Screening of domain-specific target proteins of polo-like kinase 1: construction and application of centrosome/kinetochore-specific targeting peptide

Mammalian polo-like kinase 1 (Plk1) acts at various stages in early and late mitosis. Plk1 localizes at the centrosome and maintains this position through mitosis. Thereafter Plk1 moves to the kinetochore and midbody region, important sites during chromosome separation and cytokinesis. The catalytic domain of Plk1 is in the Nterminus region, whereas the non-catalytic region in the Cterminus of Plk1 has a conserved motif, named the Polobox. This motif is critical for Plk localization. EGFP proteins fused with the N-terminus and C-terminus of Plk1 localize in the nucleus and centrosomes, respectively. The core sequences of the polo-box (50 amino acids) also localize in Plk1 target organelles. To screen for domainspecific target proteins of Plk1, we constructed an Nterminal domain and a tandem repeat polo-box motif, and used them as templates in a yeast two-hybrid screen. The HeLa cell cDNA library indicated several proteins including the centrosome/kinetochore components or regulators, to be characterized as positive clones. Through in vitro protein binding analyses, we confirmed an interaction between these proteins and Plk1. The data reported from this study indicate that the N- and Ctermini of Plk1 may function through recruitment and/or activation of domain-specific target proteins in dividing cells. Additionally, tandem repeats of the conserved core motif of the polo-box are sufficient for targeting and may be useful as a centrosome/kinetochore-specific targeting peptide.     

CLIC4 is enriched at cell-cell junctions and colocalizes with AKAP350 at the centrosome and midbody of cultured mammalian cells

CLIC4 is a member of the chloride intracellular channel (CLIC) protein family whose principal cellular functions are poorly understood. Recently, we demonstrated that several CLIC proteins, including CLIC4, interact with AKAP350. AKAP350 is concentrated at the Golgi apparatus, centrosome, and midbody and acts as a scaffolding protein for several protein kinases and phosphatases. In this report, we show that endogenous CLIC4 and AKAP350 colocalize at the centrosome and midbody of cultured cells by immunofluorescence microscopy. Unlike AKAP350, CLIC4 is not enriched in the Golgi apparatus but is enriched in mitochondria, actin-based structures at the cell cortex, and the nuclear matrix, indicating that CLIC4-AKAP350 interactions are regulated at specific subcellular sites in vivo. In addition to the centrosome and midbody, CLIC4 colocalizes with AKAP350 and the tight junction protein ZO-1 in the apical region of polarized epithelial cells, suggesting that CLIC4 may play a role in maintaining apical-basolateral membrane polarity during mitosis and cytokinesis. Biochemical studies show that CLIC4 behaves mainly as a soluble cytosolic protein and can associate with proteins of the microtubule cytoskeleton. The localization of CLIC4 to the cortical actin cytoskeleton and its association with AKAP350 at the centrosome and midbody suggests that CLIC4 may be important for regulating cytoskeletal organization during the cell cycle. These findings lead to the conclusion that CLIC4 and possibly other CLIC proteins have alternate cellular functions that are distinct from their proposed roles as chloride channels.     

Polo-box motif targets a centrosome regulator, RanGTPase

Mammalian polo-like kinase (Plk) acts at various stages in early and late mitosis. Plk1 localizes in the centrosome, the central spindle, the midbody as well as the kinetochore. The non-catalytic region in the C-terminus of Plk1 has conserved sequence motifs, named polo-boxes. These motifs are important for Plk localization. GFP protein fused with the core sequences of polo-box (50 amino acids) localized Plk to target organelles. We screened for Plk interacting proteins by constructing a tandem repeat of the polo-box motif, and used it as bait in the two-hybrid system with HeLa cell cDNA library. RanGTPase was detected as a positive clone. Through in vitro and in vivo protein binding analysis in synchronized cells by thymidine block and by nocodazole treatment, we confirmed the interaction between endogenous Ran and Plk1. We showed that endogenous Ran and Plk1 proteins were co-localized to centrosomes, which is a major target organelle of endogenous Plk1, in early mitotic cells by immunofluorescence. Finally, we demonstrated that Plk1 phosphorylated RanBPM, a Ran-binding protein in microtubule organizing center, through the interaction with Ran. These data suggested that the core motif of polo-box is sufficient for Plk1-targeting, and that Plk1 may play roles in centrosome through recruitment and/or activation of Ran/RanBPM proteins.     

