Article |
Address correspondence to Trina A. Schroer, Department of Biology/220A Mudd Hall, Johns Hopkins University, Charles and 34th Streets, Baltimore, MD 21218. Tel.: (410) 516-5373. Fax: (410) 516-5375. E-mail: schroer{at}jhu.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: dynactin; dynein; microtubule; cell cycle; centrosome
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The precise mechanisms by which microtubules remain focused and anchored at centrosomes in fibroblasts, and how this organization becomes altered in nonfibroblastic cells, are still being defined. In G1 cells that contain only one centriole pair, microtubule-anchoring activity appears to be predominantly associated with the older of the two centrioles (designated the mother centriole; Piel et al., 2000). A number of proteins, including the proposed microtubule-anchoring protein, ninein, are selectively bound to the mother centriole (for review see Doxsey, 2001). We found previously that dynactin was necessary for maintenance of the normal radial microtubule array (Quintyne et al., 1999). Dynactin is concentrated at centrosomes (Gill et al., 1991; Clark and Meyer, 1992; Paschal et al., 1993; Dictenberg et al., 1998), but it is not known with which centriole it associates.
Dynactin is best characterized as an "activator" of the minus enddirected microtubule motor, cytoplasmic dynein (Gill et al., 1991; Schroer and Sheetz, 1991). Dynactin facilitates dynein-based movement by acting as both a processivity factor (King and Schroer, 2000) and an adaptor that mediates dynein binding to subcellular cargoes and the cell cortex (for reviews see Karki and Holzbaur, 1999; Allan, 2000; Dujardin and Vallee, 2002). This dual function takes advantage of dynactin's bipartite structure. A projecting p150Glued sidearm binds both microtubules and dynein (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995; Waterman-Storer et al., 1995; Quintyne et al., 1999; Vaughan et al., 2001), whereas a backbone element comprised mostly of the actin-related protein Arp1 is thought to bind cargo (Schafer et al., 1994; for review see Allan, 2000; Muresan et al., 2001).
Cytoplasmic dynein and dynactin are found on endomembranes (Roghi and Allan, 1999; Habermann et al., 2001), the cell cortex (Dujardin and Vallee, 2002), and kinetochores and mitotic spindle poles (Pfarr et al., 1990; Steuer et al., 1990; Echeverri et al., 1996). The importance of the dyneindynactin motor in microtubule minus end focusing at spindle poles (Compton, 2000; Heald, 2000) suggested that these proteins might provide a similar function at centrosomes during interphase. In keeping with this hypothesis, overexpression of a series of dominant negative inhibitors that interfered with dynein and dynactin function in distinct ways resulted in disorganization of fibroblastic microtubule arrays (Quintyne et al., 1999). All of the inhibitors prevented proper targeting of dynein to cargo, but none altered dynactincargo binding or, presumably, the ability of dynein itself to move on microtubules.
Dynamitin had the broadest effect on cellular architecture. Dynamitin disrupts the endogenous pool of cellular dynactin, yielding a "free" pool of p150Glued that can still bind dynein but not cargo. In addition to its expected effects on dynactin structure and the Golgi complex (Echeverri et al., 1996; Burkhardt et al., 1997), dynamitin overexpression causes defocusing of the radial microtubule array and a redistribution of the pericentriolar proteins -tubulin and dynactin. Full-length p150Glued or a dynein-binding fragment, p150217548, have no effect on endogenous dynactin structure, but act as competitive inhibitors of the dyneindynactin interaction by binding dynein and preventing it from binding dynactin and cargo. Because these three inhibitors all interfere with dyneincargo targeting, they have similar effects on endomembrane, microtubule, and centrosome organization (Quintyne et al., 1999).
Two other inhibitors, p24 and a second p150Glued fragment, p1509261049, are significantly more selective in their effects. Neither perturbs cytosolic or membrane-associated dynactin, dyneindynactin binding, or dynein targeting, as endomembrane localization, motility, and dynactin structural integrity are unaffected. These inhibitors appear to disrupt only the centrosomal pool of dynactin, causing the loss of p150Glued from Arp1, which results in microtubule disorganization and compromised centrosome integrity (Quintyne et al., 1999). This suggested that centrosomal p150Glued was the dynactin subunit most important for microtubule anchoring and/or focusing during interphase. However, it was not clear whether p150Glued was acting directly by anchoring microtubules or indirectly by binding dynein, which could then focus microtubules.
