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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.018176

Journal of Cell Science 121, 1334-1343 (2008)
Published by The Company of Biologists 2008
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Centrosome separation driven by actin-microfilaments during mitosis is mediated by centrosome-associated tyrosine-phosphorylated cortactin

Wenqi Wang*, Luyun Chen*, Yubo Ding, Jing Jin and Kan Liao{ddagger}

State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China


Figure 1
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  		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 ({alpha}-PY421C) or anti-cortactin antibody ({alpha}-Cort). (C) Co-immunoprecipitation of P-Tyr421-cortactin with {gamma}-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-{gamma}-tubulin antibody ({alpha}-{gamma}Tu), anti-P-Tyr421-cortactin antibody ({alpha}-PY421C) or anti-cortactin antibody ({alpha}-Cort).

 

Figure 2
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  		Fig. 2. Centrosome and/or spindle-pole localization of P-Tyr421-cortactin, P-Tyr466-cortactin and P-Tyr482-cortactin in mitotic 3T3-L1 cells. (A) Double-immunofluorescence staining of cortactin using {alpha}-tubulin or actin antibodies. (B) Double-immunofluorescence staining using anti-P-Tyr421-cortactin (PY421C), anti-P-Tyr466-cortactin (PY466C) or anti-P-Tyr483-cortactin (PY482C) antibodies and anti-{gamma}-tubulin antibody ({gamma}-Tu). (C) Double-immunofluorescence staining using anti-P-Tyr421-cortactin (PY421C) or anti-P-Tyr466-cortactin (PY466C) antibodies and anti-{alpha}-tubulin ({alpha}-Tu) antibody (D) Phosphotyrosine proteins and spindle poles. Metaphase 3T3-L1 cell was stained with anti-phosphotyrosine antibody (PY), anti-{gamma}-tubulin antibody ({gamma}-Tu) and DAPI. D, DAPI; C, anti-cortactin antibody; A, anti-actin antibody; {alpha}-Tu, anti-{alpha}-tubulin antibody; M, the merged picture of panels D, C and A (or {alpha}-Tu). Scale bars: 10 µm.

 

Figure 3
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  		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-{alpha}-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-{gamma}-tubulin antibody ({gamma}-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-{alpha}-tubulin ({alpha}-Tu). (D) Localization of P-Tyr421-cortactin, P-Tyr466-cortactin and P-Tyr482-cortactin in pericentriolar material. 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.

 

Figure 4
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  		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-{gamma}-tubulin ({gamma}T) 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.

 

Figure 5
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  		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 {alpha}-tubulin as loading control. +Cytochalasin B, thymidine-release in the presence of cytochalasin B. (B) Immunofluorescence staining of P-cortactin and {gamma}-tubulin in HeLa cells. Proliferating HeLa cells were simultaneously stained for P-Tyr421-cortactin (PY421C) and {gamma}-tubulin ({gamma}-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.

 

Figure 6
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  		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-terminus mutants. N-terminus, amino acids (aa) 1-329; C-terminus, aa 330-546; CT(Y/F), aa 330-546 with Phe substitutions for Tyr421, Tyr466, Tyr475 and Tyr482. (B) HeLa cells transfected with GFP-CT or GFP-CT(Y/F). Arrowheads indicate the centrosomes. The 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. (D) Inhibition of centrosome separation brought upon by the Myc-tagged C-terminus of wild-type cortactin (CT) but not the cortactin C-terminus containing mutated Tyr residues [CT(Y/F)]. The centrosome-separation ratio (d/D) was measured from 100 Myc-positive cells and plotted as described for Fig. 4. (E) Average centrosome separation 10 hours after thymidine-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 (d) and corresponding nuclear diameters (D) of 100 cells were averaged for each group of cells.

 

Figure 7
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  		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 ({alpha}-Cort) or anti-phosphotyrosine421 cortactin antibody ({alpha}-PY421C). IB, western blot; T, total cell lysate; IP, immunoprecipitated sample.

 

Figure 8
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  		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.

 

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© The Company of Biologists Ltd 2008