, the microtubules did not show the outward progression but instead remained near the centrosome or dispersed throughout the cytoplasm. On the basis of these results, we conclude that cytoplasmic dynein and dynactin are essential for the transport of microtubules from the centrosome into the axon.