Centrosome-intrinsic mechanisms modulate centrosome integrity during fever

The centrosome is critical for cell division, ciliogenesis, membrane trafficking, and immunological synapse function. The immunological synapse is part of the immune response, which is often accompanied by fever/heat stress (HS). Here we provide evidence that HS causes deconstruction of all centrosome substructures primarily through degradation by centrosome-associated proteasomes. This renders the centrosome nonfunctional. Heat-activated degradation is centrosome selective, as other nonmembranous organelles (midbody, kinetochore) and membrane-bounded organelles (mitochondria) remain largely intact. Heat-induced centrosome inactivation was rescued by targeting Hsp70 to the centrosome. In contrast, Hsp70 excluded from the centrosome via targeting to membranes failed to rescue, as did chaperone inactivation. This indicates that there is a balance between degradation and chaperone rescue at the centrosome after HS. This novel mechanism of centrosome regulation during fever contributes to immunological synapse formation. Heat-induced centrosome inactivation is a physiologically relevant event, as centrosomes in leukocytes of febrile patients are disrupted.     

vAL-1, a novel polysaccharide lyase encoded by chlorovirus CVK2

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubule and the kinetochore. Our recent ultrastructural studies demonstrated a dynamic distribution of TTK, from the kinetochore to the centrosome, as cell enters into anaphase. Here, we show that a centrosomal protein TACC2 is phosphorylated in mitosis by TTK signaling pathway. TACC2 was pulled down by wild type TTK but not kinase death mutant, suggesting the potential phosphorylation-mediated interaction between these two proteins. Our immunofluorescence studies revealed that both TTK and TACC2 are located to the centrosome. Interestingly, expression of kinase death mutant of TTK eliminated the centrosomal localization of TACC2 but not other centrosomal proteins such as gamma-tubulin and NuMA, a phenotype seen in TTK-depleted cells. In these centrosomal TACC2-liberated cells, chromosomes were lagging and mis-aligned. In addition, the distance between two centrosomes was markedly reduced, suggesting that centrosomal TACC2 is required for mitotic spindle maintenance. The inter-relationship between TTK and TACC2 established here provides new avenue to study centrosome and spindle dynamics underlying cell divisional control.     

GFP tagging reveals human Polo-like kinase 1 at the kinetochore/centromere region of mitotic chromosomes

Polo-like kinases (Plks) have been implicated in various aspects of M-phase progression in organisms ranging from yeast to man. In vertebrates, Plks participate in centrosome maturation and spindle assembly, as well as the activation of the Cdk1/cyclin B complex. Moreover, Plks are required for the destruction of mitotic cyclins, indicating that they play an important role in the regulation of the ubiquitin-dependent proteolytic degradation machinery that controls exit from M-phase. Here, we have fused Green Fluorescent Protein (GFP) to the N-terminus of human Plk1, and expressed this chimeric construct in human cells. We found that GFP-Plk1 associates with centrosomes, the equatorial spindle midzone and the postmitotic bridge of dividing cells, confirming and extending previous results obtained with conventional immunofluorescence microscopy. In addition, however, we observed fluorescence emanating from the midbody between dividing cells, and from discrete dots associated with mitotic chromosomes. This latter staining pattern being reminiscent of centromeres, we performed double-labeling experiments with antibodies against the centromeric marker CENP-B, and reexamined the subcellular localization of endogenous Plk1 using different fixation procedures. Our data clearly show that both GFP-tagged Plk1 and endogenous Plk1 associate with the kinetochore/centromere region of human mitotic chromosomes. This novel localization of Plk1 suggests that substrates and/or regulators of Plks may be found among kinetochore-associated proteins with important functions in chromosome segregation and/or spindle checkpoint mechanisms. Received: 22 August 1998; in revised form: 1 September 1998 / Accepted: 2 September 1998     