Like the cell's genome, the centrosome must reproduce once per cell cycle. Centrosome doubling involves centriole pair splitting or disorientation during G1, centriole duplication during S, and the complete separation of the two centriole pairs to yield spindle poles at the onset of mitosis (for reviews see Doxsey, 2001; Hinchcliffe and Sluder, 2001). In parallel with centriole duplication, the pericentriolar material (PCM)* becomes amplified. Some PCM components, such as pericentrin, -tubulin, and PCM-1, are recruited to the centrosome in a microtubule- and dyneindynactin-dependent manner (for review see Zimmerman and Doxsey, 2000). A variety of other proteins, many of them regulatory kinases, are selectively recruited to the centrosome at particular stages of the cell cycle (for review see Lange, 2002), possibly via microtubule-based transport as well. The activities of such kinases and phosphatases are proposed to underlie the transition from G1 to S and exit from cytokinesis, both of which require centrosomes (Hinchcliffe et al., 2001; Khodjakov and Rieder, 2001; Piel et al., 2001).
In our original analysis of centrosomal dynactin function we noted that centrosomes stained differentially for dynactin and dynein; most exhibited dynactin, but only some dynein. The present study is aimed at gaining a better understanding of the roles of centrosomal dynactin and dynein, specifically with respect to interphase microtubule organization and cell cycle progression. We find that dynactin is concentrated at centrosomes throughout interphase, but that dynein is detected only during S and G2. Thus, maintenance of the G1 microtubule array appears not to require centrosomally accumulated dynein. Dynactin is associated preferentially with the mother centriole in G1 cells, providing further support for its proposed role as a microtubule anchor. The functions of centrosomal dynactin and dynein were probed further by dynactin subunit overexpression. As expected, based on their inhibitory effects on centrosomal dynactin, overexpression of certain inhibitors prevented dynein recruitment but did not affect cell cycle progression until mitosis. Inhibitors that cause just p150Glued to be lost from centrosomes did not block dynein accumulation, suggesting a novel mechanism for dynein recruitment. Surprisingly, these inhibitors caused abnormal centriole splitting in G1 and delayed entry into S phase. Our findings suggest that the integrity of centrosomal dynactin contributes to proper centriole pairing and timely entry into S phase, and provide further evidence that S phase entry is regulated by centrosome-dependent events.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Cells overexpressing dynactin shoulder/sidearm subunits show relatively normal patterns of initial microtubule nucleation and PCM recruitment, but centrosomes and the microtubule array disintegrate over time (Quintyne et al., 1999). Given the unexpected behavior of dynein in cells overexpressing the latter two inhibitors, we thought it would be informative to examine centrosomal recruitment of dynactin and dynein under conditions of initial microtubule growth (Fig. 3). Cells whose microtubules have been depolymerized by nocodazole and cold no longer exhibit centrosomal dynactin (Paschal et al., 1993; Quintyne et al., 1999; Fig. 3, 0 min time points), but both dynactin and dynein reaccumulate after nocodazole washout (Fig. 3 A) with kinetics similar to microtubule regrowth (Quintyne et al., 1999). Overexpression of dynamitin or p150217548 completely prevented accumulation of dynactin and dynein at centrosomes (Fig. 3 B). This result was dramatic but expected. Because dynamitin and p150217548 are thought to block dyneindynactin binding, they will prevent dynein-based transport of dynactin to the centrosome. Dynein is not expected to bind centrosomes that lack dynactin.
|
Effects of the loss of centrosomal dynactin and dynein on progression through S, G2, and M phases
Dynein accumulates at centrosomes during S and G2 phases and is highly enriched at mitotic spindle poles, suggesting that it is recruited in preparation for mitosis. Inhibition of dyneindynactin function profoundly affects spindle formation and pole maintenance in many systems (for reviews see Compton, 1998; Heald, 2000). Dynamitin overexpression causes cells to arrest in pseudoprometaphase with fragmented or monopolar spindles (Echeverri et al., 1996; Dujardin et al., 1998), consistent with our observation that such cells lack centrosomal dynein and the consequent loss of dynein focusing activity from spindle poles. p150217548 overexpression has very similar effects to dynamitin on microtubule, centrosome, and Golgi organization, so it seemed likely that it would also interfere with mitotic progression. When we examined mitotic index and spindle morphology in unsynchronized cells overexpressing p150217548, we noted an increased percentage of mitotic cells (Table I) with malformed spindles, as expected.
