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PubMed2006

GSK-3beta-regulated interaction of BICD with dynein is involved in microtubule anchorage at centrosome.

Microtubule arrays direct intracellular organization and define cellular polarity. Here, we show a novel function of glycogen synthase kinase-3beta (GSK-3beta) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. Our data revealed that GSK-3beta is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3beta or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3beta knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3beta and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Colocalization studies of Arp1 and p150Glued to spindle microtubules during mitosis: the effect of cytochalasin on the organization of microtubules and motor proteins in PtK1 cells.

Motor proteins play a fundamental role in the congression and segregation of chromosomes in mitosis as well as the formation of the mitotic spindle. In particular, the dynein/dynactin complex is involved in the maintenance of the spindle, formation of astral microtubules, chromosome motion, and chromosome segregation. Dynactin is a multisubunit, high molecular weight protein that is responsible for the attachment of cargo to dynein. There are a number of major subunits in dynactin that are presumed to be important during mitosis. Arp1 is thought to be the attachment site for cargo to the complex while p150(Glued), a side arm of this complex regulates binding to MTs and the binding of dynactin to dynein. We performed colocalization studies of Arp1 and p150(Glued) to spindle microtubules. Both Arp1 and p150(Glued) colocalize with spindle MTs as well as cytoplasmic components. When treated with cytochalasin J, Arp1 concentrates at the centrosomes and is less co-localized with spindle MTs. Cytochalasin J has less of an effect on the colocalization of p150(Glued) with spindle MTs, suggesting that Arp1 may have a cytochalasin J sensitive site.

Animals↗

PubMed2001

The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics.

Cytoplasmic dynein is a multisubunit, minus end-directed microtubule motor that uses dynactin as an accessory complex to perform various in vivo functions including vesicle transport, spindle assembly, and nuclear distribution [1]. We previously showed that in the filamentous fungus Aspergillus nidulans, a GFP-tagged cytoplasmic dynein heavy chain (NUDA) forms comet-like structures that exhibited microtubule-dependent movement toward and back from the hyphal tip [2]. Here we demonstrate that another protein in the NUDA pathway, NUDF, which is homologous to the human LIS1 protein involved in brain development [3, 4], also exhibits such dynamic behavior. Both NUDA and NUDF are located at the ends of microtubules, and this observation suggests that the observed dynamic behavior is due to their association with the dynamic microtubule ends. To address whether NUDA and NUDF play a role in regulating microtubule dynamics in vivo, we constructed a GFP-labeled alpha-tubulin strain and used it to compare microtubule dynamics in vivo in wild-type A. nidulans versus temperature-sensitive loss-of-function mutants of nudA and nudF. The mutants showed a lower frequency of microtubule catastrophe, a lower rate of shrinkage during catastrophe, and a lower frequency of rescue. The microtubules in the mutant cells also paused longer at the hyphal tip than wild-type microtubules. These results indicate that cytoplasmic dynein and the LIS1 homolog NUDF affect microtubule dynamics in vivo.

Aspergillus nidulans↗

PubMed2002

A CH domain-containing N terminus in NuMA?

Nuclear mitotic apparatus protein (NuMA) is an essential vertebrate component in organizing microtubule ends at spindle poles. The NuMA-dynactin/dynein motor multiprotein complex not only explains the transport of NuMA along spindle fibers but also is linked to the process of microtubule focusing. The interaction sites of NuMA to dynein/dynactin have not been mapped. In the yet functionally uncharacterized N terminus of NuMA, we predict a calponin-homology (CH) domain, a motif with binding activity for actin-like molecules. We substantiate the primary sequence analysis-based prediction with secondary structure and fold recognition analysis, and we propose the N-terminal CH domain of NuMA as a likely interaction site for actin-related protein 1 (Arp1) protein of the dynactin/dynein complex.

Amino Acid Sequence↗

PubMed2002

Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function.

Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1999

Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium.

