Centrosome separation: respective role of microtubules and actin filaments

Biology of the Cell 94 (2002) 275–288 www.elsevier.com/locate/biocell

Original article

Centrosome separation: respective role of microtubules and actin filaments Rustem Uzbekov a,1, Igor Kireyev b, Claude Prigent a,* a

Groupe cycle cellulaire, UMR 6061 génétique et développement, CNRS—Université de Rennes 1, IFR 97 génomique et santé, Faculté de médecine, 2, avenue du Professeur Léon Bernard, CS 34317, 35043 Rennes cedex, France b Cell Cycle Group, Division of Electron Microscopy, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia Received 11 February 2002; accepted 4 June 2002

Abstract In mammalian cells, the separation of centrosomes is a prerequisite for bipolar mitotic spindle assembly. We have investigated the respective contribution of the two cytoskeleton components, microtubules and actin filaments, in this process. Distances between centrosomes have been measured during cell cycle progression in Xenopus laevis XL2 cultured cells in the presence or absence of either network. We considered two stages in centrosome separation: the splitting stage, when centrosomes start to move apart (minimum distance of 1 µm), and the elongation stage (from 1 to 7 µm). In interphase, depolymerisation of microtubules by nocodazole significantly inhibited the splitting stage, while the elongation stage was, on the contrary, facilitated. In mitosis, while nocodazole treatment completely blocked spindle assembly, in prophase, we observed that 55% of the centrosomes separated, versus 94% in the control. Upon actin depolymerisation by latrunculin, splitting of the interphase centrosome was blocked, and cells entered mitosis with unseparated centrosomes. Cells compensated for this separation delay by increasing the length of both prophase and prometaphase stages to allow for centrosome separation until a minimal distance was reached. Then the cells passed through anaphase, performing proper chromosome separation, but cytokinesis did not occur, and binuclear cells were formed. Our results clearly show that the actin microfilaments participate in centrosome separation at the G2/M transition and work in synergy with the microtubules to accelerate centrosome separation during mitosis. © 2002 Published by Éditions scientifiques et médicales Elsevier SAS. Keywords: Centrosome; Microtubules; Actin; Xenopus

1. Introduction During cell cycle progression, the centrosome must accomplish two important goals: it must duplicate and separate (Urbani and Stearns, 1999; Winey, 1999). Duplication of the centrosome is one of the earliest structural changes that can be observed directly in a cell that prepares for mitosis. Centrosome duplication usually starts at the end of G1 (Sluder and Hinchcliffe, 1999), with the replication of

Abbreviation: NEB, nuclear envelope breakdown. * Corresponding author. Tel.: +33-2-23-23-45-26; fax: +33-2-23-23-44-78. E-mail address: [email protected] (C. Prigent). 1 . Present address: Equipe structure et dynamique du cytosquelette, CNRS UMR 6026, CNRS—Université de Rennes 1, Faculté des sciences, Avenue du Général Leclerc, 35042 Rennes cedex,France © 2002 Published by Éditions scientifiques et médicales Elsevier SAS. PII: S 0 2 4 8 - 4 9 0 0 ( 0 2 ) 0 1 2 0 2 - 9

the centrioles. Near the surface of each centriole close to the proximal end (minus ends of centriolar microtubules), an amorphous disc of electron-dense material with a central axis appears. Later on, nine microtubules appear at the periphery of the disc, which evolve into nine duplexes and finally nine triplets of microtubules that form the procentrioles that maturate during the S and G2 phases and even during the next cell cycle for the very new one (reviewed in Vorobjev and Nadezhdina, 1987). Controls of centrosome duplication were recently identified (Hinchcliffe et al., 1999; Lacey et al., 1999; Meraldi et al., 1999). Just like DNA replication, centrosome duplication depends on the activation of cdk/cyclin kinase activities at the restriction point in G1. Both cdk2/cyclinA and cdk2/cyclinE phosphorylate pRB, allowing E2F to become an active transcription factor that triggers centrosome duplication gene expression (Meraldi et al., 1999). Thus, both

