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Orc1 Controls Centriole and Centrosome Copy Number in Human Cells

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Science  06 Feb 2009:
Vol. 323, Issue 5915, pp. 789-793
DOI: 10.1126/science.1166745

Abstract

Centrosomes, each containing a pair of centrioles, organize microtubules in animal cells, particularly during mitosis. DNA and centrosomes are normally duplicated once before cell division to maintain optimal genome integrity. We report a new role for the Orc1 protein, a subunit of the origin recognition complex (ORC) that is a key component of the DNA replication licensing machinery, in controlling centriole and centrosome copy number in human cells, independent of its role in DNA replication. Cyclin A promotes Orc1 localization to centrosomes where Orc1 prevents Cyclin E-dependent reduplication of both centrioles and centrosomes in a single cell division cycle. The data suggest that Orc1 is a regulator of centriole and centrosome reduplication as well as the initiation of DNA replication.

The assembly of a bipolar, microtubule spindle during mitosis is essential for accurate chromosome segregation. In animal cells, spindle formation is organized by centrosomes, organelles that contain a pair of centrioles surrounded by pericentriolar material (PCM) that need to be duplicated exactly once every cell division cycle, in coordination with DNA replication to maintain genome stability (1). Licensing DNA for replication is a critical regulatory step involving the origin recognition complex (ORC), the first component for assembly of a pre–replicative complex (pre-RC) at each origin (2). Accumulated evidence supports roles for ORC subunits in addition to licensing DNA replication (3). In particular, human Orc2 subunit localizes to centrosomes, and depletion of Orc2 and Orc3 causes centrosome amplification in mitosis (4).

Several regulators of the DNA licensing machinery have been reported to be involved in the control of both DNA and centriole duplication (5). Both Cyclin E and Cyclin A, as well as Cdk2 activity, are well-known positive regulators of DNA replication and also promote centrosome duplication (or reduplication) (611). Depletion of the DNA replication licensing inhibitor Geminin causes reduplication of both DNA and centrosomes in human cells (12).

In a screen using small interfering RNA (siRNA) for human ORC proteins with roles in centrosome biology, we found that depletion of the largest human ORC subunit, HsOrc1, leads to excess centrosomes (fig. S1 and Fig. 1A). Orc1 siRNA-treated U2OS cells were analyzed for centrosome defects by dual-color indirect immunofluorescence (IF) using antibodies to centrin 2 (stains centrioles) and antibodies to γ-tubulin (stains centrosomes). Seventy-two hours after siRNA treatment, 39.77 ± 3.5% of cells transfected with Orc1-1 siRNA and 25.53 ± 0.3% of cells transfected with Orc1-2 siRNA showed multiple centrosomes and centrioles, in comparison with 2.4 ± 1.2% of control cells (fig. S1C). In Orc1-1 treated cells, 33.29 ± 2.6% of the multiple centrosomes were separated rather than linked, and the reduplication occurred in different human cell lines (figs. S1 and S2).

Fig. 1.

Orc1 depletion causes centriole and centrosome reduplication. (A) Human U2OS cells treated for 72 hours with Orc1-1 siRNA duplex were coimmunostained for centrioles with antibodies to centrin 2 (green, a to e) and centrosomes with antibodies to γ-tubulin (red, a′ to e′). Insets are higher magnification. DNA was stained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue), and merged images are shown in a′′ to e′′. Scale bar, 10 μm. (B) Quantification of centrosome and centriole numbers by γ-tubulin (top) and centrin 2 (bottom) immunostaining. Cells were harvested at 12, 36, or 60 hours after nocodazole release. Error bars, 1 SE. 1C, one centrosome; 2C(L), two centrosomes linked; 2C(S), two centrosomes separated; >=3C(L), three or more centrosomes linked; >=3C(S), three or more centrosomes separated. 1Pair, one centriole pair; 2Pairs(L), two centriole pairs linked; 2Pairs(S), two centriole pairs separated; 2 Pairs (Dis.), two centriole pairs disorganized and disengaged; >=2Pairs, more than two centriole pairs.

