Orc1 controls centriole and centrosome copy number in human cells - PubMed (original) (raw)

Orc1 controls centriole and centrosome copy number in human cells

Adriana S Hemerly et al. Science. 2009.

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.

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Figures

Fig. 1

Fig. 1. Orc1 depletion causes centriole and centrosome re-duplication

(A) Human U2OS cells treated for 72 hours with Orc1-1 siRNA duplex were co-immunostained for centrioles with anti-centrin 2 (green, a to e) and centrosomes with anti--tubulin (red, a to e) antibodies. Insets are higher magnification. DNA was stained with DAPI (blue) and merged images are shown in a to e. Scale bar, 10 m. (B) Quantification of centrosome and centriole numbers by -tubulin (upper panel) and centrin 2 (Bottom panel) immunostaining, respectively. Cells were harvested at 12, 36 or 60 hours after nocodazole release. Error bars represent one standard error. 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; 2 Pairs (L), two centriole pairs linked; 2 Pairs (S), two centriole pairs separated; 2 Pairs (Dis.), two centriole pairs with disorganized disengaged; >=2 Pairs, more than two centriole pairs.

Fig. 2

Fig. 2. Over-expression of Orc1 blocks centrosome re-duplication

U2OS asynchronous cells were transfected (or not, UNT) with the indicated constructs (see text) and treated with 16mM hydroxyurea (+ or HU). Cells were harvested at 68 hours after HU-treatment. Centrosome numbers were scored by -tubulin immunostaining in YFP or Flag positive cells. (A–C) Quantification of multiple centrosomes in HU-arrested cells. (D) Cells were immunostained for centrosomes with anti -tubulin (red, a to i); YFP expression was immunostained with anti-GFP in (A) (green, a to d) or visualized directly in (B) (e, f); and Flag expression was immunostained with anti-Flag in (C) (g to i). DNA was stained with DAPI (blue) and merged images are shown in a to i. Scale bar, 10 m. Insets are higher magnification.

Fig. 3

Fig. 3. Orc1 depletion causes Cdk2 and Cyclin E-dependent centriole and centrosome re-duplication

(A) and (B) Nocodazole arrested U2OS cells were transfected with control (GL3) or Orc1-1 siRNA, then released into the next cycle and re-transfected 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 -tubulin levels were assessed by immunoblotting with specific antibodies. Cyclin E*, a longer exposure. (B) Cells were harvested at 12 hours after nocodazole release and co-immunostained with anti-Cyclin E (green) and anti-Cyclin A (red). DNA was stained with DAPI (blue) and merged images are shown in a and b. Scale bar, 30 m. (C) Quantification of centrosome and centriole numbers by -tubulin and centrin 2 immunostaining, respectively, of asynchronous U2OS cells transfected with the indicated siRNAs. Cells were analyzed 72 hours after the first transfection. Error bars represent one standard error.

Fig. 4

Fig. 4. Cyclin E suppresses Orc1 inhibition of centrosome re-duplication in S-phase-arrested cells

In A, B, C and D: U2OS asynchronous cells were transfected with the indicated constructs (see text) and treated with 16mM 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 -tubulin immunostaining in YFP positive cells. Error bars represent one standard error. (B) Transfected, HU-treated cells were immunostained for centrosomes with anti -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 m. (C) Immunoprecipitation with anti-Flag antibody from whole-cell extract from HEK293 cells transiently co-expressing 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 -tubulin immunostaining in Flag positive cells in (D), and by -tubulin immunostaining in YFP positive cells directly visualized in (E). Error bars represent one standard error.

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