1334 Research Article separation driven by - during is mediated by centrosome-associated tyrosine-phosphorylated cortactin

Wenqi Wang*, Luyun Chen*, Yubo Ding, Jing Jin and Kan Liao‡ State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, Peopleʼs Republic of China *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 31 January 2008 Journal of Cell Science 121, 1334-1343 Published by The Company of Biologists 2008 doi:10.1242/jcs.018176

Summary The regulation of tyrosine phosphorylation is an phosphorylation sites in the truncated C-terminus of cortactin important aspect during the . From G2-M transition and found that the C-terminus could no longer interfere with to mitotic , phosphorylation of Tyr421, Tyr466 and centrosome separation process. Our study shows that, cortactin Tyr482 of cortactin, an actin-filament associated protein, is phosphorylated at Tyr421, Tyr466 and Tyr482 mediates the dramatically induced. The phosphorylated cortactin is almost actin-filament-driven centrosome separation at G2-M transition exclusively associated with or spindle poles by providing a bridge between the centrosome and actin- during mitosis. At G2-M transition prior to the breakdown of filaments. the nuclear envelope, two duplicated centrosomes migrate towards opposite ends of the nucleus to form the spindle poles. This centrosome-separation process and also the start of mitosis Supplementary material available online at are inhibited or delayed by the depolymerization of actin http://jcs.biologists.org/cgi/content/full/121/8/1334/DC1 filaments. Also inhibited is the separation of centrosomes when a truncated form of cortactin is expressed, whose C-terminus Key words: Cortactin, Cortactin tyrosine phosphorylation, contains the tyrosine phosphorylation region but lacks the actin- Centrosome separation, Actin-filament, points, Src

Journal of Cell Science binding domains. We introduced mutations at the

Introduction regulation of cell motility and formation of lamellipodia and Cortactin is an actin-filament-binding protein (Olazabal and membrane ruffles (Lua and Low, 2005). Machesky, 2001; Weed and Parsons, 2001). It has initially been During mitosis the complex sequence of events is dominated by described as a tyrosine-phosphorylated protein in cells infected with the formation and activity of the microtubule spindle. The initiation v-Src and is a direct substrate of Src kinase (Kanner et al., 1990; of the microtubule spindle is governed by the centrosome, also Wu et al., 1991). Its association with F-actin stabilizes the known as the microtubule-spindle-organization center (Bornens, network (Huang et al., 1997; Olazabal and Machesky, 2002; Rieder et al., 2001; Kellogg et al., 1994; Joshi, 1993; Mishima 2001; Pant et al., 2006). Of the all the different domains in cortactin, et al., 2004). Consequently, the separation and positioning of the N-terminal acidic region, the F-actin-binding repeats, the helix centrosomes determines the formation and orientation of spindles. domain and the SH3 domain at the C-terminus are relatively The centrosome cycle progresses in a precise order: (1) the conserved from invertebrates to vertebrates (Weed and Parsons, centrosome is duplicated at G1-S transition, (2) cohesion between 2001). The proline-rich domain between the helix domain and C- two duplicated centrosomes is broken in , (3) two terminal SH3 domain, however, is only conserved in vertebrates, centrosomes migrate apart in G2-M transition and the separated but not between vertebrates and invertebrates (Weed and Parsons, centrosomes initiate the spindle microtubules to form bipolar 2001). The proline-rich domain of Drosophila melanogaster has spindle in early mitotic phase (Meraldi and Nigg, 2002; Tsou and only 15% homology with its counterpart in mouse. The proline- Stearns, 2006). Therefore, the separation and positioning of rich domain of murine cortactin has six identified phosphorylation centrosomes are crucial for the correct formation of a bipolar spindle, sites (four tyrosine sites and two serine sites) (Huang et al., 1997; which governs the segregation and the fate of daughter Head et al., 2003; Lua and Low, 2005; Martin et al., 2006). All cells. four identified tyrosine phosphorylation sites are missing in In order to drive the centrosomes to opposite ends of nucleus, Drosophila cortactin (Genebank: AAF55840). The current motion force needs to be generated. It has been reported that actin knowledge regarding the function of cortactin and its tyrosine microfilaments and myosin II are required for centrosome separation phosphorylation mostly concerns actin-network organization, and positioning during mitosis, thus making them ideal candidates Cortactin and centrosome separation 1335

