Dynactin is required for microtubule anchoring at centrosomes.

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.     

Nudel contributes to microtubule anchoring at the mother centriole and is involved in both dynein-dependent and -independent centrosomal protein assembly

The centrosome is the major microtubule-organizing center in animal cells. Although the cytoplasmic dynein regulator Nudel interacts with centrosomes, its role herein remains unclear. Here, we show that in Cos7 cells Nudel is a mother centriole protein with rapid turnover independent of dynein activity. During centriole duplication, Nudel targets to the new mother centriole later than ninein but earlier than dynactin. Its centrosome localization requires a C-terminal region that is essential for associations with dynein, dynactin, pericentriolar material (PCM)-1, pericentrin, and gamma-tubulin. Overexpression of a mutant Nudel lacking this region, a treatment previously shown to inactivate dynein, dislocates centrosomal Lis1, dynactin, and PCM-1, with little influence on pericentrin and gamma-tubulin in Cos7 and HeLa cells. Silencing Nudel in HeLa cells markedly decreases centrosomal targeting of all the aforementioned proteins. Silencing Nudel also represses centrosomal MT nucleation and anchoring. Furthermore, Nudel can interact with pericentrin independently of dynein. Our current results suggest that Nudel plays a role in both dynein-mediated centripetal transport of dynactin, Lis1, and PCM-1 as well as in dynein-independent centrosomal targeting of pericentrin and gamma-tubulin. Moreover, Nudel seems to tether dynactin and dynein to the mother centriole for MT anchoring.     

Distinct cell cycle-dependent roles for dynactin and dynein at centrosomes

Centrosomal dynactin is required for normal microtubule anchoring and/or focusing independently of dynein. Dynactin is present at centrosomes throughout interphase, but dynein accumulates only during S and G2 phases. Blocking dynein-based motility prevents recruitment of dynactin and dynein to centrosomes and destabilizes both centrosomes and the microtubule array, interfering with cell cycle progression during mitosis. Destabilization of the centrosomal pool of dynactin does not inhibit dynein-based motility or dynein recruitment to centrosomes, but instead causes abnormal G1 centriole separation and delayed entry into S phase. The correct balance of centrosome-associated dynactin subunits is apparently important for satisfaction of the cell cycle mechanism that monitors centrosome integrity before centrosome duplication and ultimately governs the G1 to S transition. Our results suggest that, in addition to functioning as a microtubule anchor, dynactin contributes to the recruitment of important cell cycle regulators to centrosomes.     

Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo.

We have investigated the role of cytoplasmic dynein in microtubule organizing center (MTOC) positioning using RNA-mediated interference (RNAi) in Caenorhabditis elegans to deplete the product of the dynein heavy chain gene dhc-1. Analysis with time-lapse differential interference contrast microscopy and indirect immunofluorescence revealed that pronuclear migration and centrosome separation failed in one cell stage dhc-1 (RNAi) embryos. These phenotypes were also observed when the dynactin components p50/dynamitin or p150(Glued) were depleted with RNAi. Moreover, in 15% of dhc-1 (RNAi) embryos, centrosomes failed to remain in proximity of the male pronucleus. When dynein heavy chain function was diminished only partially with RNAi, centrosome separation took place, but orientation of the mitotic spindle was defective. Therefore, cytoplasmic dynein is required for multiple aspects of MTOC positioning in the one cell stage C. elegans embryo. In conjunction with our observation of cytoplasmic dynein distribution at the periphery of nuclei, these results lead us to propose a mechanism in which cytoplasmic dynein anchored on the nucleus drives centrosome separation.     