Timing of centrosome separation is important for accurate chromosome segregation

Spindle assembly, establishment of kinetochore attachment, and sister chromatid separation must occur during mitosis in a highly coordinated fashion to ensure accurate chromosome segregation. In most vertebrate cells, the nuclear envelope must break down to allow interaction between microtubules of the mitotic spindle and the kinetochores. It was previously shown that nuclear envelope breakdown (NEB) is not coordinated with centrosome separation and that centrosome separation can be either complete at the time of NEB or can be completed after NEB. In this study, we investigated whether the timing of centrosome separation affects subsequent mitotic events such as establishment of kinetochore attachment or chromosome segregation. We used a combination of experimental and computational approaches to investigate kinetochore attachment and chromosome segregation in cells with complete versus incomplete spindle pole separation at NEB. We found that cells with incomplete spindle pole separation exhibit higher rates of kinetochore misattachments and chromosome missegregation than cells that complete centrosome separation before NEB. Moreover, our mathematical model showed that two spindle poles in close proximity do not "search" the entire cellular space, leading to formation of large numbers of syntelic attachments, which can be an intermediate stage in the formation of merotelic kinetochores.     

Characterization of the structures involved in localization of the SUN proteins to the nuclear envelope and the centrosome

The nuclear envelope forms a selective barrier that separates the cytoplasm from the nucleus. During mitosis the nuclear envelope breaks down so that the microtubule network can form contacts with the kinetochore and guide chromosome segregation. Previous studies have suggested a model in which the centrosome and the microtubule network may play a role in nuclear envelope breakdown through as yet unidentified interactions with proteins localized to the nuclear envelope. In the current study we characterized a nuclear envelope protein SUN2 and identified a substructure involved in its localization to the nuclear envelope. We found that a structurally related protein, SUN1, may be localized to the nuclear envelope through a different mechanism. Furthermore, the SUN2 protein can form different assemblies, including homodimers and heterodimers with SUN1. Finally, we provide evidence indicating that SUN1 and SUN2 may form a physical interaction between the nuclear envelope and the centrosome.     

The Toxoplasma gondii kinetochore is required for centrosome association with the centrocone (spindle pole)

The kinetochore is a multi‐protein structure assembled on eukaryotic centromeres mediating chromosome attachment to spindle microtubules. Here we identified the kinetochore proteins Nuf2 and Ndc80 in the apicomplexan parasite Toxoplasma gondii. Localization revealed that kinetochores remain clustered throughout the cell cycle and colocalize with clustered centromeres at the centrocone, a structure containing the spindle pole embedded in the nuclear envelope. Pharmacological disruption of microtubules resulted in partial loss of some kinetochore and centromere clustering, indicating microtubules are necessary but not strictly required for kinetochore clustering. Generation of a TgNuf2 conditional knock‐down strain revealed it is essential for chromosome segregation, but dispensable for centromere clustering. The centromeres actually remained associated with the centrocone suggesting microtubule binding is not required for their interaction with the spindle pole. The most striking observation upon TgNuf2 depletion was that the centrosome behaved normally, but that it lost its association with the centrocone. This suggests that microtubules are essential to maintain contact between the centrosome and chromosomes, and this interaction is critical for the partitioning of the nuclei into the two daughter parasites. Finally, genetic complementation experiments with mutated TgNuf2 constructs highlighted an apicomplexan‐specific motif with a putative role in nuclear localization.     

Urochordate Ascidians Possess a Single Isoform of Aurora Kinase That Localizes to the Midbody via TPX2 in Eggs and Cleavage Stage Embryos

Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner) and INCENP (a vertebrate AURKB partner) and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody in eggs and cleavage stage embryos.     

The peptidyl prolyl isomerase cyclophilin A localizes at the centrosome and the midbody and is required for cytokinesis