for providing the migration force (Rosenblatt et al., 2004; Burakov constant throughout the cell cycle (Fig. 1B). Similar phosphorylation et al., 2003; Waters et al., 1993). When F-actin is depolymerized and dephosphorylation on Tyr466 and Tyr482 was also observed by addition of latrunculin, centrosome separation is blocked and (data not shown). cells enter mitosis with unseparated centrosomes (Uzbekov et al., In the mitotic phase, the spindle structure is predominantly 2002). In HeLa cells, one major pathway for centrosome separation formed by microtubules, whereas no distinct structure is formed by that occurs prior to nuclear-envelope breakdown is dependent on actin-filaments. As an F-actin-associated protein, cortactin was an intact actin (Whitehead et al., 1996). These studies evenly distributed in of mitotic cells (Fig. 2A and indicate or imply that actin-filaments provide the essential driving supplementary material Fig. S1). However, P-Tyr-cortactin was force for centrosome separation, which means that a regulatory almost exclusively located at the mitotic spindle poles, identified attachment of actin-filaments to centrosome is required at G2-M by staining for γ-tubulin (Fig. 2B) (Joshi, 1993). Individual transition. Here we show that cortactin phosphorylation at tyrosine antibodies against cortactin phosphorylated at Tyr421 (P-Tyr421- residues is induced in mitotic cells and phospho-cortactin (P- cortactin), Tyr466 (P-Tyr466-cortactin) or Tyr482 (P-Tyr466- cortactin) is associated with centrosomes or spindle poles at G2-M cortactin) (anti-P-Tyr421-cortactin, anti-P-Tyr466-cortactin or anti- transition until mitotic anaphase. We found that the cortactin C- P-Tyr482-cortactin antibodies, respectively) were used to stain terminal region, which can bind to centrosomes but does not mediate spindle poles (Fig. 2B). When mitotic spindles were revealed using the F-actin association, inhibited the centrosome separation prior anti-α-tubulin antibody, P-Tyr-cortactin was found to be located to nuclear envelope breakdown. We also found P-cortactin to be exactly at the spindle poles (Fig. 2C). P-cortactin, which is anchored at the tips of actin fibers in interphase cell. These associated with the spindle poles, was phosphorylated at least on observations indicate that centrosome or spindle-pole-associated P- one of the three Src kinase sites, at Tyr421, Tyr466 or Tyr482 (Fig. cortactin provide the anchor on the centrosome or spindle pole for 2B) (Kanner et al., 1990; Wu et al., 1991; Huang et al., 1997). F-actin attachment. However, this association did not enrich the spindle poles with just phosphorylated tyrosine residues (Fig. 2D). The spindle poles were Results distinct for P-Tyr-cortactin (Fig. 2B,C). Even the overexposed Induction of tyrosine phosphorylation of cortactin, and immunofluorescence images show that P-Tyr-cortactin was only association of P-Tyr-cortactin with centrosomes or spindle detected at spindle poles (supplementary material Fig. S1). poles in G2-M cells Besides the staining for P-Tyr-cortactin at centrosomes and In our study of hormone-induced 3T3-L1 adipocyte differentiation spindle poles, we detected an interaction between P-Tyr-cortactin and mitotic clonal expansion (Jin et al., 2000), an accumulated and the centrosomal protein γ-tubulin (Fig. 1C). Anti-P-Tyr421- tyrosine-phosphorylated protein was identified as P-Tyr-cortactin cortactin antibody immunoprecipitated γ-tubulin in mitotic cells and (data not shown). In hydroxylurea synchronized 3T3-L1 cells, we anti-γ-tubulin antibody immunoprecipitated P-Tyr-cortactin (Fig. observed cell-cycle-dependent phosphorylation of cortactin at 1C). In addition, centrosomal fraction isolated by sucrose-density Tyr421 (Fig. 1A,B). Levels of P-Tyr-cortactin were low in gradient from nocodazole-arrested mitotic cells contained P-Tyr- interphase, G1-S transition or S phase cells. However, it was cortactin (Fig. 1D) (Moudjou and Bornens, 1994). Furthermore, in dramatically increased after cells reached G2 phase. The P-cortactin cells expressing eGFP-tagged cortactin, GFP-cortactin fusion

Journal of Cell Science was then dephosphorylated when the cells finished mitosis and protein was visualized at the spindle pole (supplementary material returned to (Fig. 1B). Cortactin protein levels were Fig. S1).

Fig. 1. Cell-cycle dependent cortactin phosphorylation at Tyr421. (A) Hydroxylurea-synchronized 3T3-L1 cells. Int, control interphase cells; HU, cells arrested by hydroxylurea. (B) Cortactin phosphorylation at Tyr421 during the cell cycle. Whole-cell extracts were blotted withanti-P-Tyr421-cortactin antibody (α-PY421C) or anti-cortactin antibody (α-Cort). (C) Co-immunoprecipitation of P-Tyr421-cortactin with γ-tubulin in nocodazole-arrested mitotic cells. IP, immunoprecipitation; IB, western blot. Total, cell extract before immunoprecipitation. (D) Co-purification of P-Tyr421-cortactin with centrosomes of mitotic cells. Centrosomes were isolated from nocodazole-arrested mitotic cells by using sucrose-density centrifugation. Samples of each fraction were blotted with anti-γ-tubulin antibody (α-γTu), anti-P-Tyr421-cortactin antibody (α-PY421C) or anti-cortactin antibody (α-Cort). 1336 Journal of Cell Science 121 (8)