Dynein and dynactin as organizers of the system of cell microtubules

A review of the role of the microtubule motor dynein and its cofactor dynactin in the formation of a radial system of microtubules in the interphase cells and of mitotic spindle. Deciphering of the structure, functions, and regulation of activity of dynein and dynactin promoted the understanding of mechanisms of cell and tissue morphogenesis, since it turned out that these cells help the cell in finding its center and organize microtubule-determined anisotropy of intracellular space. The structure of dynein and dynactin molecules has been considered, as well as possible pathways of regulation of the dynein activity and the role of dynein in transport of cell components along the microtubules. Attention has also been paid to the functions of dynein and dynactin not related directly to transport: their involvement in the formation of an interphase radial system of microtubules. This system can be formed by self-organization of microtubules and dynein-containing organelles or via organization of microtubules by the centrosome, whose functioning requires dynein. In addition, dynein and dynactin are responsible for cell polarization during its movement, as well as for the position of nucleus, centrosomes, and mitotic spindle in the cell.     

Excess centrosomes disrupt endothelial cell migration via centrosome scattering

Supernumerary centrosomes contribute to spindle defects and aneuploidy at mitosis, but the effects of excess centrosomes during interphase are poorly understood. In this paper, we show that interphase endothelial cells with even one extra centrosome exhibit a cascade of defects, resulting in disrupted cell migration and abnormal blood vessel sprouting. Endothelial cells with supernumerary centrosomes had increased centrosome scattering and reduced microtubule (MT) nucleation capacity that correlated with decreased Golgi integrity and randomized vesicle trafficking, and ablation of excess centrosomes partially rescued these parameters. Mechanistically, tumor endothelial cells with supernumerary centrosomes had less centrosome-localized γ-tubulin, and Plk1 blockade prevented MT growth, whereas overexpression rescued centrosome γ-tubulin levels and centrosome dynamics. These data support a model whereby centrosome–MT interactions during interphase are important for centrosome clustering and cell polarity and further suggest that disruption of interphase cell behavior by supernumerary centrosomes contributes to pathology independent of mitotic effects.     

Dynein and dynactin as organizers of the system of cell microtubules

A review of the role of the microtubule motor dynein and its cofactor dynactin in the formation of a radial system of microtubules in the interphase cells and of mitotic spindle. Deciphering of the structure, functions, and regulation of activity of dynein and dynactin promoted the understanding of mechanisms of cell and tissue morphogenesis, since it turned out that these cells help the cell in finding its center and organize microtubule-determined anisotropy of intracellular space. The structure of dynein and dynactin molecules has been considered, as well as possible pathways of regulation of the dynein activity and the role of dynein in transport of cell components along the microtubules. Attention has also been paid to the functions of dynein and dynactin not related directly to transport: their involvement in the formation of an interphase radial system of microtubules. This system can be formed by self-organization of microtubules and dynein-containing organelles or via organization of microtubules by the centrosome, whose functioning requires dynein. In addition, dynein and dynactin are responsible for cell polarization during its movement, as well as for the position of nucleus, centrosomes, and mitotic spindle in the cell.     

A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization

We present evidence that vimentin intermediate filament (IF) motility in vivo is associated with cytoplasmic dynein. Immunofluorescence reveals that subunits of dynein and dynactin are associated with all structural forms of vimentin in baby hamster kidney-21 cells. This relationship is also supported by the presence of numerous components of dynein and dynactin in IF-enriched cytoskeletal preparations. Overexpression of dynamitin biases IF motility toward the cell surface, leading to a perinuclear clearance of IFs and their redistribution to the cell surface. IF-enriched cytoskeletal preparations from dynamitin-overexpressing cells contain decreased amounts of dynein, actin-related protein-1, and p150Glued relative to controls. In contrast, the amount of dynamitin is unaltered in these preparations, indicating that it is involved in linking vimentin cargo to dynactin. The results demonstrate that dynein and dynactin are required for the normal organization of vimentin IF networks in vivo. These results together with those of previous studies also suggest that a balance among the microtubule (MT) minus and plus end-directed motors, cytoplasmic dynein, and kinesin are required for the assembly and maintenance of type III IF networks in interphase cells. Furthermore, these motors are to a large extent responsible for the long recognized relationships between vimentin IFs and MTs.     

