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MASTL promotes cyclin B1 destruction by enforcing Cdc20-independent binding of cyclin B1 to the APC/C.

Voets E, Wolthuis R - Biol Open (2015)

Bottom Line: However, we found that cyclin B1, through Cdk1 and Cks, is targeted to the phosphorylated APC/C(Cdc20) at the start of prometaphase, when the spindle checkpoint is still active.Here, we show that MASTL is essential for cyclin B1 recruitment to the mitotic APC/C and that this occurs entirely independently of Cdc20.Importantly, MASTL-directed binding of cyclin B1 to spindle checkpoint-inhibited APC/C(Cdc20) critically supports efficient cyclin B1 destruction after checkpoint release.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology I (B5) and Division of Molecular Carcinogenesis (B7), The Netherlands Cancer Institute (NKI-AvL), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

MASTL depletion increases the frequency of anaphase bridges that may depend on the activity of separase.(A) DNA bridges formed during anaphase are specific to MASTL silencing. Asynchronously growing HeLa cells were transfected with indicated siRNAs, formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using anti-α-Tubulin and DAPI. Arrows indicate DNA bridges connecting the daughter cells in G1 phase. (B) Combined depletion of MASTL and PPP2CA prevents the formation of DNA bridges. HeLa cells stably infected with an shRNA targeting PPP2CA (shPPP2CA) or a control shRNA (shGFP) were transfected with indicated siRNAs. Cells were formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using DAPI. Representative images show a regular cell or two daughter cells that are connected to each other by a DNA bridge (indicated by the red arrow). The amount of cells containing a DNA bridge were scored and quantified for each condition [n = 3; mean±standard deviation (s.d.)]. (C) Western blot analysis to show the effects of indicated shRNAs on total protein levels. A reduction in the protein levels of PP2A-A and PP2A-Cα demonstrates the knockdown efficiency of shPPP2CA. (D) MASTL silencing does not alter the mitotic chromosome morphology. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by a thymidine block followed by subsequent nocodazole treatment for 5 hours. Cells were processed for chromosome spreads 48 hours after transfection (or 24 hours in case of SGOL1 RNAi). Where indicated, cells were treated for 13 hours with 5 µM ICRF-193. Chromosomes were stained using DAPI. The different treatments are categorised based on the mitotic chromosome morphology. Insets demonstrate the morphology of a representative chromosome for each condition. (E) Cyclin B1 and separase physically interact during mitosis. HeLa cells were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 µM of RO-3306 for 2 hours to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a mouse anti-separase and aliquots of the immunoprecipitates were analysed by western blotting. (F) Absence of MASTL impairs the activation of separase in mitosis. HeLa cells transfected with indicated siRNAs were synchronised in G2 and M phase by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the separase cleavage product signal was quantified for each condition, starting in mitosis, and corrected for the signal of the same product present in mitotic lysates. The resulting amount was corrected for the total level of separase (full length plus cleavage product). Finally, these values were normalised to 100% at the onset (30 min G1 condition) of mitotic exit (n = 3; mean±s.e.m.). (G) Schematic working model of cyclin B1-mediated separase inhibition. The activation of APC/CCdc20 results in the destruction of cyclin B1 and securin (indicated by the ubiquitin chains), thereby liberating separase from its inhibitors. Subsequently, activated separase triggers the induction of anaphase by cleaving centromeric cohesin (red dots). When cyclin B1 degradation is impaired, its binding to separase will prevent proper sister chromatid separation, leading to the DNA bridges observed in MASTL RNAi cells.