The detection of both cortactin and P-Tyr-cortactin in isolated and supplementary material Fig. S1) supported the conclusion that centrosomes (Fig. 1C,D) and at centrosomes or spindle poles (Fig. 2 it is the tyrosine-phosphorylated form of cortactin, revealed by anti- P-cortactin antibodies in immunofluorescence staining. To further verify the specificity of anti-P-cortactin antibodies for P-Tyr- cortactin, four identified tyrosine phosphorylation sites were mutated to phenylalanine (Tyr421Phe, Tyr466Phe, Tyr475Phe and Tyr482Phe) in murine cortactin (Huang et al., 1997; Head et al., 2003; Martin et al., 2006) (supplementary material Fig. S2). Anti- P-Tyr421-cortactin and anti-P-Tyr466-cortactin antibodies (both from Sigma) were able to detect tyrosine-phosphorylated exogenous wild-type cortactin fused to GFP (GFP-cortactin) as well as endogenous cortactin (supplementary material Fig. S2). Exogenous mutated GFP-cortactin fusion protein containing the four Phe substitutions was not detected by either antibody. Most importantly, tyrosine phosphorylation on exogenous GFP-cortactin containing only Tyr421 was detected by anti-P-Tyr421-cortactin antibody but not anti-P-Tyr466-cortactin antibody, whereas tyrosine phosphorylation on exogenous GFP-cortactin containing only Tyr466 was detected by anti-P-Tyr466-cortactin antibody but not anti-P-Tyr421-cortactin antibody (supplementary material Fig. S2). These results unequivocally established the specificity of anti- P-Tyr421-cortactin and anti-P-Tyr466-cortactin antibodies for their respective P-Tyr-cortactin. Moreover, two additional antibodies against P-Tyr421-cortactin and P-Tyr466-cortactin from BioSource, which have been characterized (Head et al., 2003), were used for immunofluorescence staining and compared with the antibodies in our current study. As shown in supplementary material Fig. S2, the use of antibodies from either supplier (Sigma nad BioSource) resulted in identical staining of centrosomes or spindle poles. Our observation that GFP-cortactin localizes at spindle poles together with the confirmed antibody specificity (as characterized by the mutated cortactins; supplementary material Figs S1, S2), it seemed extremely unlikely that the described P- Tyr-cortactin immunostaining found at centrosomes or spindle

Journal of Cell Science poles was due to non-specific staining of other . In fact, no centrosome-associated P-cortactin was observed by immunofluorescence in 3T3-L1 cells whose cortactin expression had been suppressed (supplementary material Fig. S3). This observation clearly points against non-specific centrosomal staining when using anti-P-cortactin antibodies in immunofluorescence analysis. The centrosome has its own duplication cycle: at G1-S transition one centrosome is duplicated into two centrosomes; from S to early G2 phase the duplicated centrosomes are cohered; at G2-M transition the cohesion between two centrosomes is broken and two centrosomes migrate apart to form the spindle poles at opposite ends of nucleus (Meraldi and Nigg, 2002; Tsou and Stearns, 2006; Mishima et al., 2004). Accordingly, there was little P-Tyr-cortactin in G1-phase, G1-S-transition and S-phase cells (Fig. 1B). Neither was centrosome(s) in G1 and S phase associated by P-cortactin (Fig. Fig. 2. Centrosome and/or spindle-pole localization of P-Tyr421-cortactin, P- 3A,B). At G2-M transition, when chromatin was visibly condensed, Tyr466-cortactin and P-Tyr482-cortactin in mitotic 3T3-L1 cells. (A) Double- the nuclear envelope was still intact and centrosomes had not immunofluorescence staining of cortactin using α-tubulin or actin antibodies. separated, P-cortactin was already associated with centrosomes (Fig. (B) Double-immunofluorescence staining using anti-P-Tyr421-cortactin 3B). From G2-M transition to metaphase the P-cortactin association (PY421C), anti-P-Tyr466-cortactin (PY466C) or anti-P-Tyr483-cortactin (PY482C) antibodies and anti-γ-tubulin antibody (γ-Tu). (C) Double- was increased as centrosomes were separated and spindles were immunofluorescence staining using anti-P-Tyr421-cortactin (PY421C) or anti- formed (Fig. 3A,B). When spindles gradually disintegrated during P-Tyr466-cortactin (PY466C) antibodies and anti-α-tubulin (α-Tu) antibody the transition from anaphase to telophase, the association of P- (D) Phosphotyrosine proteins and spindle poles. Metaphase 3T3-L1 cell was cortactin with spindle poles was also diminished (Fig. 3A,B). stained with anti-phosphotyrosine antibody (PY), anti-γ-tubulin antibody (γ- Tu) and DAPI. D, DAPI; C, anti-cortactin antibody; A, anti-actin antibody; α- Dissociation of P-cortactin from telophase centrosomes correlated Tu, anti-α-tubulin antibody; M, the merged picture of panels D, C and A (or α- with the dephosphorylation of P-cortactin after mitosis (Fig. 1B). Tu). Scale bars: 10 μm. The same association and dissociation for P-Tyr466-cortactin and Cortactin and centrosome separation 1337

P-Tyr482-cortactin was also observed during mitosis in 3T3-L1 cell during mitosis, Src kinase is activated and associated with (data not shown). centrosomes (David-Pfeuty and Nouvian-Dooghe, 1990; David- Of four tyrosine phosphorylation sites (Tyr421, Tyr466, Tyr475, Pfeuty et al., 1993; Kuga et al., 2007). We found also that activated Tyr482) in murine cortactin (Kanner et al., 1990; Wu et al., 1991; Src kinase is dynamically associated with mitotic spindle poles Huang et al., 1997), we detected phosphorylation at all three Src during mitosis in 3T3-L1 cells (supplementary material Fig. S3). kinase sites (Tyr421, Tyr466, Tyr482) in centrosome-associated P- The association of P-cortactin with centrosomes or spindle poles cortactin (Fig. 2B). The tyrosine phosphorylation pattern that correlated closely with the association of activated Src kinase determines centrosomal association for P-cortactin is currently under (supplementary material Fig. S3). This suggested that regulation of investigation. Src kinase is activated by autophosphorylation on tyrosine phosphorylation of cortactin occurs on the centrosomes or Tyr418 (Roskoski, Jr, 2004). It has been described previously that, spindle poles. In the spindle pole, the associated P-cortactin was Journal of Cell Science