Lamins position the nuclear pores and centrosomes by modulating dynein

Lamins, the type V nuclear intermediate filament proteins, are reported to function in both interphase and mitosis. For example, lamin deletion in various cell types can lead to an uneven distribution of the nuclear pore complexes (NPCs) in the interphase nuclear envelope, whereas deletion of B-type lamins results in spindle orientation defects in mitotic neural progenitor cells. How lamins regulate these functions is unknown. Using mouse cells deleted of different combinations or all lamins, we show that lamins are required to prevent the aggregation of NPCs in the nuclear envelope near centrosomes in late G2 and prophase. This asymmetric NPC distribution in the absence of lamins is caused by dynein forces acting on NPCs via the dynein adaptor BICD2. We further show that asymmetric NPC distribution upon lamin depletion disrupts the distribution of BICD2 and p150 dynactin on the nuclear envelope at prophase, which results in inefficient dynein-driven centrosome separation during prophase. Therefore lamins regulate microtubule-based motor forces in vivo to ensure proper NPC distribution in interphase and centrosome separation in the mitotic prophase.     

Analysis of the dynein-dynactin interaction in vitro and in vivo

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells

Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150^Glued subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.     

The dynactin complex maintains the integrity of metaphasic centrosomes to ensure transition to anaphase

The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, we used RNA interference to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, we observed cells with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. We attribute the figure eight chromosome alignment to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. Our findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase.     

Amphiastral mitotic spindle assembly in vertebrate cells lacking centrosomes

The role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays--termed an amphiaster ("a star on both sides")--that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB). Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP--the small GTPase Ran in its GTP bound state--could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.     

Dynein motor regulation stabilizes interphase microtubule arrays and determines centrosome position

Cytoplasmic dynein is a microtubule-based motor protein responsible for vesicle movement and spindle orientation in eukaryotic cells. We show here that dynein also supports microtubule architecture and determines centrosome position in interphase cells. Overexpression of the motor domain in Dictyostelium leads to a collapse of the interphase microtubule array, forming loose bundles that often enwrap the nucleus. Using green fluorescent protein (GFP)-alpha-tubulin to visualize microtubules in live cells, we show that the collapsed arrays remain associated with centrosomes and are highly motile, often circulating along the inner surface of the cell cortex. This is strikingly different from wild-type cells where centrosome movement is constrained by a balance of tension on the microtubule array. Centrosome motility involves force-generating microtubule interactions at the cortex, with the rate and direction consistent with a dynein-mediated mechanism. Mapping the overexpression effect to a C-terminal region of the heavy chain highlights a functional domain within the massive sequence important for regulating motor activity.     

Geminin is partially localized to the centrosome and plays a role in proper centrosome duplication

Background information. Centrosome duplication normally parallels with DNA replication and is responsible for correct segregation of replicated DNA into the daughter cells. Although geminin interacts with Cdt1 to prevent loading of MCMs (minichromosome maintenance proteins) on to the replication origins, inactivation of geminin nevertheless causes centrosome over‐duplication in addition to the re‐replication of the genome, suggesting that geminin may play a role in centrosome duplication. However, the exact mechanism by which loss of geminin affects centrosomal duplication remains unclear and the possible direct interaction of geminin with centrosomal‐localized proteins is still unidentified. Results. We report in the present study that geminin is physically localized to the centrosome. This unexpected geminin localization is cell‐cycle dependent and mediated by the actin‐related protein, Arp1, one subunit of the dynein—dynactin complex. Disruption of the integrity of the dynein—dynactin complex by overexpression of dynamitin/p50, a well‐characterized inhibitor of dynactin, reduces the centrosomal localization of both geminin and Arp1. Enrichment of geminin on centrosomes was enhanced when cellular ATP production was suppressed in the ATP‐inhibitor assay, whereas the accumulation of geminin on the centrosome was disrupted by depolymerization of the microtubules using nocodazole. We further demonstrate that the coiled‐coil motif of geminin is required for its centrosomal localization and the interaction of geminin with Arp1. Depletion of geminin by siRNA (small interfering RNA) in MDA‐MB‐231 cells led to centrosome over‐duplication. Conversely, overexpression of geminin inhibits centrosome over‐duplication induced by HU in S‐phase‐arrested cells, and the coiled‐coil‐motif‐mediated centrosomal localization of geminin is required for its inhibition of centrosome over‐duplication. Centrosomal localization of geminin is conserved among mammalian cells and geminin might perform as an inhibitor of centrosome duplication. Conclusions. The results of the present study demonstrate that a fraction of geminin is localized on the centrosome, and the centrosomal localization of geminin is Arp1‐mediated and dynein—dynactin‐dependent. The coiled‐coil motif of geminin is required for its targeting to the centrosome and inhibition of centrosome duplication. Thus the centrosomal localization of geminin might perform an important role in regulation of proper centrosome duplication.     