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f05: MASTL depletion increases the frequency of anaphase bridges that may depend on the activity of separase.(A) DNA bridges formed during anaphase are specific to MASTL silencing. Asynchronously growing HeLa cells were transfected with indicated siRNAs, formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using anti-α-Tubulin and DAPI. Arrows indicate DNA bridges connecting the daughter cells in G1 phase. (B) Combined depletion of MASTL and PPP2CA prevents the formation of DNA bridges. HeLa cells stably infected with an shRNA targeting PPP2CA (shPPP2CA) or a control shRNA (shGFP) were transfected with indicated siRNAs. Cells were formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using DAPI. Representative images show a regular cell or two daughter cells that are connected to each other by a DNA bridge (indicated by the red arrow). The amount of cells containing a DNA bridge were scored and quantified for each condition [n = 3; mean±standard deviation (s.d.)]. (C) Western blot analysis to show the effects of indicated shRNAs on total protein levels. A reduction in the protein levels of PP2A-A and PP2A-Cα demonstrates the knockdown efficiency of shPPP2CA. (D) MASTL silencing does not alter the mitotic chromosome morphology. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by a thymidine block followed by subsequent nocodazole treatment for 5 hours. Cells were processed for chromosome spreads 48 hours after transfection (or 24 hours in case of SGOL1 RNAi). Where indicated, cells were treated for 13 hours with 5 µM ICRF-193. Chromosomes were stained using DAPI. The different treatments are categorised based on the mitotic chromosome morphology. Insets demonstrate the morphology of a representative chromosome for each condition. (E) Cyclin B1 and separase physically interact during mitosis. HeLa cells were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 µM of RO-3306 for 2 hours to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a mouse anti-separase and aliquots of the immunoprecipitates were analysed by western blotting. (F) Absence of MASTL impairs the activation of separase in mitosis. HeLa cells transfected with indicated siRNAs were synchronised in G2 and M phase by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the separase cleavage product signal was quantified for each condition, starting in mitosis, and corrected for the signal of the same product present in mitotic lysates. The resulting amount was corrected for the total level of separase (full length plus cleavage product). Finally, these values were normalised to 100% at the onset (30 min G1 condition) of mitotic exit (n = 3; mean±s.e.m.). (G) Schematic working model of cyclin B1-mediated separase inhibition. The activation of APC/CCdc20 results in the destruction of cyclin B1 and securin (indicated by the ubiquitin chains), thereby liberating separase from its inhibitors. Subsequently, activated separase triggers the induction of anaphase by cleaving centromeric cohesin (red dots). When cyclin B1 degradation is impaired, its binding to separase will prevent proper sister chromatid separation, leading to the DNA bridges observed in MASTL RNAi cells.