Fig. 3. The dynamic association of P-Tyr-cortactin with centrosome or spindle poles during mitosis in 3T3-L1 cells. (A) Association of P-Tyr421-cortactin with spindle poles in mitotic phases. Nuclei were stained with DAPI (blue), anti-P-Tyr421-cortactin (red) and anti-α-tubulin (green) antibodies. The insertion in the G2- M panel is an enlarged spindle pole or centrosome. (B) Dynamic association of P-cortactin with centrosomes or spindle poles. Arrowheads indicate the staining of centrosomes by using anti-γ-tubulin antibody (γ-Tu) and P-Tyr421-cortactin (PY421C). Insertions are enlarged views of the centrosomes. (C) Spindle poles. Staining was for P-Tyr421-cortactin (PY421C) and anti-α-tubulin (α-Tu). (D) Localization of P-Tyr421-cortactin, P-Tyr466-cortactin and P-Tyr482-cortactin in . Cells expressing transfected GFP tagged centrin 2 (GFP-Cetn2) were stained with anti-P-Tyr421-cortactin, P-Tyr466-cortactin or P-Tyr482- cortactin antibodies. GFP-Centrin 2 was visualized by eGFP fluorescence. Scale bars: 10 μm. 1338 Journal of Cell Science 121 (8)

located at the centrosome but not in the peripheral α-tubulin mass entry and can be indicated by increased phosphorylation of histone (Fig. 3C). As revealed by eGFP-tagged centrin 2, P-cortactin within 3 (Ajiro et al., 1996). Histone 3 phosphorylation was clearly blocked the centrosome was in the pericentriolar material but not associated by treatment with cytochalasin B (Fig. 5A). Twelve hours after directly to the pair of (Fig. 3D). thymidine release, control HeLa cells completed the mitosis as cyclin B1, which is highly expressed in G2-M cells and degraded Centrosome separation is blocked by F-actin depolymerization after the completion of mitosis (Smits and Medema, 2001), started and correlates with P-cortactin association to decrease (Fig. 5A). However, cyclin B1 levels in cytochalasin- Centrosome separation prior to nuclear envelope breakdown is B-treated cells remained high 12 hours after thymidine release (Fig. driven by actin-filaments but not microtubules (Rosenblatt et al., 5A). P38, which is activated at spindle-assembly check point 2004; Burakov et al., 2003; Uzbekov et al., 2002; Whitehead et al., (Takenaka et al., 1998), was less activated in cytochalasin-B-treated 1996; Waters et al., 1993). Since cortactin is an F-actin-associated cells (Fig. 5A). Thus, actin-filaments depolymerization inhibited protein and its tyrosine phosphorylated form was associated with the mitotic entry at stage of centrosome separation and chromatin centrosomes prior to their separation (Fig. 3B), the function of condensation prior to spindle assembly. centrosome-associated P-cortactin in centrosome separation driven In the temporal order, cortactin phosphorylation was induced in by actin-filaments was examined. HeLa cells were arrested at G1- G2 phase and concomitantly associated with centrosome (Fig. 1B, S transition by thymidine (Fig. 4A). By 8 hours after thymidine Fig. 3B and Fig. 5B). It might be phosphorylated by activated Src release, the synchronized cells progressed through S phase and kinase at the centrosomes (supplementary material Fig. S3). The reached G2 phase (Fig. 4A). During this time the centrosomes were centrosomes isolated from HeLa cells that were blocked at G1-S not separated and the average distance between two cohesive transition by thymidine contained no P-cortactin, whereas centrosomes was less than 2 μm (Fig. 4C). Ten hours after centrosomes isolated from mitotic HeLa cells showed association thymidine release cells progressed into mitosis and centrosome with P-cortactin (Fig. 5C). Since the biochemical isolation of separation was observed (Fig. 4A). The average distance between centrosomes could not completely eliminate other cellular two separated centrosomes in the synchronized cell population was organelles, the association of P-cortactin with cohesive centrosomes ~10 μm (Fig. 4C). The centrosome separation process in a and separated centrosomes was further analyzed in synchronized cell population can be better illustrated by plotting immunofluorescence studies using anti-P-cortactin antibodies. The the centrosome-separation ratio (distance between two centrosomes average distance between two cohesive centrosomes was 1.8 μm vs nuclear diameter) for individual cells (Fig. 4B,D). Before (Fig. 4C). If P-cortactin mediates the attachment of actin-filaments reaching G2-M transition (4-8 hours after thymidine release), the that pull centrosomes apart, most centrosomes that are separated centrosome-separation ratio was less than 0.1 in most cells (Fig. by more than 1.8 μm should be associated with P-cortactin. As 4D). At G2-M transition (10 hours after thymidine release), shown in Fig. 5D, there is a clear correlation between P-cortactin centrosome separation was clearly visible and the centrosome- association and centrosome separation. Almost all separated separation ratio was shifted towards 1.0 (Fig. 4A,D). centrosomes were associated with P-cortactin, whereas two-thirds However, in the presence of cytochalasin B, which of the cohered centrosomes were not associated with P-cortactin depolymerizes actin-filaments, centrosome separation and mitotic (Fig. 5D).