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DNA replication and centrosome duplication are under the control of the same restriction point. The “licensing” concept (Chong and Blow, 1996) allowing only one round of DNA replication per cell cycle is also true for centrosome duplication. Nucleophosmin may ensure this function; this protein is present in the unduplicated centrosome and leaves the centrosome only when phosphorylated by cdk2/cyclinE, giving the signal for centrosome duplication (Tokuyama et al., 2001). When duplicated, the centrosomes must separate to reach a position in metaphase that allows the bipolar spindle to assemble. Parental centrioles are connected to each other by fibrillar linkage (Tournier et al., 1991, Paoletti and Bornens, 1997), implying that separation must start by centriole disconnection. When the duplicated centrosomes begin to separate, it is the splitting stage. Although the cell cycle control of this initial stage of separation remains poorly understood, some players have been identified, such as the duo protein kinase Nek2/protein phosphatase PP1 and their physiological substrate C-Nap1 (Fry et al., 1998a, 1998b; Mayor et al., 2000; Meraldi and Nigg, 2001). Eventually, the two independent centrosomes separate to metaphasespindle-size distance: this is the elongation stage (Jean et al., 1999). Because the centrosome is the major microtubuleorganising centre of the cell, microtubules were early suspected to participate in centrosome separation. Microtubule dependent movement in the cell is carried out by minus-end and plus-end directed motors. Both types of motors have been found in the centrosome and demonstrated to be important for bipolar mitotic spindle formation: the cytoplasmic dynein–dynactin–NuMA (Gaglio et al., 1995, 1996, 1997; Echeverri et al., 1996), ncd (or Kar3) (Walczak et al., 1997; Matthies et al., 1996) and kinesins from the bimC family (Eg5) (Sawin et al., 1992; Heck et al., 1993; Blangy et al., 1995; Wilson et al., 1997, Giet et al., 1999). Classically, centrosome separation is presented as the sliding of anti-parallel bundles of microtubules controlled by various motors. But microtubules are not flexible enough to bend in such a way that they can ensure, alone, the curved movement of the centrosome around the nucleus in early mitosis. And indeed, centrosome separation also requires motor molecules (Mountain et al., 1999; Sharp et al., 1999) and other cytoskeleton network (Gavin, 1997). Actin filaments, for instance, have been reported to be involved in centrosome separation and spindle assembly (Whitehead et al., 1996; Forer and Pickett-Heaps, 1998; Silverman-Gavrila and Forer, 2000). The participation of actin in the process of centrosome separation was experimentally shown by Euteneuer and Schliwa (1985) using artificial centrosome separation in granulocytes treated with the tumour-promoter drug 12-O-tetradecanoylphozbol-13acetate (TPA). Hence, these data directly show that the position of the centrosome and centrioles in the cell relies not only on the microtubule network, but also on actin microfilaments. Moreover, depolymerisation of actin fila-

ments in yeast leads to aberrant spindle–pole-body migration and spindle mis-orientation (Theesfeld et al., 1999). In the present work, we revisited the respective roles of microtubules and actin filaments in the process of centrosome separation during cell cycle progression. We concentrated on the centrosome separation that precedes bipolar spindle assembly, from centrosome duplication to metaphase. The duplicated centrosomes were visualised using Xenopus aurora-A (pEg2) staining, because the protein localised to centrosomes just after duplication (Roghi et al., 1998). We measured the distance between pEg2-positive centrosomes from late S phase to metaphase. Although our data suggest distinct roles for microtubule and actin networks in centrosome separation, they also indicate a cooperation between both networks in this process.

2. Materials and methods 2.1. Cell culture XL2 cell lines (Anizet et al., 1981) were cultured in Leibovitz medium (L15) with 10% of foetal calf serum and penicillin (100 U/ml), streptomycin (100 µg/ml) and amphotericin (25 µg/ml) in 25 cm2 cell flasks (Falcon) or 12-well cell culture plates (Corning Inc.). Cells were incubated at 25 °C and maintained in exponential growth as described by Smith and Tata (1991). 2.2. Indirect immunofluorescence microscopy Xenopus laevis XL2 cells were grown on round coverslips in 12-well plates (Corning Inc.) for 48 h, washed with phosphate-buffered saline (PBS: 136 mM NaCl, 26 mM KCl, 2 mM Na2HPO4, 2 mM KH2PO4, pH 7.2) and fixed by immersion in cold (–20 °C) absolute methanol for 8 min. Following washes in PBS, cells were blocked in PBS containing 3% BSA for 30 min and then incubated with both mouse anti-pEg2 monoclonal antibody 1C1 (20 µg/ml) (Roghi et al., 1998) and rabbit anti-γ-tubulin polyclonal antibody (dilution 1:100) (Komarova et al., 1997). The antibodies were sequentially revealed by fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (dilution 1:100) (Interchim) and Texas red-conjugated goat anti-mouse IgG (dilution 1:100) (Interchim). All antibody reagents were diluted in PBS containing 1% BSA, and incubations were performed at room temperature for 60 min. Cells were rinsed in PBS containing 1% BSA between each incubation (3 × 10 min). Actin filaments were stained with FITC-conjugated phalloidin, and living cells were permeabilised by incubation in 50 mM imidazole–HCl (pH 6.8), 50 mM KCl, 0.1 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 0.1 mM β-mercaptoethanol, 30% glycerol and 1% Triton X-100 for 3 min at room temperature and fixed in 3% paraformaldehyde for 30 min at room temperature. Then, the cells were