To study the kinetics of centriole and centrosome reduplication after Orc1 depletion, U2OS cells were synchronized in mitosis by nocodazole treatment and released into the next cycle. Centriole and centrosome numbers were assessed 12 hours, 36 hours, and 60 hours after nocodazole release (Fig. 1B and fig. S3). Disorganized disengagement of duplicated centrioles (2Pairs Dis.) was observed 12 hours after release when most of the cells were in G1 or early S phase (Fig. 1B and fig S3, centrin 2). This was followed by an increase in the percentage of cells showing centriole reduplication (>2Pairs). The complete centrosome structure, as evidenced by γ-tubulin staining, was observed later, about 60 hours after release (γ-tubulin). The very early effect of Orc1 depletion on centriole disengagement and reduplication suggests that Orc1 might have a more direct effect on these processes, whereas complete centrosome formation follows centriole reduplication. Similar dynamics of centriole reduplication after Orc1 depletion were observed in HeLa cells (fig. S4). It has been proposed that centriole engagement blocks reduplication (13). In Orc1-depleted cells, however, the duplicated centrioles were found disengaged and underwent reduplication, suggesting that Orc1 may block reduplication by participating in processes of centriole engagement of the newly duplicated centrioles.

Different mechanisms for centrosome amplification have already been reported. Apparent multiple centrosomes can arise by centrosome fragmentation, centriole splitting that occurs during early mitosis after DNA damage (14), or authentic reduplication. Analysis of multiple parameters—including DNA content (fig. S1B), cell cycle markers (fig. S5), centriolin staining of mature centrosomes (figs. S6 and S7), and the numbers of centrioles per PCM (Fig. 1A)—all indicate that Orc1 depletion causes bona fide centriole and centrosome reduplication in U2OS cells (see supporting material).

To address whether overexpression of Orc1 would affect centrosome reduplication caused by prolonged S-phase arrest in the presence of hydroxyurea (HU) (9, 15), U2OS cells were transiently transfected with YFP-Orc1WT, YFP-Orc2, or a vector carrying only yellow fluorescent protein (YFP) in the presence of HU (Fig. 2, A and D, and fig. S8). Centrosome reduplication after HU treatment, but not normal duplication, was substantially inhibited in YFP-Orc1WT cells, whereas YFP or YFP-Orc2 did not have any effect, suggesting a role for Orc1 as an inhibitor of centrosome reduplication.

Fig. 2.

Overexpression of Orc1 blocks centrosome reduplication. U2OS asynchronous cells were transfected with the indicated constructs and treated with 16 mM hydroxyurea (+HU). Untransfected cells were treated or not with HU (UNT +HU and UNT -HU, respectively). Cells were harvested at 68 hours after HU treatment. Centrosome numbers were scored by γ-tubulin immunostaining in YFP- or Flag-positive cells. (A to C) Quantification of multiple centrosomes in HU-arrested cells. Error bars, 1 SE. (D) Cells were immunostained for centrosomes with antibody to γ-tubulin (red, a′ to i′); YFP expression was immunostained with antibody to green fluorescent protein (GFP) in (A) (green, a to d) or visualized directly in (B) (e and f); and Flag expression was immunostained with antibody to Flag in (C) (g to i). DNA was stained with DAPI (blue), and merged images are shown in a′′ to i′′. Scale bar, 10 μm. Insets are higher magnification.

YFP-Orc1 associated with centrosomes in addition to its nuclear localization in ∼35% of transfected cells (fig. S9A and movie S1). Furthermore, endogenous Orc1 and other ORC subunits cofractionated with purified centrosomes (fig. S9B). Transiently expressed Orc1 fused to the pericentrin-AKAP450 centrosomal targeting (PACT) domain, a conserved motif that targets proteins to centrosomes (16), resulted in inhibition of centrosome reduplication in HU-treated U2OS cells (Fig. 2, B and D), suggesting that Orc1 controls centrosome copy number when located at centrosomes.

Centrosome reduplication was inhibited by the full-length and N-terminal region of Orc1 (amino acids 1 to 500; Orc1WT.Nterm.Flag) (Fig. 2C), the latter of which is deficient in ORC assembly (17). Both Orc1 versions also inhibited centrosome reduplication when expressed after the application of the HU block, indicating that the inhibition was not caused by a G1 arrest in these cells (fig. S10). Thus, Orc1 control of centrosome copy number is separate from its DNA replication role and does not require assembly of ORC, but such assembly may facilitate a more efficient block.

Orc1 depletion in synchronized U2OS cells did not trigger early responses of DNA damage checkpoint pathways that are known to affect centriole and centrosome copy number (18, 19). Activation of Chk1 by phosphorylation on serine 317 was not detected, and p27 and p53 pathways were activated well after centriole amplification was initiated (Fig. 1B and fig. S11).

