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1.
Figure 1

Figure 1. Plk1 activity is required for localization of γ-tubulin and GCP-WD to centrosomes and spindle microtubules.. From: Plk1-Dependent Recruitment of ?-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components.

(A) HeLa cells treated with monastrol or BI2536 were fixed and immunostained for γ-tubulin and pericentrin.DAPI was used to label DNA. (B) HeLa cells treated with monastrol, BI2536 or siRNA against Plk1 were fixed and immunostained for GCP-WD and α-tubulin. DNA was visualised using DAPI.

Laurence Haren, et al. PLoS ONE. 2009;4(6):e5976.
2.
Figure 3

Figure 3. The S418A mutation does not affect centrosome localization of GCP-WD.. From: Plk1-Dependent Recruitment of ?-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components.

HeLa cells depleted of endogenous GCP-WD and transiently expressing RNAi-resistant EGFP-tagged GCP-WD wt (A) or S418A mutant (B) were treated with monastrol or BI2536, fixed and immunostained with antibodies against GFP and γ-tubulin. DNA was visualised using DAPI.

Laurence Haren, et al. PLoS ONE. 2009;4(6):e5976.
3.
Figure 5

Figure 5. RNAi-mediated depletion of GCP-WD, pericentrin and Cep215, respectively, does not affect the levels of other centrosome proteins.. From: Plk1-Dependent Recruitment of ?-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components.

After RNAi-mediated depletion of the indicated proteins in Hela cells, whole cell extracts were analyzed by westernblotting using specific antibodies as indicated. The p38 protein was used as a loading control.

Laurence Haren, et al. PLoS ONE. 2009;4(6):e5976.
4.
Figure 4

Figure 4. Plk1 inhibition interferes with the recruitment of Cep192, Cep215 and pericentrin to mitotic centrosomes.. From: Plk1-Dependent Recruitment of ?-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components.

HeLa cells were treated with monastrol or BI2536, fixed and immunostained for GCP-WD, γ-tubulin, Cep192, Cep215 or pericentrin. Fluorescence intensities at the centrosomes were quantified in interphase and in mitosis (n≥10 cells/20 centrosomes per condition). Intensities were plotted as a percentage of the intensities measured in mitotic cells treated with monastrol. Error bars: SEM, 95% CI.

Laurence Haren, et al. PLoS ONE. 2009;4(6):e5976.
5.
Figure 7

Figure 7. Interdependencies of components involved in the recruitment of γ-tubulin complexes to mitotic centrosomes.. From: Plk1-Dependent Recruitment of ?-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components.

During centrosome maturation Plk1 phosphorylates PCM components, which results in the accumulation of PCM including γTuRC recruitment factors at mitotic centrosomes. Plk1-dependent phosphorylation of GCP-WD does not affect centrosome recruitment of γTuRCs, which is regulated upstream of GCP-WD. Upstream regulators and potential Plk1 substrates include Cep192, pericentrin, and Cep215. Pericentrin and Cep215 contribute to each other's centrosome localization and to γTuRC binding. Pericentrin is also important for centrosome localization of Cep192, which has the strongest impact on centrosome recruitment of GCP-WD/γTuRC [24], [25]. Solid black arrows indicate an essential role in the recruitment of the component they are pointing at, dashed black arrows indicate a partial role. Red arrows indicate Plk1-dependent regulation.

Laurence Haren, et al. PLoS ONE. 2009;4(6):e5976.
6.
Figure 6

Figure 6. Cep215 and pericentrin depend on each other for localizing to mitotic centrosomes and function upstream of GCP-WD in γ-tubulin recruitment.. From: Plk1-Dependent Recruitment of ?-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components.

(A) HeLa cells transfected with plasmids expressing shRNA targeting Cep215 or GCP-WD, were fixed and stained for GCP-WD and Cep215 or γ-tubulin and pericentrin. DAPI was used to label DNA. As a control cells were transfected with empty vector. In some panels one of the two centrosomes is out of focus and only one centrosome can be seen. The arrowhead marks the position of the out-of-focus centrosome, which in both cases was labelled with similar intensity as the one shown. (B) HeLa cells transfected with siRNA targeting pericentrin were processed for immunofluorescence with antibodies against γ-tubulin and Cep215 or GCP-WD and pericentrin. DAPI was used to visualize DNA. Unspecific RNA was transfected as a control. (C) After depletion of the indicated proteins by RNAi cells were fixed and stained with antibodies against γ-tubulin, GCP-WD, pericentrin and Cep215. Fluorescence intensities at mitotic centrosomes were quantified (n≥10 cells/20 centrosomes per condition). Intensities were plotted as a percentage of the intensities measured in control cells. Error bars: SEM, 95% CI.

Laurence Haren, et al. PLoS ONE. 2009;4(6):e5976.
7.
Figure 2

Figure 2. Plk1 interacts with GCP-WD in mitosis and contributes to its mitotic phosphorylation.. From: Plk1-Dependent Recruitment of ?-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components.

(A) Hela cell lysates immunoblotted for GCP-WD. Cells were synchronised in mitosis by nocodazole treatment. BI2536 was added for 90 min before harvesting the cells by shake-off. The asterisk indicates the position of unphosphorylated GCP-WD. (B) Coimmunoprecipitation of endogenous GCP-WD and Plk1. Proteins were immunoprecipitated from mitotic lysates obtained from nocodazole-arrested HeLa cells after shake-off with antibodies against HA (Control IP), Plk1 and GCP-WD, as indicated. Precipitates were separated by gel electrophoresis, blotted and probed with anti-GCP-WD and anti-Plk1 antibodies. (C) Myc-tagged Plk1 was immunoprecipitated from extracts prepared from asynchronously growing HeLa cells after transient transfection. Unspecific mouse IgG was used as a control (Control IP). Precipitates were treated either with buffer alone or with λ-phosphatase and analyzed by westernblotting with anti-Myc and anti-GCP-WD antibodies. (D) After cotransfection of Myc-tagged Plk1 with C-terminal EGFP-tagged wildtype or mutant fragments of GCP-WD (EGFP-C300) in HeLa cells, Myc-tagged Plk1 was immunoprecipitated from non-synchronous cultures and precipitates were immunoblotted using anti-GFP antibody. (E) EGFP-tagged C-terminal GCP-WD wt or mutant fragments were transiently expressed in HeLa cells and immunoprecipitated after nocodazole treatment. Precipitates were subjected to an in vitro phosphorylation assay in the absence or presence of BI2536 to inhibit Plk1 activity. After SDS-PAGE the immunoprecipitated proteins were detected by coomassie staining, incorporation of [γ-32P] was detected by autoradiography. (F) Lysates from U2OS cells expressing EGFP-tagged GCP-WD wt or T557A mutant were immunoblotted for GCP-WD. Cells were synchronized in mitosis by nocodazole treatment. BI2536 was added for 90 min before harvesting the cells by shake-off. The asterisk indicates the position of unphosphorylated GCP-WD-EGFP.

Laurence Haren, et al. PLoS ONE. 2009;4(6):e5976.

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