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washed with PBS and incubated for 20 min in 1.6 µM FITC-conjugated phalloidin, followed by PBS wash. After each staining procedure, the coverslips were rinsed in PBS and mounted in a Mowiol containing anti-fade DABCO (diazabicyclo [2.2.2]octan, Sigma, 1 mg/ml) and DAPI (4’,6-diamidino-2-phenylindole, Sigma, 1 µg/ml) for DNA staining. Samples were observed using a Leica DMRXA fluorescent microscope; images were captured with a blackand-white camera (COHU) and processed with Leica QFISH software. 2.3. Drug treatment For microtubule depolymerisation, cells were incubated in a medium containing 5 µg/ml of nocodazole (Sigma Chemicals). For actin microfilament depolymerisation, cells were incubated in a medium containing 0.2 µg/ml latrunculin-A (molecular probe). Incubation times were 2, 4, 6 and 9 h.

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2.6. Measurement of mitotic sub-phase length in living cells Live observations were performed at 25 °C with an inverted microscope (Leica DMIRBE) using a 40× objective and interferential contrast (Hoffman). The length of the mitotic sub-phases was measured starting with a cell culture containing a large number of G2 cells (cells with large nucleus and nucleolus with reduced density). The cells were periodically observed by interferential contrast with a minimal level of illumination. The transition between sub-phases was identified as follows: in the beginning of prophase, the chromosome started to condense; passage into prometaphase corresponded to nuclear envelope breakdown (NEB); upon entry into metaphase, chromosomes aligned; chromosomes started to separate in anaphase; and telophase lasted from the beginning of chromosome decondensation to the separation of the two daughter cells

3. Results 2.4. Measurement of the distances between centrosomes Usually, anti-γ-tubulin antibodies are used for specific centrosome staining, but γ-tubulin decorates centrosomes all along cell cycle progression (Komarova et al., 1997). In this report, we used the anti-pEg2 monoclonal antibody 1C1, which decorates only duplicated centrosomes (Roghi et al., 1998). Our aim was to follow centrosome separation from duplication to cell division, and pEg2 precisely decorates centrosomes only during this period. Distances between pEg2 stained centrosomes were measured using an ocular micrometer calibrated with an object micrometer. The estimated accuracy of measurement was 0.5 µm, so we considered that cells with an inter-centrosome distance of more than 1 µm had separated centrosomes, and less than 1 µm, unseparated ones. 2.5. Identification of mitotic phases Cells were fixed with cold methanol and the DNA stained with 1 µg/ml DAPI (4’,6-diamidino-2-phenylindole, Sigma). A combination of phase contrast and fluorescent microscopy (DAPI) (Leica DMRXA) was used to distinguish the percentage of cells at each phase of the cell cycle. The following criteria were used to identify mitotic subphases: prophase cells contained visible condensed chromosome and a nuclear envelope; prometaphase cells lacked the nuclear envelope and showed randomly positioned chromosomes; metaphase cells showed all chromosomes aligned in a metaphase plate; anaphase cells had initiated chromosome separation; and telophase cells remained attached by a cytoplasm bridge and the chromosomes had started to decondense.