Journal of Cell Science entry were delayed or blocked (Fig. 4A, Fig. 5A). Cells progressed normally through S phase to G2 phase in the presence of Centrosome separation is blocked by the wild-type cortactin cytochalasin B (4-8 hours after thymidine release), but could not C-terminus but not by the cortactin C-terminus containing reach G2-M transition at 10 hours after thymidine release. The mutated Tyr residues centrosome separation in the presence of cytochalasin B was The Tyr phosphorylation sites in cortactin are located at the C- blocked as most cells in the synchronized cell population had low terminal proline-rich domain. We therefore created a truncated a centrosome-separation ratio (Fig. 4D). F-actin depolymerization version of the cortactin C-terminus lacking its F-actin-binding appeared to inhibit centrosome separation prior to nuclear envelope repeats (Fig. 6A). Thus, the phosphorylated cortactin C-terminus breakdown. The same results were also obtained with the treatment can still bind to centrosomes albeit without providing binding for of latrunculin A, another F-actin depolymerization reagent (data F-actin. By competing-off wild-type P-cortactin from centrosomes, not shown). the truncated cortactin C-terminus should have a dominant-negative Interestingly, the cohesion between two duplicated centrosomes effect on P-cortactin-mediated centrosome separation (Fig. 6C). was broken when cells were treated with nocodazole to Centrosomes labeled with the eGFP-tagged cortactin C-terminus depolymerize microtubules (Fig. 4A). In the presence of nocodazole, were observed in HeLa cells expressing the fusion protein (Fig. two centrosomes were randomly separated even through thymidine- 6B), and centrosome separation was clearly also inhibited (Fig. synchronized cells were still in S phase (Fig. 4A,C,D). Without the 6D,E). restriction from intact microtubule system the centrosome separation Since the association of cortactin with centrosome depends on appeared to be a default process, because depolymerization of tyrosine phosphorylation, the substitution of tyrosine residues for microtubules by using nocodazole or of both microtubules and phenylalanine prevents interaction of the cortactin C-terminus with microfilaments by using nocodazole and cytochalasin B induced the centrosome and should abolish its dominant-negative effect on centrosome separation at any cell-cycle phases (Fig. 4A,C,D). centrosome separation (Fig. 6C). In HeLa cell expressing the eGFP- Microtubules appeared to provide the cohesion between two tagged mutated cortactin C-terminus, no association with duplicated centrosomes and restrict the premature centrosome centrosomes was observed (Fig. 6B), and centrosome separation separation. progressed normally when they were thymidine-synchronized (Fig. Actin-filament depolymerization by cytochalasin B not only 6D,E). Therefore, only the wild-type cortactin C-terminus but not blocked centrosome separation, but also inhibited mitotic entry in that containing mutated Tyr residues has the ability to inhibit HeLa cells (Fig. 5A). Chromatin condensation signals the mitotic centrosome separation. Cortactin and centrosome separation 1339 Journal of Cell Science

Fig. 4. Centrosome separation is blocked by depolymerization of actin-filaments in HeLa cells. (A) HeLa cells that had been through two rounds of thymidine synchronization were released in the presence of cytochalasin B (+cyto B), nocodazole (+noco) or both (+nono & ctyo B). At indicated times (4 hours, 6 hours, 8 hours and 10 hours), control cells and regeant-treated cells were harvested for flow cytometry and immunofluorescence staining with anti-γ-tubulin (γT) and DAPI. Scale bars: 10 μm. (B) Measurements of the distance between (d) two centrosomes and (D) the corresponding nuclear diameter. (C) Average distance of two centrosomes and nuclear diameter of 200 cells. Measurements were conducted using fluorescence images similar to those shown in A. (D) Plotting of the centrosome-separation ratio (distance between two centrosomes per nuclear diameter, d/D). In each experiment, 200 cells were plotted. 1340 Journal of Cell Science 121 (8)

Anchorage of actin fibers on P-Tyr-cortactin exposure time for P-cortactin at spindle poles was not enough to The dominant-negative effect of the cortactin C-terminus on reveal its presence at actin fiber tips (Fig. 7D and supplementary centrosome separation indicated that the F-actin-binding capacity material Fig. S5). Although no direct attachment of visible actin of cortactin is essential for P-cortactin in centrosome separation fibers to mitotic centrosomes or spindle poles was visualized – (Fig. 6D,E), because P-cortactin associated with a centrosome might because of the disintegration of actin stress fibers in mitotic phases serve as the binding site for actin-filaments in order to pull – we detected interaction between P-Tyr-cortactin and actin by centrosomes. In COS-7 or NIH3T3 cells, actin stress fibers can be immunoprecipitation, and the centrosome or spindle pole was the readily stained by phalloidin and visualized (Fig. 7 and most prominent site of P-cortactin in mitotic cells (Fig. 7D,E). supplementary material Fig. S5). Cortactin per se did not exhibit Inhibition of centrosome separation by the wild-type cortactin C- any distinct pattern of actin fiber association (Fig. 7A,C). However, terminus – which can associate with centrosomes but not with F- P-cortactin was exclusively present at the ends of actin fibers (Fig. actin – clearly supports the role of P-cortactin as an anchor for 7A,C). P-cortactin anchored the actin fibers to the focal adhesion actin-filaments in order to pull centrosomes apart. points, which were revealed by staining (Fig. 7B). All three Src kinase sites in cortactin (Tyr421, Tyr466, Tyr482) were Discussion phosphorylated in the P-cortactin that had associated with focal Owing to the high visibility of the microtubule spindle during adhesion points (Fig. 7B). Results from cortactin knockdown cells, mitosis, the function of centrosome in spindle formation is well Src-kinase-transfected cells and cells expressing GFP-cortactin elucidated (Bornens, 2002; Rieder et al., 2001; Kellogg et al., 1994; fusion protein provided evidence for the specificity of P-cortactin Joshi, 1993; Mishima et al., 2004). However, much less studied is at focal adhesion points (supplementary material Fig. S4). how the duplicated centrosomes migrate and position themselves During mitosis, P-cortactin was no longer associated with on opposite sides of the nucleus before the microtubule spindle is paxillin because focal adhesion points were most probably assembled. In contrast to the hypothesis that microtubules are disintegrated (Fig. 7D). Instead, most P-cortactin was localized at extended between two centrosomes to push them apart, our results the spindle poles (Fig. 7D). In fact, the spindle-pole-associated P- from the nocodazole treatment suggests that the microtubules cortactin was so concentrated that the suitable microscopic stabilize the cohesion between two duplicated centrosomes and Journal of Cell Science