Dynein recruitment to centrosomes slightly precedes centriole duplication (Fig. 1), suggesting that dynein function might also contribute to centrosome doubling or another late cell cycle event. To address this question we evaluated the effects of dyneindynactin inhibitors on late cell cycle progression. Cells were synchronized at the G1S boundary by double thymidine block, microinjected with dynamitin or p150217548 cDNAs, and then released from the block (Fig. 4). Neither DNA synthesis, as assessed by BrdU incorporation, nor centriole duplication, as determined by centrin staining, was affected.
|
Loss of centrosomal p150Glued inhibits S phase entry and induces G1 centriole splitting
Overexpression of dynactin shoulder/sidearm subunits profoundly destabilizes centrosomes (Quintyne et al., 1999), but progression through S and G2 appears unaffected (Fig. 4). This is not necessarily surprising, because the centrosome-associated surveillance mechanism that governs S phase entry may already be satisfied in cells synchronized at the G1S boundary. To determine how the loss of centrosomal dynactin might impact this mechanism, we evaluated cell cycle progression from G1 into S (Fig. 5). The proportion of unsynchronized cells in G1 versus later in the cell cycle was determined by examining BrdU incorporation, centrin staining, PCNA (a protein that accumulates in S phase nuclei; Bravo and Celis, 1985), and the protein kinases Nek2 and IAK-1, two potential regulators of cell cycle progression that accumulate at centrosomes in S phase (Schultz et al., 1994; Gopalan et al., 1997). Approximately 30% of unsynchronized control cells revealed no BrdU incorporation or nuclear PCNA, indicating that they were still in G1. 5060% of cells showed no evidence of centriole duplication (two centrin foci or no centrosomal Nek2 or IAK-1). When we repeated this analysis in cells overexpressing dynactin subunits, dynamitin and p150217548 were seen to have no effect. However, significantly more cells overexpressing p1509261049 or p24 appeared to be G1, as judged by the behavior of PCNA and the three centriole markers. p1509261049 had a particularly potent effect on centrosomal Nek2 recruitment; <20% of overexpressing cells stained for this marker compared with 50% of controls. These results were strongly suggestive of a G1S delay in these cells.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Targeting of dynein to centrosomes
The dyneindynactin motor is of critical importance for mitotic spindle pole stability (for review see Compton, 2000), but the lack of dynein accumulation at centrosomes during G1 and early S suggests that its microtubule focusing activity is not required across the cell cycle. A number of structural and regulatory proteins, some of which are required for the G2 to M transition or early mitotic events, are recruited to centrosomes during S and G2 phases. Given its importance in mitosis, it comes as no surprise that dynein also binds centrosomes in a cell cycledependent manner. This may involve modification of dynein, dynactin, or some other component of the PCM.
When analyzed at steady-state, cells overexpressing some dynactin inhibitors can target dynein to centrosomes despite the absence of centrosomal p150Glued (Table I), but do so more slowly (Fig. 3), suggesting a different mechanism. The "slow" mode of binding may involve pericentrin, a centrosomal protein that can bind dynein directly (Purohit et al., 1999; Tynan et al., 2000). Any pericentrin-dependent binding mechanism must be complex because centrosomal dynein is not observed in cells overexpressing other dynactin inhibitors whose centrosomes contain pericentrin (Quintyne et al., 1999). For example, dynein binding might utilize pericentrin that is recently trafficked to centrosomes via the dyneindynactin motor itself (Young et al., 2000). In any case, our data indicate that dynactin provides the primary mechanism by which dynein associates with centrosomes under normal circumstances.