3.1. Centrosome separation in control XL2 cells We investigated centrosome separation in Xenopus XL2 cells by the measurement of inter-centrosome distance in fixed cells immunostained by the pEg2 antibody, which decorates only duplicated centrosomes (Roghi et al., 1998) (Fig. 1). The mitotic cycle progression was monitored by chromatin staining with DAPI and by observation of NEB by phase contrast microscopy. We estimated that 26.7% of the interphase cells containing pEg2-decorated centrosomes had already started separation (inter-centrosome distance d ≥ 1). The average distance in all pEg2-positive interphase cells was around 1.3 µm, whereas the average distance in cells containing separated centrosomes was 3.4 µm. Ninetyfour percent of the prophase cells exhibited separated centrosomes, with an average inter-centrosome distance of about 5 µm. Ninety-nine percent of the prometaphase cells exhibited separated centrosomes with an average intercentrosomal distance of 5.75 µm, while metaphase cells had fully assembled spindles 7.4 µm long, with a distinct metaphase plate (Table 1). pEg2 kinase localised around the pericentriolar material of the centrosomes only after duplication from the end of the S phase to the beginning of the following G1 in XL2 cells (Roghi et al., 1998); therefore, because we used pEg2 staining as a centrosome marker, we excluded G1 and early S cells from our measurements. In XL2 cells, the inter-centrosome distance during interphase (late S–G2) was short on average, about 1.3 µm (Table 1). Centrosome separation starts somewhere between the end of S and the beginning of the M phase. To reveal more accurate dynamics of centrosome separation, we created a frequency histogram of inter-centrosomal distances in interphase cells and estimated the proportion of cells (pEg2 positive) with separated and unseparated centrosomes (Fig. 2A). We

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distance over 10 µm). 26.7% of the pEg2-positive interphase cells have started centrosome separation, though only 7.4% show the centrosome separated by more than 3.5 µm, indicating that they were close to completing this process—to reach metaphase spindle inter-pole distance between centrosomes. Centrosomes become pEg2 positive about 2 h 45 min before the end of S; according to the length of the XL2 cell cycle, we estimated a window of interphase pEg2-positive centrosome to last about 5 h 15 min (end of S phase = 2 h 45 min and G2 = 2 h 30 min). Seventy-five percent of these cells show unseparated centrosomes, which indicates that centrosome separation (elongation phase) starts in mid-G2 (75% of 5 h 15 min ≈ 4 h, being 1 h 15 min before the M phase) (Fig. 2C). In prophase, the average inter-centrosome distance increased dramatically, and only 6% of the cells had unseparated centrosomes; this percentage kept decreasing while cells entered prometaphase to reach 1%. We investigated the respective roles of microtubules and actin filaments in centrosome separation using the microtubule depolymerising drug nocodazole and the actin depolymerising drug latrunculin-A. Fig. 1. pEg2 decorates only the duplicated centrosome. Two cells (one in G1 and one in G2) were stained with anti-pEg2 monoclonal antibody and anti-γ-tubulin polyclonal antibodies. The right panels show the centrosome in G1 cells, while the left panels show the centrosomes in G2 cells.

considered that the cells with an inter-centrosome distance from 1 to 3.5 µm had started centrosome separation. Then, we also specified cells with centrosome separated to metaphase spindle distance or more (more than 7.5 µm) and cells with moderately separated centrosomes (from 4 to 7 µm) (Fig. 2B). From Table 1 and the histogram, it is clearly seen that about 26.7% of the pEg2-decorated interphase cells show separated centrosomes (19.4% from 1 to 3.5 µm, 4.4% from 4 to 7 µm, 1.5% from 7.5 to 10 µm, and 1.5% with a

3.2. Depolymerisation of microtubules or actin filaments inhibits the splitting of the centrosome during interphase The number of interphase cells showing separated centrosome was estimated in cells cultivated for 2, 4, 6 and 9 h in the presence of either 5 µg/ml nocodazole or 0.2 µg/ml latrunculin. In XL2 cells, 2 h of nocodazole treatment depolymerised all mitotic microtubules and a large majority of the interphase microtubules; 4, 6 and 9 h of nocodazole treatment depolymerised all cytoplasmic microtubules (Fig. 3, panel f). Nocodazole treatment stopped cell cycle progression between prometaphase and metaphase, because C-mitosis (colchicine-like-mitosis) took place (Hamilton and Snyder, 1982). After 6 h of nocodazole treatment, the actin filament network remained similar to that in control cells (Fig. 3, panel h).