Cytoplasmic dynein intermediate chain (IC) mediates dynein-dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806-28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507-1516). To investigate the physiological role of IC and dynein-dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICDeltaC associated with dynactin but not with dynein heavy chain, whereas ICDeltaN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

Animals↗

PubMed1998

Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment.

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.

Animals↗

PubMed2001

beta III spectrin binds to the Arp1 subunit of dynactin.

Cytoplasmic dynein is an intracellular motor responsible for endoplasmic reticulum-to-Golgi vesicle trafficking and retrograde axonal transport. The accessory protein dynactin has been proposed to mediate the association of dynein with vesicular cargo. Dynactin contains a 37-nm filament made up of the actin-related protein, Arp1, which may interact with a vesicle-associated spectrin network. Here, we demonstrate that Arp1 binds directly to the Golgi-associated betaIII spectrin isoform. We identify two Arp1-binding sites in betaIII spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to betaIII spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and betaIII spectrin co-purify on vesicles isolated from rat brain, and betaIII spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, betaIII spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, betaIII spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for betaIII spectrin. We hypothesize that the interaction between betaIII spectrin and Arp1 recruits dynein and dynactin to intracellular membranes and provides a direct link between the microtubule motor complex and its membrane-bounded cargo.

Actins↗

PubMed1999

Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170.

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170's putative cargo-binding domain. Overexpression studies using p150(Glued), the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

Animals↗

PubMed2001

Immunolocalization of cytoplasmic dynein and dynactin subunits in cultured macrophages: enrichment on early endocytic organelles.

Cytoplasmic dyneins and their cofactor, dynactin, work together to mediate the movement of numerous cargo organelles toward the minus-ends of microtubules. In many cases, there is compelling evidence that dynactin functions in part to attach dyneins to cargo organelles, but this may not always be the case. We have localized three dynactin subunits (Arp1, p62 and p150(Glued)) and two subunits of conventional cytoplasmic dynein (dynein intermediate chain and dynein heavy chain 1) in murine macrophages using immunogold labeling of thawed cryosections. Using stereological techniques, we have quantified the relative distributions of each of these subunits on specific membrane organelles to generate a comprehensive analysis of the distribution of these proteins in a single cell type. Our results show that each of the subunits tested exhibits the same distribution with respect to different membrane organelles, with highest levels present on early endosomes, and lower levels present on later endocytic organelles, the mitochondrial outer membrane, the plasma membrane and vesicles in the Golgi region. An additional pool of punctate dynactin labeling was detected in the cell periphery, in the absence of dynein labeling. Even when examined closely, membrane organelles could not be detected in association with these dynactin-positive sites; however, double labeling with anti-tubulin antibody revealed that at least some of these sites represent the ends of microtubules. The similarities among the labeling profiles with respect to membrane organelles suggest that dynein and dynactin bind to membrane organelles as an obligate unit. In contrast, our results show that dynactin can associate with microtubule ends in the absence of dynein, perhaps providing sites for subsequent organelle and dynein association to form a functional motility complex.

Animals↗

PubMed2004

Protein 4.1R, a microtubule-associated protein involved in microtubule aster assembly in mammalian mitotic extract.

Non-erythroid protein 4.1R (4.1R) consists of a complex family of isoforms. We have shown that 4.1R isoforms localize at the mitotic spindle/spindle poles and associate in a complex with the mitotic-spindle organization proteins Nuclear Mitotic Apparatus protein (NuMA), dynein, and dynactin. We addressed the mitotic function of 4.1R by investigating its association with microtubules, the main component of the mitotic spindles, and its role in mitotic aster assembly in vitro. 4.1R appears to partially co-localize with microtubules throughout the mitotic stages of the cell cycle. In vitro sedimentation assays showed that 4.1R isoforms directly interact with microtubules. Glutathione S-transferase (GST) pull-down assays using GST-4.1R fusions and mitotic cell extracts further showed that the association of 4.1R with tubulin results from both the membrane-binding domain and C-terminal domain of 4.1R. Moreover, 4.1R, but not actin, is a mitotic microtubule-associated protein; 4.1R associates with microtubules in the microtubule pellet of the mitotic asters assembled in mammalian cell-free mitotic extract. The organization of microtubules into asters depends on 4.1R in that immunodepletion of 4.1R from the extract resulted in randomly dispersed microtubules. Furthermore, adding a 135-kDa recombinant 4.1R reconstituted the mitotic asters. Finally, we demonstrated that a mitotic 4.1R isoform appears to form a complex in vivo with tubulin and NuMA in highly synchronized mitotic HeLa extracts. Our results suggest that a 135-kDa non-erythroid 4.1R is important to cell division, because it participates in the formation of mitotic spindles and spindle poles through its interaction with mitotic microtubules.