Fig. 5. Correlation between P-cortactin association and centrosome separation. (A) Analysis of cell-cycle marker proteins. Cell extracts from thymidine- synchronized HeLa cells (isolated after thymidine-release at the time points indicated) was western blotted for cyclin B, phosphorylated histone 3 (P-histone3), p38/MAP kinase (p38), phosphorylated p38 (P-p38) or α-tubulin as loading control. +Cytochalasin B, thymidine-release in the presence of cytochalasin B. (B) Immunofluorescence staining of P-cortactin and γ-tubulin in HeLa cells. Proliferating HeLa cells were simultaneously stained for P-Tyr421-cortactin (PY421C) and γ-tubulin (γ-Tu), nuclei were stained with DAPI. Scale bars: 10 μm. (C) P-cortactin in isolated centrosomes from mitotic cells or G1-S cells. Centrosomes were isolated from thymidine-arrested G1-S-phase HeLa cells or from nocodazole-arrested mitotic cells by using sucrose-density centrifugation. T, total cell extract; P, last fraction with the bottom pellet; 1-11, fractions from the gradient. (D) Association of P-cortactin in centrosomes. HeLa cells released from thymidine arrest for 10 hours were stained as described for B. Cells with intact nuclear envelopes were analyzed for P-cortactin association and centrosome separation. In each group 50 cells were analyzed and the results of three experiments were averaged. d, distance between two centrosomes. Cortactin and centrosome separation 1341

prevent premature centrosome separation (Fig. 4). Other research has also found that the movement of centrosome separation is not owing to the interaction of microtubules from opposing asters (Waters et al., 1993). That actin-filaments drive centrosome separation before spindle assembly has been suggested by other researchers and is supported by our current study (Rosenblatt et al., 2004; Burakov et al., 2003; Uzbekov et al., 2002; Whitehead et al., 1996; Waters et al., 1993) (Fig. 4). Since the association of P-Tyr- cortactin with centrosomes precedes centrosome separation (Fig. 5) and actin fibers anchor on P-Tyr- cortactin (Fig. 7), actin-filaments might attach to P- cortactin on centrosomes and then provide the force to drive centrosomes apart. Inhibition of centrosome separation by the wild-type cortactin C-terminus – which can associate with centrosomes but cannot bind to actin-filaments – strongly supports the idea that P-cortactin anchors actin-filaments to centrosomes (Fig. 6). The accumulation of P-Tyr- cortactin on centrosomes attracts actin-filaments during mitosis (Figs 2,3). Based on these observations, we conclude that the actin-filaments attached to P-cortactin on unseparated centrosomes pull the centrosomes apart before the nuclear envelope breaks down (Fig. 8). Centrosome separation happens at G2-M transition and, presumably, the exact temporal regulation of F-actin attachment to the centrosome is crucial for mitotic progression. Association of P- Tyr-cortactin but not cortactin per se with the centrosome meets this prerequisite for the regulated F-actin attachment. Both tyrosine phosphorylation