Table 1 Distance between centrosomes in XL-2 cells during interphase and during the different mitotic sub-phases Phase of the cell cycle

Distance between centrosomes (µm)

Percentage of cells with separated centrosomes

Min–max

Average All

Separated (d ≥ 1)

Interphase (Eg2+) (N = 206)

0.5–23

1.28 ± 0.17

3.44 ± 0.53

26.7

Prophase (N = 100)

0.5–14

4.76 ± 0.28

5.03 ± 0.27

94

Prometaphase (N = 100)

0.5–10.5

5.73 ± 0.21

5.78 ± 0.21

99

Metaphase (N = 100)

6.0–10.0

7.39 ± 0.09

7.39 ± 0,09

100

Anaphase (N = 100)

7.5–18

11.35 ± 0.20

11.35 ± 0.20

100

Telophase (N = 100)

11.5–30

17.9 ± 0.41

17.9 ± 0.41

100

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A

279

C

B

Fig. 2. Centrosome splitting and separation during interphase in Xenopus laevis XL2 cells. The distance has been measured between centrosomes decorated by the Xenopus aurora-A kinase (pEg2). (A) Frequency histogram of inter-centrosome distances in interphase cells containing pEg2-decorated centrosomes. (B) Interphase pEg2-positive cells stained with anti-pEg2 monoclonal antibodies and DAPI. The centrosome appears in red (pEg2) and the nucleus in blue (DNA). The five images shown illustrate the five classes of centrosome distances we chose. The bar is 1 µm. (C) Timing of centrosome splitting and separation. Separation was considered when the centrosomes were separated by more than 1 µm. The period between duplication and separation (3 h 45 min) is in blue and the period of separation is in yellow. R stands for restriction point.

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Fig. 3. Depolymerisation of microtubules and actin filaments in Xenopus XL2 cells by nocodazole and latrunculin-A treatment, respectively. Panels a–d: control cells; panels e–h: cells were treated with 5 µg/ml nocodazole; panels i–l: cells were treated with 0.2 µg/ml latrunculin-A. Panels a, e, i, c, g and k: DNA was stained with DAPI. Panels b, f and j: microtubules were stained with anti-tubulin antibodies revealed using a TRITC-conjugated secondary antibody. Panels d, h and l: actin filaments were stained with FITC-conjugated phalloidin. Cells were observed under a DMRXA fluorescent microscope (Leica); images were taken using a black-and-white camera (COHU) and processed using QFISH software (Leica). The bar is 20 µm.

Two hours of incubation in the presence of latrunculin was sufficient to completely depolymerise actin filaments (Fig. 3, panel l), whereas the interphase microtubule network was conserved even after 6 h of latrunculin treatment (Fig. 3, panel j). While depolymerisation of microtubules arrested cells (C-mitosis), depolymerisation of actin filaments blocked cytokinesis only, leading to the formation of binucleated cells (mitosis looked normal until telophase). Upon incubation with either nocodazole or latrunculin for 9 h, the distance between centrosomes in interphase cells remained under 1 µm in more than 93% of the cells (Fig. 4), indicating that both drugs inhibited centrosome splitting. Latrunculin seemed to be more effective in inhibiting centrosome separation than was nocodazole, although similar inhibition levels were reached after 9 h of contact with

either drug. These results show that both networks, microtubules and actin filaments, are necessary for centrosome splitting before mitosis onset. 3.3. During interphase, once centrosomes have split, depolymerisation of microtubules accelerates separation, whereas depolymerisation of actin filament inhibits separation In order to investigate the different effect of both nocodazole and latrunculin-A on centrosome separation during interphase, we compared the distance between centrosomes that have already started to separate (inter-centrosomal distance of 1 µm and more). Three classes of intercentrosome distances were considered: class I from 1 to

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Upon nocodazole treatment, the number of cells in class I progressively decreased, while the number of cells in the other classes increased (Fig. 5). After 6 h of nocodazole treatment, almost 50% of the cells of which the centrosomes had started to separate showed an inter-centrosomal distance over 7.5 µm (Fig. 5), over the spindle size. Latrunculin treatment led to the opposite effect: it was the percentage of cells with small inter-centrosomal distance that increased, from 72.7% in control cells to almost 100% after 6 h of treatment (Fig. 5). All cells eventually showed inter-centrosomal distances between 1 and 1.5 µm (average inter-centrosomal distance 1.2 µm). Centrosome separation was completely blocked after 6 h of incubation in latrunculin-A, strongly indicating that actin filaments are required for separation. Fig. 4. Percentage of interphase cells with pEg2/aurora-A-positive separated centrosomes after nocodazole or latrunculin-A treatments. The number of cells with separated centrosomes was estimated by pEg2/aurora-A staining in interphase cells. The measures were performed in cells treated for 0 (control cells) 2, 4 or 6 h with either 5 µg/ml of nocodazole (black bars) or 0.2 µg/ml of latrunculin-A (grey bars).