Failed cytokinesis leads to tetraploidy, which is an important intermediate preceding aneuploidy and the onset of tumorigenesis. The centrosome is required for the completion of cytokinesis through the transport of important components to the midbody; however, the identity of molecular components and the mechanism involved remains poorly understood. In this study, we report that the peptidyl prolyl isomerase cyclophilin A (cypA) is a centrosome protein that undergoes cell cycle-dependent relocation to the midzone and midbody during cytokinesis in Jurkat cells implicating a role during division. Depletion of cypA does not disrupt mitotic spindle formation or progression through anaphase; however, it leads to cytokinesis defects through an inability to resolve intercellular bridges, culminating in delayed or failed cytokinesis. Defective cytokinesis is also evident by an increased prevalence of midbody-arrested cells. Expression of wild-type cypA reverses the cytokinesis defect in knockout cells, whereas an isomerase mutant does not, indicating that the isomerisation activity of cypA is required for cytokinesis. In contrast, wild-type cypA and the isomerase mutant localize to the centrosome and midbody, suggesting that localization to these structures is independent of isomerase activity. Depletion of cypA also generates tetraploid cells and supernumerary centrosomes. Finally, colony formation in soft agar is impaired in cypA-knockout cells, suggesting that cypA confers clonogenic advantage on tumor cells. Collectively, this data reveals a novel role for cypA isomerase activity in the completion of cytokinesis and the maintenance of genome stability.     

Kinetochore localization and microtubule interaction of the human spindle checkpoint kinase Mps1

Members of the Mps1 protein kinase family have been implicated in the regulation of the kinetochore-mediated spindle assembly checkpoint in species ranging from yeast to man. However, conflicting data have been reported on the subcellular localization of vertebrate Mps1 kinases and their possible roles in centrosome duplication. Moreover, little is presently known about the regulation of Mps1 kinases during the cell cycle. Here, we have used immunofluorescence microscopy, immunoblotting and siRNA-mediated depletion of hMps1 to re-investigate the subcellular localization of this kinase. Our data confirm the kinetochore association of hMps1 but suggest that the centrosome staining produced by some anti-hMps1 antibodies could be due to cross-reactivity with other proteins. We also show that the kinetochore association of hMps1 is mediated by the amino-terminal, non-catalytic domain and specifically requires the presence of the Hec1/Ndc80-Nuf2 complex at the kinetochore. Finally, we have combined in vitro binding studies and kinase assays to explore the influence of microtubules on hMps1 activity. Our data indicate that the catalytic domain of hMps1 displays affinity for microtubules and that microtubule binding could contribute to the regulation of kinase activity.Electronic Supplementary Material Supplementary material is available for this article at .Abbreviations DAPI 4,6-Diamidino-2-phenylindole - EGFP Enhanced green fluorescent protein - Mab Monoclonal antibody - MBP Myelin basic protein - PBS Phosphate-buffered saline - RT Room temperature     

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.     

New insights into subcomplex assembly and modifications of centrosomal proteins

ABSTRACT: This review provides a brief overview of the recent work on centrosome proteomics, protein complex identification and functional characterization with an emphasis on the literature of the last three years. Proteomics, genetic screens and comparative genomics studies in different model organisms have almost exhaustively identified the molecular components of the centrosome. However, much knowledge is still missing on the protein-protein interactions, protein modifications and molecular changes the centrosome undergoes throughout the cell cycle and development. The dynamic nature of this large multi-protein complex is reflected in the variety of annotated subcellular locations and biological processes of its proposed components. Some centrosomal proteins and complexes have been studied intensively in different organisms and provided detailed insight into centrosome functions. For example, the molecular, structural and functional characterization of the gamma-Tubulin ring complex (gamma-TuRC) and the the discovery of the Augmin/HAUS complex has advanced our understanding of microtubule (MT) capture, nucleation and organization. Surprising findings revealed new functions and localizations of proteins that were previously regarded as bona fide centriolar or centrosome components, e.g. at the kinetochore or in the nuclear pore complex regulating MT plus end capture or mRNA processing. Many centrosome components undergo posttranslational modifications such as phosphorylation, SUMOylation and ubiquitylation that are critical in modulating centrosome function and biology. A wealth of information has recently become available driven by new developments in technologies such as mass spectrometry, light and electron microscopy providing more detailed molecular and structural definition of the centrosome and particular roles of proteins throughout the cell cycle and development.     