Several reports have shown that Cyclin E overexpression in mammalian cells is linked to genome and centrosome abnormalities (20, 21) and that centrosome reduplication requires Cdk2 activity (79, 11). Elevated levels of Cyclin E were identified as early as 12 hours after release of synchronized Orc1-depleted U2OS cells in Cyclin A–negative cells (Fig. 3, A and B, and fig. S5, A and B) and continued to increase. We thus hypothesized that Orc1 control of centriole and centrosome reduplication involved Cyclin E and Cdk2. The Cdk2 inhibitor roscovitine suppressed centriole and centrosome reduplication in Orc1-depleted cells (fig. S12). In a second experiment, simultaneous depletion of Orc1 and Cyclin E blocked centriole and centrosome reduplication, whereas normal duplicated centriole and centrosome numbers were observed (Fig. 3C and fig. S13A). Cyclin E siRNA did not change the number of cells with a 4C DNA content after Orc1-1 treatment (fig. S13B), which implies that the block of centrosome overduplication in Orc1-1–depleted cells by Cyclin E siRNA was not due to a compete G1 arrest. In contrast, Cyclin A siRNA did not abolish centrosome reduplication in Orc1-depleted cells (Fig. 3C).

Fig. 3.

Orc1 depletion causes Cdk2 and Cyclin E–dependent centriole and centrosome reduplication. (A and B) Nocodazole-arrested U2OS cells were transfected with control (GL3) or Orc1-1 siRNA, then released into the next cycle and retransfected with the same siRNA duplexes. (A) Immunoblot of whole-cell extract of cells harvested at the indicated time points. Orc1, Cyclin E, Cyclin A, and α-tubulin levels were assessed by immunoblotting with specific antibodies. CycE*, a longer exposure. (B) Cells were harvested at 12 hours after nocodazole release and coimmunostained with antibody to Cyclin E (green) and antibody to Cyclin A (red). DNA was stained with DAPI (blue), and merged images are shown in a′′ and b′′. Scale bar, 30 μm. (C) Quantification of centrosome and centriole numbers by γ-tubulin (top) and centrin 2 (bottom) immunostaining, respectively, of asynchronous U2OS cells transfected with the indicated siRNAs. Cells were analyzed 72 hours after the first transfection. Error bars, 1 SE.

We tested the ability of elevated levels of cyclins to cause centrosome reduplication in HU-treated cells expressing YFP.Orc1WT (Fig. 4A). Coexpression of Cyclin E with YFP.Orc1WT blocked YFP.Orc1WT inhibition of HU-induced centrosome reduplication, whereas neither Cyclin A nor Cyclin B had any effect (Fig. 4, A and B). HU-treated cells coexpressing YFP.Orc1WT with Cyclin A showed a cytoplasmic pool of overexpressed YFP.Orc1WT in addition to the nuclear localization and an enhancement of YFP-Orc1 located at centrosomes (Fig. 4B, b to b′′) (The number of YFP-Orc1–positive centrosomes in the presence of Cyclin A increased six-fold compared with YFP-Orc1 alone). Coexpression of Cyclin E with YFP.Orc1WT.PACT also eliminated the ability of YFP.Orc1WT.PACT to block centrosome reduplication, whereas Cyclin A or Cyclin B had no effect (Fig. 4, A and B). Altogether, the results suggest that Orc1 might directly regulate centriole and centrosome copy number in human cells and that the mechanism involves Cyclin E.

Fig. 4.

Cyclin E suppresses Orc1 inhibition of centrosome reduplication in S-phase–arrested cells. In (A), (B), (D), and (E), U2OS asynchronous cells were transfected with the indicated constructs and treated with 16 mM hydroxyurea (+HU). Untransfected cells were treated or not with HU (UNT +HU and UNT–HU, respectively). Cells were harvested at 68 hours after transfection and HU treatment. (A) Quantification of multiple centrosomes in transfected HU-arrested cells. Centrosome numbers were scored by γ-tubulin immunostaining in YFP-positive cells. Error bars, 1 SE. (B) Transfected, HU-treated cells were immunostained for centrosomes with antibody to γ-tubulin (red, a′ to d′), and YFP expression was visualized directly (green, a to d). DNA was stained with DAPI (blue), and merged images are shown in a′′ to d′′. Scale bar, 10 μm. (C) Immunoprecipitation with antibody to Flag from whole-cell extract from HEK293 cells transiently coexpressing the indicated constructs and immunoblotting with the indicated antibodies. Vector, full-length Orc1WT.Flag (wild type) or full-length Orc1A-A.Flag (mutant) were transiently transfected alone (–) or with either T7-Cyclin E, T7-Cyclin A, or T7-Cyclin. Right panel: 10% of the immunoprecipitates. (D and E) Quantification of multiple centrosomes in transfected HU-arrested cells. Centrosome numbers were scored by γ-tubulin immunostaining in Flag-positive cells in (D) and by γ-tubulin immunostaining in YFP-positive cells directly visualized in (E). Error bars, 1 SE.