3.5 µm (from unseparated to half the spindle size); class II from 4 to 7 µm (from half the spindle size to spindle size), class III over 7 µm (more than spindle size). In control cells, the average distance between these centrosomes was 1.3 ± 0.6 µm, and among cells containing separated centrosomes, 72% were in class I, 16.7% in class II and 10.9% in class III.

3.4. In prophase, depolymerisation of microtubules blocked centrosome splitting while depolymerisation of actin filament blocked elongation In the presence of nocodazole, the number of prophase cells with unseparated centrosomes increased dramatically (Fig. 6A, black bars), indicating that centrosome separation was blocked at a very early stage, the splitting stage. However, the number of prophase cells containing centrosomes separated by a long distance, either half the spindle size (Fig. 6A, dark grey bars) or larger than spindle size (Fig. 6A, white bars), was relatively stable in the presence of the drug. These series of results suggest that in prophase, microtubules are necessary for centrosomes to

Fig. 5. Distribution of distances between centrosomes in interphase cells after treatment with nocodazole or latrunculin-A. Three different classes of inter-centrosome distances were considered: from 1 to 3.5 µm (black bars), from 4 to 7 µm (grey bars) and over 7.5 µm (white bars). The measures were performed in control cells or in cells treated for 2, 4 or 6 h with either 5 µg/ml of nocodazole or 0.2 µg/ml of latrunculin-A.

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This set of results seems to indicate that in prophase, the microtubules are required for centrosome splitting, while the actin filament network is necessary for the centrosome to separate from half the spindle size to spindle size (3.5–7 µm), to move around the nucleus. 3.5. In mitosis, depolymerisation of the actin filament provokes a defect in centrosome separation only during prophase and prometaphase

Fig. 6. Distribution of distances between centrosomes in prophase cells after treatment with nocodazole or latrunculin-A. Four different classes of inter-centrosome distances were considered: under 1 µm (black bars) for the unseparated centrosome, from 1 to 3.5 µm (dark grey bars), from 4 to 7 µm (light grey bars) and over 7.5 µm (white bars). (A) Cells were treated with nocodazole for 0 (control), 2, 4 and 6 h. (B) Cells were treated with latrunculin-A for 0 (control), 2, 4 and 6 h.

reach a position in which they are separated by a distance that corresponds to the spindle size. The effect of latrunculin-A on the inter-centrosome distance during prophase differed from that of nocodazole. The number of cells containing unseparated centrosomes and centrosomes separated by distances between 1 and 3.5 µm (half the spindle size) increased, whereas, on the contrary, the number of cells containing centrosomes separated by distances between 4 and 7 µm decreased. Centrosomes separated by more than 7.5 µm were undetectable as early as 2 h after latrunculin treatment (Fig. 6B, white bars). It shows firstly that in prophase, actin filaments, unlike in interphase, are not required for the splitting of the centrosome (Fig. 6B). And secondly, the number of cells with centrosomes separated by a distance that corresponds to half the spindle size increased significantly, as if, without actin filaments, the centrosomes were blocked at this position, unable to initiate a curving movement around the nucleus. This inter-centrosome distance (half the spindle size) corresponds approximately to the time when the centrosomes must initiate a curving movement due to the presence of the nuclear envelope: turning around the nucleus.

In the presence of latrunculin-A, prometaphase cells as well as prophase ones had an average inter-centrosome distance shorter than that in control cells (Fig. 7A and B, and F, panels h and i). However, in metaphase, the spindle of latrunculin-treated cells had a normal length (Fig. 7F, panels d and j). We found the same distribution frequency of distances between centrosomes in the control and latrunculin-treated cells (Fig. 7D). Latrunculin-treated cells underwent normal anaphase (Fig. 7F, panel k), though after that the two daughter cells did not separate (Fig. 7F, panel l), and the two separated centrosomes (d > 10 µm) migrated rapidly towards each other (d < 1 µm) (data not shown). Again, this suggests that actin filaments are required because of the presence of the nuclear envelope. In metaphase, when the nuclear envelope has been disrupted, spindle assembly did not require actin anymore, and normal spindle could be assembled.