Centrosomal dynactin function
Our findings suggest that centrosomal dynactin plays important roles in microtubule anchoring, dynein binding, and recruitment and maintenance of cell cycle regulators. That dynein cannot be detected at centrosomes during G1 strongly suggests that p150Glued anchors microtubules directly. Aside from dynactin, few candidate microtubule anchors exist (for review see Bornens, 2002). The -TuRC can nucleate and cap microtubule minus ends but is not thought to serve as an anchor (Doxsey, 2001). Other proteins that are selectively associated with the mother centriole include ninein (Mogensen et al., 2000), ODF2/cenexin (Nakagawa et al., 2001), and
-tubulin (Chang and Stearns, 2000). Ninein is a large coiled-coil protein that lacks defined microtubule binding motifs (Bouckson-Castaing et al., 1996). Although the existing data support our hypothesis that p150Glued provides a key microtubule-anchoring activity at centrosomes, it is possible that dynactin is just one component of a microtubule-anchoring complex or matrix that contains other structural and/or regulatory components. Overexpression of the dynactin inhibitors used here would interfere with the recruitment of any protein that is targeted to centrosomes via p150Glued, so the exact nature of the anchoring mechanism remains an open question.
Centrosome duplication involves amplification of the PCM, a process that depends on dyneindynactin-dependent transport (for review see Zimmerman and Doxsey, 2000). Overexpression of inhibitors of the dyneindynactin interaction would be predicted to interfere with the centrosome cycle but, remarkably, they have no effect until mitosis. Even more surprising is the fact that dynactin inhibitors that have no measurable effect on dynein-based motility (Quintyne et al., 1999) somehow delay S phase entry. Overexpression of p24 also drives cells into apoptosis just before mitosis. This may reflect a normal biological function of p24, but is more likely an artifact of overexpression.
Centrosomal dynactin, centriole duplication, and S phase entry
The daughter centriole in some cells moves independently of the mother in G1 but the two become linked during S and G2 (Piel et al., 2000), demonstrating that formation of a single, coherent centrosomal unit correlates with centriole duplication. Our observations suggest that centriole coupling is required for centriole duplication and S phase entry. That the centrosome must behave as a single copy organelle during duplication is an appealing notion, as this would allow concerted and efficient recruitment of PCM components and ensure that centrosome-associated signaling molecules (for review see Lange, 2002) become equally apportioned via the spindle poles into the two daughter cells.
Insight into how overexpression of different dynactin subunits might cause such distinct effects on the cell cycle can be gained by considering how each class of inhibitor affects centrosome structure and dynamics (Fig. 7; Quintyne et al., 1999). In control cells, microtubules are anchored by dynactin, centrosome components are transported to the centrosome via dynein as usual, and G1S progression occurs normally. When dyneindynactin binding is blocked, microtubule nucleation persists but microtubules are no longer retained. Dynein-based trafficking of other centrosomal components is prevented, although some PCM proteins may reach their target in other ways. Despite this, the centriole coupling mechanism is maintained, centriole duplication proceeds, and cells enter S phase at the expected time. In cells overexpressing p1509261049 or p24, the story is more complex. Centrosomes nucleate microtubules and dynein-based movement of dynactin and other centrosome components continues on the newly assembled pool (Fig. 3; Quintyne et al., 1999). However, p150Glued, microtubules, and any associated proteins are gradually released, leaving behind the naked Arp1 filament and residual PCM. This may result in an inappropriate balance of centriole cohesion factors, centriole splitting factors, G1 stabilizers, and/or S phase activators.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression constructs
To make DsRed-p150217548, a fragment containing p150217548 was amplified from CMV-p150 (Quintyne et al., 1999) by PCR, inserted directly into the pTA vector, and then subcloned into pDsRed-N1 (CLONTECH Laboratories, Inc.) using EcoRI. p1509261049GFP was made by subcloning the region encoding p1509261049 from CMV-p1509261049 (Quintyne et al., 1999), using BglII and EcoRI, into pEGFP-C1 (CLONTECH Laboratories, Inc.). The clones were transiently transfected into Cos-7 cells to confirm that the effects of overexpression of these constructs were identical to those previously reported with untagged p150217548 and p1509261049 (Quintyne et al., 1999) by scoring for microtubule disruption, Golgi dispersal, -tubulin fragmentation, and centrosomal dynactin. cDNAs encoding p24GFP and dynamitinGFP were described previously (Quintyne et al., 1999).