Journal of Cell Science of cortactin and the association of P-cortactin with the centrosome starts at G2-M transition, just before the centrosomes separate (Figs 1, 3, 5). All three identified Src kinase phosphorylation sites in cortactin (Tyr421, Tyr466, Tyr482) are phosphorylated in centrosome-associated P-cortactin (Fig. 2B). Activated Src kinase was also dynamically associated with the centrosome and/or spindle poles during mitosis (supplementary material Fig. S3) (David-Pfeuty and Nouvian-Dooghe, 1990; David- Pfeuty et al., 1993; Kuga et al., 2007). Kinetically, Fig. 6. Centrosome separation is blocked by the wild-type cortactin C-terminus but not by the cortactin C-terminus containing mutated Tyr residues. (A) Wild-type cortactin and cortactin C- the association and dissociation of P-Tyr-cortactin terminus mutants. N-terminus, amino acids (aa) 1-329; C-terminus, aa 330-546; CT(Y/F), aa with the centrosome or spindle pole was closely 330-546 with Phe substitutions for Tyr421, Tyr466, Tyr475 and Tyr482. (B) HeLa cells correlated to that of activated Src kinase during transfected with GFP-CT or GFP-CT(Y/F). Arrowheads indicate the centrosomes. The mitosis (supplementary material Fig. S3). The role insertions are the enlarged views of the centrosomal area. Scale bars: 10 μm. (C) Illustration of the dominant-negative effect the cortactin C-terminal domains exert on centrosome separation. of Src kinase as a regulator of cell growth is well (D) Inhibition of centrosome separation brought upon by the Myc-tagged C-terminus of wild- established. As an oncogene product, high Src kinase type cortactin (CT) but not the cortactin C-terminus containing mutated Tyr residues [CT(Y/F)]. activity promotes cell transformation. In cancer The centrosome-separation ratio (d/D) was measured from 100 Myc-positive cells and plotted as cells, many oncogene kinases are highly activated described for Fig. 4. (E) Average centrosome separation 10 hours after thymidine- and their regulation is disrupted. As such, the synchronization release in HeLa cells expressing Myc-tagged C-terminus of wild-type cortactin (CT) C-terminus containing mutated Tyr residues [CT(Y/F)]. The distances of two centrosomes regulation of cortactin phosphorylation can also be (d) and corresponding nuclear diameters (D) of 100 cells were averaged for each group of cells. expected to be disrupted. With abnormal cortactin phosphorylation, the incidences of abnormal centrosome separation and abnormal spindle formation would be The amino acid sequence of murine cortactin contains a stretch increased, which would lead to abnormal chromosome segregation. of ten Tyr residues in the C-terminal region (amino acids 421-499; It is well-known that polyploidy or aneuploidy is a common event positions 421, 433, 442, 460, 466, 475, 482, 485, 497, 499). These in cancer cells. Tyr residues are relatively conversed between mouse, human and 1342 Journal of Cell Science 121 (8)

Fig. 7. P-cortactin anchors actin stress fibers in COS-7 and NIH3T3 cells at interphase. (A) NIH3T3 cells were stained for P-Tyr421-cortactin (PY421C), cortactin (Cort) or F-actin (using phalloidin); nuclei were stained with DAPI. M, merged images. Scale bars: 50 μm. (B) NIH3T3 cells were stained for P-Tyr421-cortactin (PY421C), P-Tyr466- cortactin (PY466C) or P-Tyr482-cortactin (PY482C) together with anti-paxillin (PAX) and F-actin (using phalloidin); nuclei were stained with DAPI. M, merged images. Scale bars, 50 μm. (C) Merged images of HIN3T3 cells stained for F-actin together with cortactin or P-cortactin. Scale bars, 10 μm. (D) COS-7 cells stained for P-Tyr421- cortactin (PY421C), paxillin (PAX), or F-actin (using phalloidin); nuclei were stained with DAPI. M, merged images. Arrowheads indicate centrosomes associated with P-cortactin. Scale bars, 50 μm (in interphase cells, top panels), 10 μm (in metaphase cells, bottom panels). (E) Co-immunoprecipitation of P-cortactin and actin. 3T3-L1 cells were homogenized and sonicated. The cell lysate was immunoprecipitated (IP) using anti-cortactin antibody (α-Cort) or anti- phosphotyrosine421 cortactin antibody (α-PY421C). IB, western blot ; T, total cell lysate; IP, immunoprecipitated sample. Journal of Cell Science chicken, but not Drosophila or other invertebrates. There are only on certain tyrosine residues determines the cellular localization of five Tyr residues (Tyr449, Tyr486, Tyr495, Tyr507, Tyr509) in P-cortactin and regulates its function in vertebrate . Drosophila. However, none of those positions correspond to the In conclusion, our results indicate a new mechanism and Tyr phosphorylation sites identified in murine cortactin (Tyr421, regulation of F-actin-mediated centrosome separation, as well as a Tyr466, Tyr475, Tyr482). Given the important regulatory new function for cortactin and the regulation of its phosphorylation. functions for Tyr421, Tyr466 and Tyr482 phosphorylation, the Cortactin tyrosine phosphorylation is often associated with dynamics phosphorylation of other tyrosine residues requires further of actin networks (Weed and Parsons, 2001; Daly, 2004; Lua and investigation. It may well be that the combination of phosphorylation Low, 2005). Regulation of cortactin phosphorylation might be

Fig. 8. Model of P-cortactin- mediated actin attachment for centrosome separation. At G2-M transition, centrosome-associated P- cortactin provides the anchor for actin filament attachment. The attached actin filaments provide the motion force to pull centrosomes apart. Cortactin and centrosome separation 1343