3.6. Duration of mitosis in the presence of latrunculin

In order to understand how the cell is able to overcome the delay of centrosome separation caused by the lack of actin filaments in the early stages of mitosis, we estimated the duration of mitotic phases using fixed control and latrunculin-treated cells. Compared to the normal situation, in the presence of latrunculin, the proportion of cells in prophase and prometaphase increased, the proportion of cells in metaphase decreased, and the proportion of cells in anaphase was unchanged (Fig. 8A). This result suggested that in the presence of latrunculin, the length of both prophase and prometaphase increased. To show this directly, we measured the duration of mitotic phases in living cells. We have previously measured the length of each mitotic stage of the XL2 cell line in normal conditions (Uzbekov et al., 1998). The average time of prophase was 5.7 ± 1.4 min, prometaphase 6.7 ± 1.6 min, metaphase 19.4 ± 5.3 min and anaphase 5.7 ± 1.2 min. The duration of the various mitotic stages was estimated in the presence of latrunculin by live cell observation. The

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Fig. 7. Centrosome separation in the different sub-phases of mitosis in cells treated with latrunculin-A for 6 h. Five different classes of centrosome distances were considered: I: d < 1 µm; II: 1 ≤ d ≤ 3.5 µm; III: 4 ≤ d ≤ 7; IV: 7.5 ≤ d ≤ 10; V: d > 10. The number of cells containing centrosomes separated by distances corresponding to these classes was expressed as a percentage of mitotic cells. The controls (untreated cells) are shown as grey bars and latrunculin-A-treated cells as black bars. A – prophase, B – prometaphase, C – metaphase, D – anaphase, E – telophase.

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Fig. 7 (suite). F – pEg2-positive cells stained with anti-pEg2 monoclonal antibodies and DAPI. The centrosome appears in red (pEg2) and the nucleus in blue (DNA). Panels a, g and m are interphase cells. Panels b–f, h–l and n–s show cells in various mitotic sub-phases. Panels a–f are control cells, panels g–l latrunculin-treated cells (6 h) and panels m–s nocodazole-treated cells. Panels h and i show centrosome separation delay in the presence of latrunculin and panel l shows the cytokinesis defect. Panels n and o show an example of the two main distances observed in the presence of nocodazole (unseparated d < 1 µm and d ∼ 7 µm). Panels p–s show typical c-mitosis obtained after nocodazole treatment.

time of prophase was difficult to measure in such cells, because in the presence of latrunculin, the majority of cells were spherical, and the beginning of chromosome condensation was really undetectable. In order to solve this problem, when we found cells in prophase, we waited for NEB and then measured the length of prometaphase, metaphase and anaphase. The length of anaphase was measured from the beginning of chromosome separation until the chromosomes reached the cell edge. We found that the length of anaphase was practically unchanged, though the length of prometaphase increased three times and the length of metaphase decreased two times. The duration of prophase was estimated from the proportion of prophases in fixed cells, and we found that prophase length increased approximately twice (Fig. 8B).

Surprisingly, although the length of prophase, prometaphase and metaphase changed significantly, the time between NEB and anaphase onset (beginning of chromosome separation) was constant, 25–26 min in both control and latrunculin-treated cells (Fig. 8B). It seems from our data that when the inter-centrosome distance has reached a value that corresponds to half the metaphase spindle size, the cell waits for the centrosomes to start to turn around the nucleus. As this movement is not initiated without actin filaments, the cell eventually takes the decision to trigger NEB. The data strongly indicate that latrunculin delays centrosome separation when they should start a curving movement around the nuclear envelope, causing elongation of the prophase–prometaphase stages.

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separation. These results suggest that interphase microtubules (1) participate in the splitting stage and (2) slow down the elongation stage until mitosis. In physiological conditions, increase of the p34cdc2 activity that precedes G2/M transition leads to interphase microtubule depolymerisation (Verde et al., 1990). This might contribute to accelerate centrosome separation upon entry into mitosis, just like nocodazole. In contrast, actin filaments seem to actively participate in centrosome separation in interphase, because latrunculin treatment inhibited both splitting and elongation stages of centrosome separation. Centrosome separation in interphase might be controlled by actin-based motor activities.

Fig. 8. Mitosis in the presence of latrunculin-A. (A) The percentage of cells in each mitotic sub-phase (except telophase) has been estimated in control cells or after treatment with latrunculin-A for 6 h. (B) The length of each mitotic sub-phase (excepted telophase) has been estimated by following cells in real time in controls or after treatment with latrunculin-A for 6 h.