Cell culture, transfection, and microinjection
COS-7 cells were grown in DME (GIBCO BRL; Life Technologies) supplemented with 10% FCS (Atlas). For transient transfections, cells were electroporated and seeded as previously described (Quintyne et al., 1999). For microinjection, cells were seeded onto gridded 18 x 18 mm2 coverslips (Bellco) and either grown overnight or synchronized as described below. Dynactin subunit expression vector cDNAs (0.1 mg/ml in buffer containing 2 mM KH2PO4, 8 mM K2PO4, and 100 mM KCl) were injected into nuclei using an Eppendorf micromanipulator. Cells were incubated at 37°C for 424 h before being fixed and processed for immunofluorescence. Overexpression could be detected by GFP fluorescence after 2 h, and the characteristic effects of overexpression on Golgi complex morphology could be detected as early as 4.5 h after injection. For cell cycle experiments, cells were either injected 25 h before release from thymidine block (Fig. 4), or between 14 and 18 h after release (Fig. 6).
Immunofluorescence microscopy
Immunofluorescence was performed as previously described (Quintyne et al., 1999). In brief, cells were fixed for 5 min in -20°C methanol, treated with blocking solution, treated with primary antibodies, washed, and then treated with secondary antibodies and DAPI. Samples were scored using an Axiovert 35 microscope (ZEISS). For experiments involving electroporated or synchronized cells, at least 200 overexpressing (or control) cells were scored per construct per experiment or time point, and each experiment was repeated at least twice. For experiments involving microinjected cells, 5070 cells were scored per construct per time point, and each experiment was repeated at least twice. Stacks for deconvolution were acquired and processed using a DeltaVision deconvolving microscope system (Applied Precision). All images were imported into Adobe Photoshop® (Adobe Systems) as TIFFs for contrast manipulation and figure assembly.
Microtubule regrowth assay
Microtubule regrowth assays were performed as previously described (Quintyne et al., 1999). In brief, transfected cells were seeded on coverslips, grown overnight, and treated with 33 µM nocodazole (Sigma-Aldrich) on ice for 25 min to depolymerize microtubules. Cells were washed with nocodazole-free medium, refed, and incubated at room temperature for varying times before being fixed and processed for immunofluorescence.
Cell synchronization and release
Cells were seeded onto coverslips at an initial density of 1.5 x 107 cells per 10-cm dish and grown overnight. A double thymidine block was performed by treating cells with fresh DME containing 2 mM thymidine (Sigma-Aldrich) for 12 h, releasing for 12 h in normal medium, and then incubating them again in 2 mM thymidine for 1214 h. Essentially, all cells were synchronized at the G1S boundary, as determined by the presence of two centrin foci, before release from the block. For BrdU incorporation, cells were incubated in DME + 10 µM BrdU (BD Biosciences) at 37°C for 3 h before fixing and processing for immunofluorescence as described above.
![]() |
Footnotes |
---|
* Abbreviations used in this paper: CNAP-1, centrosomal Nek2-associated protein-1; PAR, poly-ADP-ribose; PCM, pericentriolar material.
![]() |
Acknowledgments |
---|
This work was supported by National Institutes of Health grants GM44589 and DK44375 to T.A. Schroer.
Submitted: 19 March 2002
Revised: 13 September 2002
Accepted: 17 September 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bornens, M. 2002. Centrosome composition and microtubule anchoring mechanisms. Curr. Opin. Cell Biol. 14:2534.[CrossRef][Medline]
Bouckson-Castaing, V., M. Moudjou, D.J. Ferguson, S. Mucklow, Y. Belkaid, G. Milon, and P.R. Crocker. 1996. Molecular characterisation of ninein, a new coiled-coil protein of the centrosome. J. Cell Sci. 109:179190.