Blood Proteins↗

PubMed1999

Role for microtubules in centrosome doubling in Chinese hamster ovary cells.

The centrosome must be replicated once, and only once, during each cell cycle. To achieve this somatic cells need to synthesize centrosome proteins, target those centrosome proteins to the parental centrosome, and then assemble the centrosome subunits into a functional organelle. The mechanisms that underlie each of these processes are not known. Studies were performed to investigate whether cellular microtubules are involved in centrosome doubling events. For these experiments, CHO cells were arrested in either hydroxyurea (HU) alone or in HU plus a microtubule inhibitor for 3640 h. The cells then were induced to enter mitosis and the numbers of spindle poles/centrosomes were counted following processing of the cells for immunofluorescence microscopy using anticentrosome antiserum. These studies demonstrated that centrosome replication events occurred in cells arrested with either HU alone or HU and taxol while centrosome replication did not occur in cells treated with HU and either nocodazole or colcemid. Immunoblot analysis determined that centrosome proteins were synthesized in HU/nocodazole-arrested cells and demonstrated that the role of microtubules in the centrosome replication process is not to ensure the synthesis of centrosome subunits. Rather, our results suggest that microtubules may be involved in the transport/targeting of centrosome subunits to the parental centrosome during duplication events. For microtubules to contribute to the transport of centrosome subunits during centrosome doubling, centrosome subunits would need to be able to bind to microtubules. To test this, co-sedimentation studies were performed and it was determined that the centrosome proteins, though overproduced under these conditions, remained soluble in HU/nocodazole-treated cells and co-pelleted with taxol-stabilized microtubules in the presence of GTP and AMP-PNP. Moreover, co-sedimentation of one of the centrosome proteins, PCM-1, with microtubules could be inhibited by pre-incubation of extracts with antibodies against dynactin. Together, these data suggest that during centrosome replication in somatic mammalian cells, PCM-1, and perhaps other centrosome components, are targeted to the centrosome via transport along microtubules by motor complexes that include dynein/dynactin.

Animals↗

PubMed2005

Sequence and comparative genomic analysis of actin-related proteins.

Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4.

Actins↗

PubMed2005

Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles.

Cytoskeletal dynamics at the Golgi apparatus are regulated in part through a binding interaction between the Golgi-vesicle coat protein, coatomer, and the regulatory GTP-binding protein Cdc42 (Wu, W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000. Nature. 405:800-804; Fucini, R.V., J.L. Chen, C. Sharma, M.M. Kessels, and M. Stamnes. 2002. Mol. Biol. Cell. 13:621-631). The precise role of this complex has not been determined. We have analyzed the protein composition of Golgi-derived coat protomer I (COPI)-coated vesicles after activating or inhibiting signaling through coatomer-bound Cdc42. We show that Cdc42 has profound effects on the recruitment of dynein to COPI vesicles. Cdc42, when bound to coatomer, inhibits dynein binding to COPI vesicles whereas preventing the coatomer-Cdc42 interaction stimulates dynein binding. Dynein recruitment was found to involve actin dynamics and dynactin. Reclustering of nocodazole-dispersed Golgi stacks and microtubule/dynein-dependent ER-to-Golgi transport are both sensitive to disrupting Cdc42 mediated signaling. By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42. We propose a model for how proper temporal regulation of motor-based vesicle translocation could be coupled to the completion of vesicle formation.

Actins↗

PubMed2005

[Huntington's disease: intracellular signaling pathways and neuronal death].