Orc1 coprecipitated with Cyclin E and Cyclin A, as previously shown (22), whereas no association was observed with Cyclin B (Fig. 4C). Association in a complex with Cyclin E was stronger than with Cyclin A; however, so far we have not been able to find evidence that Orc1 binds Cyclin E directly. Nevertheless, purified wild-type Orc1 inhibited Cyclin E-Cdk2 and Cyclin A-Cdk2 kinase activity using histone H1 as substrate (fig. S14B).

The Orc1 “RXL” mutant (K235A-L237A, Orc1A-A) impairs the direct interaction with Cyclin A but does not affect Cyclin E binding (Fig. 4C and fig. S14A) and cannot block Cyclin A–associated kinase activity (fig. S14B). Although some inhibition was observed, Orc1A-A and Orc1A-A.Nterm did not block centrosome reduplication as efficiently as did wild-type Orc1, which suggests that Orc1-Cyclin A interaction is required for optimal centrosome copy number control (Fig. 4D). Orc1A-A.PACT blocked centrosome reduplication to the same extent as Orc1WT.PACT (Fig. 4E). Because Cyclin A function has been previously linked to the cytoplasmic localization of Orc1 (23), possibly Orc1A-A and Orc1A-A.Nterm mutants were not exported efficiently to centrosomes to block HU-induced reduplication, whereas Orc1A-A.PACT was directly targeted to centrosomes. Accordingly, Cyclin A stimulation of Orc1 association with centrosomes was significantly reduced when coexpressed with the RXL Orc1A-A mutant. Altogether, the data indicate that both Cyclin E and Cyclin A play a role in Orc1 mediated control of centrosome numbers.

ORC in human cells is dynamically assembled and disassembled during the cell cycle and participates in many aspects of chromosome duplication and segregation, including functions at kinetochores, centromeres, and heterochromatin (3). Orc2 is localized to centrosomes, but its depletion affects centrosome copy number in mitosis in addition to controlling DNA replication, centromere activity, and chromosome structure (4). In contrast, Orc1 controls Cyclin E–dependent centrosome copy number by directly preventing reduplication of centrioles immediately after centrioles duplicate upon commitment to cell division. Without Orc1, duplicated centrioles are disengaged and extensive Cyclin E–dependent centriole reduplication occurs. In the absence of Orc1 and Cyclin E, centrosome reduplication does not occur; however, centrioles duplicate, which suggests that such duplication is Cyclin A–dependent, consistent with previous studies (24). Similarly, Cyclin E, but not Cyclin A, overrides the Orc1 inhibition of centrosome reduplication.

Cyclin E is required for centrosome duplication and reduplication and coprecipitates with Orc1 (7, 11, 2527). We have not been able to detect direct binding between Cyclin E and Orc1, which suggests that a mediator protein may facilitate the binding. The mediator may be another DNA replication protein implicated in centrosome biology (12, 28). Furthermore, depletion of Orc1 increases Cyclin E protein levels in cells that may contribute to centriole reduplication. Both proteins are degraded by SKP1/cullin/F-box protein (SCF), which localizes to centrosomes and has also been implicated in the control of centrosome duplication (29, 30).

There is a short time during the cell division cycle after centrioles have duplicated when Cyclin E is still present in cells (late G1 and early S phase). During this time, if Cyclin E activity is not checked, centriole reduplication might occur. We suggest that a mechanism involving Cyclin A–dependent localization of Orc1 to centrosomes prevents Cyclin E–dependent centriole reduplication during this time window (fig. S15). In this manner, Orc1 plays a dual role in coordinating DNA replication and centrosome copy number.

Supporting Online Material

www.sciencemag.org/cgi/content/full/323/5915/789/DC1

Materials and Methods

Figs. S1 to S15

References

Movie S1

References and Notes

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