involved in the reorganization of the cytoskeleton during mitosis. and 2006CB910700 from the Ministry of Sciences and Technology of As actin fibers were also found to be anchored at focal adhesion China. points through P-cortactin (Fig. 7), P-cortactin could be a general anchoring site for actin fibers. In addition, P-cortactin association References was highest in metaphase spindle poles (Fig. 3B and supplementary Ajiro, K., Yoda, K., Utsumi, K. and Nishikawa, Y. (1996). Alteration of cell cycle- dependent histone phosphorylations by okadaic acid. J. Biol. Chem. 271, 13197-13201. material Fig. S3). Its function at mitotic spindles requires further Bornens, M. (2002). Centrosome composition and microtubule anchoring mechanisms. investigation. Curr. Opin. Cell Biol. 14, 25-34. Burakov, A., Nadezhdina, E., Slepchenko, B. and Rodionov, V. (2003). Centrosome positioning in interphase cells. J. Cell Biol. 162, 963-969. Materials and Methods Daly, R. J. (2004). 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Journal of Cell Science ethanol or 4% paraformaldehyde, treated with RNaseA, and stained with propidium 119, 2851-2853. iodide. Immunofluorescence staining, immunoprecipitation and western blot analysis Meraldi, P. and Nigg, E. A. (2002). The centrosome cycle. FEBS Lett. 521, 9-13. were carried out following a previously described protocol (Huo et al., 2003). Mishima, M., Pavicic, V., Gruneberg, U., Nigg, E. A. and Glotzer, M. (2004). Cell cycle regulation of central spindle assembly. Nature 430, 908-913. Centrosome isolation Moudjou, M. and Bornens, M. (1994). Cell Biology: A Laboratory Handbook. New York: μ 3T3-L1 cells were arrested at mitotic phase by treating them with 1 M nocodazole Academic Press. for 24 hours and 1 μg/ml cytochalasin B for the final 1 hour. Centrosomes were Olazabal, I. M. and Machesky, L. M. (2001). Abp1p and cortactin, new “hand-holds” isolated by one round of 60% sucrose cushion centrifugation and the second round for actin. J. Cell Biol. 154, 679-682. of 30%, 50% and 70% sucrose step-gradient centrifugation (Moudjou and Bornens, Pant, K., Chereau, D., Hatch, V., Dominguez, R. and Lehman, W. (2006). Cortactin 1994). The second gradient was 5 ml and the fractions were collected from the top binding to F-actin revealed by electron microscopy and 3D reconstruction. J. Mol. Biol. by 1 ml/fraction for fractions 1-3, and 0.2 ml/fraction for fractions 4-12. 359, 840-847. For isolation of centrosomes at G1-S transition or of mitotic centrosomes from Rieder, C. L., Faruki, S. and Khodjakov, A. (2001). The centrosome in vertebrates: more HeLa cell, cells were blocked at G1-S transition by addition of 2 mM thymidine for than a microtubule-organizing center. Trends Cell Biol. 11, 413-419. 24 hours or by addition of 100 ng/ml nocodazole for 24 hours. At the final hour, 1 Rosenblatt, J., Cramer, L. P., Baum, B. and Mcgee, K. M. (2004). Myosin II-dependent μg/ml cytochalasin B and 100 ng/ml nocodazole were added to depolymerize the cortical movement is required for centrosome separation and positioning during mitotic cytoskeleton. spindle assembly. Cell 117, 361-372. Roskoski, R., Jr (2004). Src protein-tyrosine kinase structure and regulation. Biochem. Biophys. Res. Commun. 324, 1155-1164. Construction and expression of cortactin mutants Smits, V. A. J. and Medema, R. H. (2001). Checking out the G2/M transition. Biochim. Murine cortactin cDNA was isolated from 3T3-L1 cells. The cortactin N-terminal Biophys. Acta 1519, 1-12. comprises amino acids 1-329 that contain the N-terminal acidic region and six-and- Takenaka, K., Moriguchi, T. and Nishida, E. (1998). Activation of the protein kinase a-half actin binding repeats. The cortactin C-terminal comprises amino acids 330- p38 in the spindle assembly checkpoint and mitotic arrest. Science 280, 599-602. 546, containing the helix, proline-rich region and SH3 domain. CT(Y/F) is the Tsou, M. B. and Stearns, T. (2006). Controlling centrosome number: licenses and blocks. cortactin C-terminus with Phe substitutions for Tyr421, Tyr466, Tyr475, Tyr482 . Curr. Opin. Cell Biol. 18, 74-78. The C-terminus was fused to GFP to yield the GFP-CT fusion protein or was Myc- Uzbekov, R., Kireyev, I. and Prigent, C. (2002). Centrosome separation: respective role tagged to yield CT-myc-tagged cortactin C-terminal protein. Thymidine-synchronized of microtubules and actin filaments. Biol. Cell 94, 275-288. HeLa cells were transfected with cortactin C-terminal fusion protein Invitrogen Waters, J. C., Cole, R. W. and Rieder, C. L. (1993). The force-producing mechanism Lipofectamin 2000 following manufacturer’s protocol. The transfected cells were for centrosome separation during spindle formation in vertebrates is intrinsic to each synchronized again and harvested for immunoflurescence staining 10 hours after aster. J. Cell Biol. 122, 361-372. the thymidine-release. GFP or Myc-tag immunofluorescence were visualized to Weed, S. A. and Parsons, J. T. (2001). Cortactin: coupling membrane dynamics to cortical identify the expression of transfected protein and γ-tubulin was stained to reveal actin assembly. Oncogene 20, 6418-6434. centrosomes or spindle poles. Whitehead, C. M., Winkfein, R. J. and Rattner, J. B. (1996). The relationship of HsEg5 and the actin cytoskeleton to centrosome separation. Cell Motil. Cytoskeleton 35, 298-308. Wu, H., Reynolds, A. B., Kanner, S. B., Vines, R. R. and Parsons, J. T. (1991). This work was supported by grants 90208007 and 30521005 from Identification and characterization of a novel cytoskeleton-associated pp60src substrate. the China National Nature Sciences Foundation, and 2002CB513000 Mol. Cell. Biol. 11, 5113-5124.