Huntington's disease (HD) is a mid-life onset neurodegenerative disorder characterized by unvoluntary movements (chorea), personality changes and dementia that progress to death within 10-20 years of onset. There are currently no treatment to delay or prevent appearance of the symptoms in the patients. The defective gene in HD contains a trinucleotide CAG repeat expansion within its coding region that is expressed as a polyglutamine (polyQ) repeat in the protein huntingtin. The exact molecular mechanims by which mutant huntingtin induces cell death as well as the function of huntingtin are not totally understood. Studying mechanisms by which polyQ-huntingtin induces neurodegeneration has shown that phosphorylation plays a key role in HD. The IGF-1/Akt/SGK pathway reduces polyQ-huntingtin induced toxicity. This anti-apopototic effect is mediated via the phosphorylation of serine 421 of huntingtin. Moreover, components of this pathway are altered in disease. What is the function of huntingtin? Several studies indicate that huntingtin is an anti-apoptotic protein that could regulate intracellular dynamic. We recently demonstrated, that huntingtin specifically enhances vesicular transport of brain-derived neurotrophic factor (BDNF) along microtubules. Huntingtin-mediated transport involves Huntingtin-Associated Protein-1 (HAP1) and the p150(Glued) subunit of dynactin, an essential component of molecular motors. BDNF transport is attenuated both in the disease context and by reducing the levels of wild-type huntingtin. The alteration of the huntingtin/HAP1/ p150(Glued) complex correlates with reduced association of motor proteins with microtubules. Finally, polyQ-huntingtin-induced transport deficit results in the loss of neurotrophic support and neuronal toxicity.

Animals↗

PubMed2003

Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport.

Bicaudal D is an evolutionarily conserved protein, which is involved in dynein-mediated motility both in Drosophila and in mammals. Here we report that the N-terminal portion of human Bicaudal D2 (BICD2) is capable of inducing microtubule minus end-directed movement independently of the molecular context. This characteristic offers a new tool to exploit the relocalization of different cellular components by using appropriate targeting motifs. Here, we use the BICD2 N-terminal domain as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate the rapid dynein-mediated transport of selected organelles. Surprisingly, unlike other cytoplasmic dynein-mediated processes, this transport shows very low sensitivity to overexpression of the dynactin subunit dynamitin. The dynein-recruiting activity of the BICD2 N-terminal domain is reduced within the full-length molecule, indicating that the C-terminal part of the protein might regulate the interaction between BICD2 and the motor complex. Our findings provide a novel model system for dissection of the molecular mechanism of dynein motility.

Drosophila Proteins↗

PubMed2001

African swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein.

Dynein is a minus-end-directed microtubule-associated motor protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus protein p54 to the light chain of cytoplasmic dynein (LC8). Interaction of p54 and LC8 occurs both in vitro and in cells, and the two proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of p54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubule-mediated virus transport.

African Swine Fever Virus↗

PubMed2004

Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules.

Polyglutamine expansion (polyQ) in the protein huntingtin is pathogenic and responsible for the neuronal toxicity associated with Huntington's disease (HD). Although wild-type huntingtin possesses antiapoptotic properties, the relationship between the neuroprotective functions of huntingtin and pathogenesis of HD remains unclear. Here, we show that huntingtin specifically enhances vesicular transport of brain-derived neurotrophic factor (BDNF) along microtubules. Huntingtin-mediated transport involves huntingtin-associated protein-1 (HAP1) and the p150(Glued) subunit of dynactin, an essential component of molecular motors. BDNF transport is attenuated both in the disease context and by reducing the levels of wild-type huntingtin. The alteration of the huntingtin/HAP1/p150(Glued) complex correlates with reduced association of motor proteins with microtubules. Finally, we find that the polyQ-huntingtin-induced transport deficit results in the loss of neurotrophic support and neuronal toxicity. Our findings indicate that a key role of huntingtin is to promote BDNF transport and suggest that loss of this function might contribute to pathogenesis.

Animals↗