Cytoplasmic dynein is required for the nuclear attachment and migration of centrosomes during mitosis in Drosophila.

Cytoplasmic dynein is a multisubunit minus-end-directed microtubule motor that serves multiple cellular functions. Genetic studies in Drosophila and mouse have demonstrated that dynein function is essential in metazoan organisms. However, whether the essential function of dynein reflects a mitotic requirement, and what specific mitotic tasks require dynein remains controversial. Drosophila is an excellent genetic system in which to analyze dynein function in mitosis, providing excellent cytology in embryonic and somatic cells. We have used previously characterized recessive lethal mutations in the dynein heavy chain gene, Dhc64C, to reveal the contributions of the dynein motor to mitotic centrosome behavior in the syncytial embryo. Embryos lacking wild-type cytoplasmic dynein heavy chain were analyzed by in vivo analysis of rhodamine-labeled microtubules, as well as by immunofluorescence in situ methods. Comparisons between wild-type and Dhc64C mutant embryos reveal that dynein function is required for the attachment and migration of centrosomes along the nuclear envelope during interphase/prophase, and to maintain the attachment of centrosomes to mitotic spindle poles. The disruption of these centrosome attachments in mutant embryos reveals a critical role for dynein function and centrosome positioning in the spatial organization of the syncytial cytoplasm of the developing embryo.     

Cytoplasmic dynein is involved in the retention of microtubules at the centrosome in interphase cells

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid ( t _1/2 ∼ 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein-inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT-anchoring proteins.     

Centrosome Function: Sometimes Less Is More

Tight regulation of centrosome duplication is critical to ensure that centrosome number doubles once and only once per cell cycle. Superimposed onto this centrosome duplication cycle is a functional centrosome cycle in which they alternate between phases of quiescence and robust microtubule (MT) nucleation and MT-anchoring activities. In vertebrate cycling cells, interphase centrioles accumulate less pericentriolar material (PCM), reducing their MT nucleation capacity. In mitosis, centrosomes mature, accumulating more PCM to increase their nucleation and anchoring capacities to form robust MT asters. Interestingly, functional cycles of centrosomes can be altered to suit the cell's needs. Some interphase centrosomes function as a microtubule-organizing center by increasing their ability to anchor MTs to form centrosomal radial arrays. Other interphase centrosomes maintain their MT nucleation capacity but reduce/eliminate their MT-anchoring capacity. Recent work demonstrates that Drosophila cells take this to the extreme, whereby centrioles lose all detectable PCM during interphase, offering an explanation as to how centrosome-deficient flies develop to adulthood. Drosophila stem cells further modify the functional cycle by differentially regulating their two centrioles – a situation that seems important for stem cell asymmetric divisions, as misregulation of centrosome duplication in stem/progenitor cells can promote tumor formation. Here, we review recent findings that describe variations in the functional cycle of centrosomes.     

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.