Burkhardt, J.K., C.J. Echeverri, T. Nilsson, and R.B. Vallee. 1997. Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution. J. Cell Biol. 139:469484.
Clark, S.W., and D.I. Meyer. 1992. Centractin is an actin homologue associated with the centrosome. Nature. 359:246250.[CrossRef][Medline]
Compton, D.A. 1998. Focusing on spindle poles. J. Cell Sci. 111:14771481.
Dictenberg, J.B., W. Zimmerman, C.A. Sparks, A. Young, C. Vidair, Y. Zheng, W. Carrington, F.S. Fay, and S.J. Doxsey. 1998. Pericentrin and -tubulin form a protein complex and are organized into a novel lattice at the centrosome. J. Cell Biol. 141:163174.
Dujardin, D., U.I. Wacker, A. Moreau, T.A. Schroer, J.E. Rickard, and J.R. De Mey. 1998. Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment. J. Cell Biol. 141:849862.
Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. Molecular characterization of 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J. Cell Biol. 132:617633.[Abstract]
Fry, A.M., T. Mayor, P. Meraldi, Y.D. Stierhof, K. Tanaka, and E.A. Nigg. 1998. C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycleregulated protein kinase Nek2. J. Cell Biol. 141:15631574.
Gill, S.R., T.A. Schroer, I. Szilak, E.R. Steuer, M.P. Sheetz, and D.W. Cleveland. 1991. Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein. J. Cell Biol. 115:16391650.[Abstract]
Gill, S.R., D.W. Cleveland, and T.A. Schroer. 1994. Characterization of DLC-A and DLC-B, two families of cytoplasmic dynein light chain subunits. Mol. Biol. Cell. 5:645654.[Abstract]
Gopalan, G., C.S. Chan, and P.J. Donovan. 1997. A novel mammalian, mitotic spindle-associated kinase is related to yeast and fly chromosome segregation regulators. J. Cell Biol. 138:643656.
Habermann, A., T.A. Schroer, G. Griffiths, and J.K. Burkhardt. 2001. Immunolocalization of cytoplasmic dynein and dynactin subunits in cultured macrophages: enrichment on early endocytic organelles. J. Cell Sci. 114:229240.
Helps, N.R., X. Luo, H.M. Barker, and P.T. Cohen. 2000. NIMA-related kinase 2 (Nek2), a cell-cycle-regulated protein kinase localized to centrosomes, is complexed to protein phosphatase 1. Biochem. J. 349:509518.[CrossRef][Medline]
Hinchcliffe, E.H., and G. Sluder. 2001. "It takes two to tango:" understanding how centrosome duplication is regulated throughout the cell cycle. Genes Dev. 15:11671181.
Hinchcliffe, E.H., F.J. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Requirement of a centrosomal activity for cell cycle progression through G1 into S phase. Science. 291:15471550.
Karki, S., and E.L. Holzbaur. 1999. Cytoplasmic dynein and dynactin in cell division and intracellular transport. Curr. Opin. Cell Biol. 11:4553.[CrossRef][Medline]
Karki, S., and E.L.F. Holzbaur. 1995. Affinity chromatography demonstrates a direct binding between cytoplasmic dynein and the dynactin complex. J. Biol. Chem. 270:2880628811.
Karki, S., B. LaMonte, and E.L.F. Holzbaur. 1998. Characterization of the p22 subunit of dynactin reveals the localization of cytoplasmic dynein and dynactin to the midbody of dividing cells. J. Cell Biol. 142:10231034.
Khodjakov, A., and C.L. Rieder. 2001. Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression. J. Cell Biol. 153:237242.
Lange, B.M. 2002. Integration of the centrosome in cell cycle control, stress response and signal transduction pathways. Curr. Opin. Cell Biol. 14:3543.[CrossRef][Medline]
Mayor, T., Y.D. Stierhof, K. Tanaka, A.M. Fry, and E.A. Nigg. 2000. The centrosomal protein C-Nap1 is required for cell cycleregulated centrosome cohesion. J. Cell Biol. 151:837846.