Cdk1/Erk2- and Plk1-dependent phosphorylation of a centrosome protein, Cep55, is required for its recruitment to midbody and cytokinesis

Centrosomes in mammalian cells have recently been implicated in cytokinesis; however, their role in this process is poorly defined. Here, we describe a human coiled-coil protein, Cep55 (centrosome protein 55 kDa), that localizes to the mother centriole during interphase. Despite its association with gamma-TuRC anchoring proteins CG-NAP and Kendrin, Cep55 is not required for microtubule nucleation. Upon mitotic entry, centrosome dissociation of Cep55 is triggered by Erk2/Cdk1-dependent phosphorylation at S425 and S428. Furthermore, Cep55 locates to the midbody and plays a role in cytokinesis, as its depletion by siRNA results in failure of this process. S425/428 phosphorylation is required for interaction with Plk1, enabling phosphorylation of Cep55 at S436. Cells expressing phosphorylation-deficient mutant forms of Cep55 undergo cytokinesis failure. These results highlight the centrosome as a site to organize phosphorylation of Cep55, enabling it to relocate to the midbody to function in mitotic exit and cytokinesis.     

Immunoelectron microscopic localization of phosphoproteins associated with the mitotic spindle.

We examined the immunogold staining of microtubules and microtubule organizing centers using an improved silver-enhancement reagent for small (1-1.4 nm) gold-conjugated secondary antibodies. First, the staining properties of different commercial preparations of gold-labeled antibodies were compared for sample penetration, label uniformity, and labeling density, and Nanogold 1.4-nm gold-conjugated F(ab') was found to be superior to the other probes examined. However, in samples examined for the localization of alpha- and beta-tubulin, gold staining did not extend through the pericentriolar material nor were the centrioles labeled. This apparent lack of centrosomal staining was not due to problems associated with penetration of the antibody probes, since staining adjacent to and within the centriolar cylinder was observed when phosphoprotein antigens recognized by the MPM-2 antibody were localized. The MPM-2 antibodies also localized to mitotic kinetochores, kinetochore fibers, and midbodies, in addition to mitotic centrosomes. The level of MPM-2 staining of the centrosome varied through the cell cycle. At interphase, this staining was restricted within the centriolar cylinder, whereas in mitotic cells extensive staining throughout the pericentriolar material was also observed. These results established the close relationship of MPM-2-reactive phosphoproteins with the centrosome, and suggest that this technique may be useful for ultrastructural localization of other cytoskeletal proteins.     

The Nup107-160 Nucleoporin Complex Promotes Mitotic Events via Control of the Localization State of the Chromosome Passenger Complex

The human Nup107-160 nucleoporin complex plays a major role in formation of the nuclear pore complex and is localized to kinetochores in mitosis. Here we report that Seh1, a component of the Nup107-160 complex, functions in chromosome alignment and segregation by regulating the centromeric localization of Aurora B and other chromosome passenger complex proteins. Localization of CENP-E is not affected by Seh1 depletion and analysis by electron microscopy showed that microtubule kinetochore attachments are intact. Seh1-depleted cells show impaired Aurora B localization, which results in severe defects in biorientation and organization of the spindle midzone and midbody. Our results indicate that a major function of the Nup107 complex in mitosis is to ensure the proper localization of the CPC at the centromere.     

Overexpression of an Aurora-C kinase-deficient mutant disrupts the Aurora-B/INCENP complex and induces polyploidy

Aurora kinases are emerging as key regulators of centrosome function, chromosome segregation and cytokinesis. We previously isolated Aurora-C (Aie1), a third type of Aurora kinase, in a screen for kinases expressed in mouse sperm and eggs. Currently, we know very little about the precise localization and function of Aurora-C. Immunofluorescence analysis of ectopically expressed GFP-Aurora-C has revealed that Aurora-C is a new member of the chromosomal passenger proteins localizing first to the centromeres and then to the central spindles during cytokinesis. In order to study the potential role of Aurora-C, we examined the effects of a kinase-deficient (KD) mutant (AurC-KD) in HeLa Tet-Off cells under tetracycline control. Our results showed that overexpression of AurC-KD causes defects in cell division and induces polyploidy and apoptosis. Interestingly, AurC-KD overexpression also inhibits centromere/kinetochore localization of Aurora-B, Bub1, and BubR1, reduces histone H3 phosphorylation, and disrupts the association of INCENP with Aurora-B. Together, our results showed that Aurora-C is a chromosomal passenger protein, which may serve as a key regulator in cell division.     

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.