In mitosis, the situation is different. First of all, the dynamics of the newly polymerised mitotic microtubules is different from that of interphase microtubules; then specific motor molecules are activated (Vernos and Karsenti, 1996). In mitosis, microtubules are absolutely required for centrosome separation. Actin filaments, on the contrary, are dispensable, but contribute to centrosome separation before NEB. The function of microtubules and actin filaments in centrosome separation during cell cycle progression is illustrated in Fig. 9.

We investigated here the respective contribution of microtubules and actin filaments in the separation of duplicated centrosomes during cell cycle progression. This work was initiated by the question of whether or not actin filaments participate in centrosome separation while they turn around the nucleus to reach opposite positions. This question was raised because microtubules were obviously not sufficient to ensure such a curved movement alone. In order to address this question, we measured the distance between the two centrosomes during cell cycle progression in cells treated with either nocodazole, which depolymerises microtubules, or with latrunculin-A, which depolymerises actin filaments (Spector et al., 1989). Centrosomes were identified by pEg2 (aurora-A) staining that decorates only duplicated centrosomes (Roghi et al., 1998). We first estimated that centrosome separation started at a time in the course of pEg2 appearance in the centrosome region at late interphase and progressed rapidly in prophase and prometaphase.

In the absence of actin filaments, centrosome separation is slowed down during both prophase and prometaphase. This phenomenon leads to an increase in the length of both these mitotic phases. After a longer prophase, during which the distance between centrosomes never exceeds 3.5 µm (half the spindle size), the cell takes the decision to trigger NEB. Why is prophase longer when actin filaments are disrupted? We speculate that progression through prophase/prometaphase transition proceeds as if the cell were waiting for the centrosomes to separate. Because no cell cycle arrest was observed in the absence of actin filaments, we suggest that redundant mechanisms participate in centrosome separation. While microtubules are absolutely required for centrosome separation in mitosis, we suggest that actin filaments accelerate the separation in prophase by allowing movement around the nucleus (Fig. 10). This is reminiscent of the phenotype observed in the Drosophila mutant of the gene twinstar encoding the actin-filament-severing protein cofilin. Mutation of this gene leads to centrosome migration delay and a failure of the two microtubule asters to associate with the nuclear envelope (Gunsalus et al., 1995).

The number of cells containing separated centrosomes dramatically decreased upon treatment with either nocodazole or latrunculin-A. Lacking microtubules or lacking actin filaments, cells were not able to split duplicated centrosomes during interphase. However, depolymerisation of microtubules in cells containing centrosomes that had already started to separate led to an acceleration of the

The most intriguing result was that once the cell took the decision to trigger nuclear envelope breakdown, the delay caused by a longer prometaphase was compensated for by a faster metaphase. During this long prometaphase, the bipolar spindle might be assembled just as in cell free extract: the centrosomes keep separating, while microtubules plus ends are captured by the chromosome kinetochores

4. Discussion

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Fig. 9. Role of microtubules and actin filaments in centrosome separation during interphase and mitosis. Interphase centrosome separation is shown on the left and mitotic centrosome separation on the right.

(Fig. 10). This would explain why the prometaphase duration increases in the absence of actin filaments. Then, when all the kinetochores are attached to microtubules, the cell can trigger anaphase.

also participate in the attachment of centrosomes to the nuclear envelope and in the movement of the centrosomes around the nucleus during prophase.

Actin filaments have been found to participate in spindle orientation and cleavage furrow positioning in yeast (Palmer et al., 1992; Yang et al., 1997). Recent results also suggest that during the syncytial blastoderm stage of Drosophila embryogenesis, centrosomes can organise actin filaments via a centrosome-associated Scrambled protein (Stevenson et al., 2001). Although this remains to be proven, we would like to suggest here that actin filaments

Acknowledgements We thank Dr. E.S. Nadezhdina for critical reading of the manuscript. Rustem Uzbekov would like to thank his daughter Olga for technical assistance. This research work was supported by the CNRS, the “Association pour la Recherche contre le Cancer” (ARC), and “la Ligue Nationale Contre le Cancer” (LNCC).

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Fig. 10. Model for actin filament function in centrosome separation during mitosis (prophase). On the left, mitosis is represented in the absence of actin filaments, compared with a normal mitosis on the right. Centrosomes are represented by red spots and movement by red arrows.

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