Meraldi, P., and E.A. Nigg. 2001. Centrosome cohesion is regulated by a balance of kinase and phosphatase activities. J. Cell Sci. 114:37493757.
Mogensen, M.M., A. Malik, M. Piel, V. Bouckson-Castaing, and M. Bornens. 2000. Microtubule minus-end anchorage at centrosomal and non-centrosomal sites: the role of ninein. J. Cell Sci. 113:30133023.
Nakagawa, Y., Y. Yaman, T. Okanoue, S. Tsukita, and S. Tsukita. 2001. Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from mother centrioles. Mol. Biol. Cell. 12:16871697.
Ou, Y.Y., G.J. Mack, M. Zhang, and J.B. Rattner. 2002. CEP110 and ninein are located in a specific domain of the centrosome associated with centrosome maturation. J. Cell Sci. 115:18251835.
Paschal, B.M., E.L.F. Holzbaur, K.K. Pfister, S. Clark, D.I. Meyer, and R.B. Vallee. 1993. Characterization of a 50-kDa polypeptide in cytoplasmic dynein preparations reveals a complex with p150Glued and a novel actin. J. Biol. Chem. 268:1531815323.
Piel, M., P. Meyer, A. Khodjakov, C.L. Rieder, and M. Bornens. 2000. The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells. J. Cell Biol. 149:317330.
Piel, M., J. Nordberg, U. Euteneuer, and M. Bornens. 2001. Centrosome-dependent exit of cytokinesis in animal cells. Science. 291:15501553.
Purohit, A., S.H. Tynan, R. Vallee, and S.J. Doxsey. 1999. Direct interaction of pericentrin with cytoplasmic dynein light intermediate chain contributes to mitotic spindle organization. J. Cell Biol. 147:481492.
Quintyne, N.J., S.R. Gill, D.M. Eckley, C.L. Crego, D.A. Compton, and T.A. Schroer. 1999. Dynactin is required for microtubule anchoring at fibroblast centrosomes. J. Cell Biol. 147:321334.
Roghi, C., and V.J. Allan. 1999. Dynamic association of cytoplasmic dynein heavy chain 1a with the Golgi apparatus and intermediate compartment. J. Cell Sci. 112:46734685.
Sanders, M.A., and J.L. Salisbury. 1994. Centrin plays an essential role in microtubule severing during flagellar excision in Chlamydomonas reinhardtii. J. Cell Biol. 124:795805.[Abstract]
Schafer, D.A., S.R. Gill, J.A. Cooper, J.E. Heuser, and T.A. Schroer. 1994. Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles f-actin. J. Cell Biol. 126:403412.[Abstract]
Schroer, T.A., and M.P. Sheetz. 1991. Two activators of microtubule-based vesicle transport. J. Cell Biol. 115:13091318.[Abstract]
Schultz, S.J., A.M. Fry, C. Sutterlin, T. Ried, and E.A. Nigg. 1994. Cell cycle-dependent expression of Nek2, a novel human protein kinase related to the NIMA mitotic regulator of Aspergillus nidulans. Cell Growth Differ. 5:625635.[Abstract]
Tynan, S.H., A. Purohit, S.J. Doxsey, and R.B. Vallee. 2000. Light intermediate chain 1 defines a functional subfraction of cytoplasmic dynein which binds to pericentrin. J. Biol. Chem. 275:3276332768.
Uto, K., and N. Sagata. 2000. Nek2B, a novel maternal form of Nek2 kinase, is essential for the assembly or maintenance of centrosomes in early Xenopus embryos. EMBO J. 19:18161826.
Vaughan, K.T., and R.B. Vallee. 1995. Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued. J. Cell Biol. 131:15071516.[Abstract]
Vaughan, P.S., J.D. Leszyk, and K.T. Vaughan. 2001. Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin. J. Biol. Chem. 276:2617126179.
Waterman-Storer, C.M., S. Karki, and E.L. Holzbaur. 1995. The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1). Proc. Natl. Acad. Sci. USA. 92:16341638.[Abstract]
Young, A., J.B. Dictenberg, A. Purohit, R. Tuft, and S.J. Doxsey. 2000. Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes. Mol. Biol. Cell. 11:20472056.