Mentions: In a different line of investigation, when we compared the effects of either CDC20 or MASTL RNAi by immunofluorescence, we had observed that only depletion of MASTL increased the incidence of DNA bridges, as detected by DAPI-positive threads connecting the G1 daughter cells (Fig. 5A). Partial CDC20 depletion increased the amount of multinucleated cells, possibly due to a delay in metaphase, leading to premature sister chromatid separation as a consequence of cohesion fatigue (Daum et al., 2011; Stevens et al., 2011). However, neither siCDC20, nor siCDH1 resulted in any detectable formation of DNA bridges (Fig. 5A; data not shown). These mitotic defects that arise upon MASTL RNAi are indicative of a premature exit from mitosis, most likely due to an increase in PP2A activity. We therefore tested whether co-depletion of MASTL and PPP2CA could restore the formation of DNA bridges between G1 daughter cells (Fig. 5B,C). Indeed, the effect of MASTL depletion was dependent on the presence of PP2A.


MASTL promotes cyclin B1 destruction by enforcing Cdc20-independent binding of cyclin B1 to the APC/C.

Voets E, Wolthuis R - Biol Open (2015)

MASTL depletion increases the frequency of anaphase bridges that may depend on the activity of separase.(A) DNA bridges formed during anaphase are specific to MASTL silencing. Asynchronously growing HeLa cells were transfected with indicated siRNAs, formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using anti-α-Tubulin and DAPI. Arrows indicate DNA bridges connecting the daughter cells in G1 phase. (B) Combined depletion of MASTL and PPP2CA prevents the formation of DNA bridges. HeLa cells stably infected with an shRNA targeting PPP2CA (shPPP2CA) or a control shRNA (shGFP) were transfected with indicated siRNAs. Cells were formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using DAPI. Representative images show a regular cell or two daughter cells that are connected to each other by a DNA bridge (indicated by the red arrow). The amount of cells containing a DNA bridge were scored and quantified for each condition [n = 3; mean±standard deviation (s.d.)]. (C) Western blot analysis to show the effects of indicated shRNAs on total protein levels. A reduction in the protein levels of PP2A-A and PP2A-Cα demonstrates the knockdown efficiency of shPPP2CA. (D) MASTL silencing does not alter the mitotic chromosome morphology. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by a thymidine block followed by subsequent nocodazole treatment for 5 hours. Cells were processed for chromosome spreads 48 hours after transfection (or 24 hours in case of SGOL1 RNAi). Where indicated, cells were treated for 13 hours with 5 µM ICRF-193. Chromosomes were stained using DAPI. The different treatments are categorised based on the mitotic chromosome morphology. Insets demonstrate the morphology of a representative chromosome for each condition. (E) Cyclin B1 and separase physically interact during mitosis. HeLa cells were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 µM of RO-3306 for 2 hours to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a mouse anti-separase and aliquots of the immunoprecipitates were analysed by western blotting. (F) Absence of MASTL impairs the activation of separase in mitosis. HeLa cells transfected with indicated siRNAs were synchronised in G2 and M phase by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the separase cleavage product signal was quantified for each condition, starting in mitosis, and corrected for the signal of the same product present in mitotic lysates. The resulting amount was corrected for the total level of separase (full length plus cleavage product). Finally, these values were normalised to 100% at the onset (30 min G1 condition) of mitotic exit (n = 3; mean±s.e.m.). (G) Schematic working model of cyclin B1-mediated separase inhibition. The activation of APC/CCdc20 results in the destruction of cyclin B1 and securin (indicated by the ubiquitin chains), thereby liberating separase from its inhibitors. Subsequently, activated separase triggers the induction of anaphase by cleaving centromeric cohesin (red dots). When cyclin B1 degradation is impaired, its binding to separase will prevent proper sister chromatid separation, leading to the DNA bridges observed in MASTL RNAi cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400591&req=5

f05: MASTL depletion increases the frequency of anaphase bridges that may depend on the activity of separase.(A) DNA bridges formed during anaphase are specific to MASTL silencing. Asynchronously growing HeLa cells were transfected with indicated siRNAs, formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using anti-α-Tubulin and DAPI. Arrows indicate DNA bridges connecting the daughter cells in G1 phase. (B) Combined depletion of MASTL and PPP2CA prevents the formation of DNA bridges. HeLa cells stably infected with an shRNA targeting PPP2CA (shPPP2CA) or a control shRNA (shGFP) were transfected with indicated siRNAs. Cells were formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using DAPI. Representative images show a regular cell or two daughter cells that are connected to each other by a DNA bridge (indicated by the red arrow). The amount of cells containing a DNA bridge were scored and quantified for each condition [n = 3; mean±standard deviation (s.d.)]. (C) Western blot analysis to show the effects of indicated shRNAs on total protein levels. A reduction in the protein levels of PP2A-A and PP2A-Cα demonstrates the knockdown efficiency of shPPP2CA. (D) MASTL silencing does not alter the mitotic chromosome morphology. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by a thymidine block followed by subsequent nocodazole treatment for 5 hours. Cells were processed for chromosome spreads 48 hours after transfection (or 24 hours in case of SGOL1 RNAi). Where indicated, cells were treated for 13 hours with 5 µM ICRF-193. Chromosomes were stained using DAPI. The different treatments are categorised based on the mitotic chromosome morphology. Insets demonstrate the morphology of a representative chromosome for each condition. (E) Cyclin B1 and separase physically interact during mitosis. HeLa cells were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 µM of RO-3306 for 2 hours to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a mouse anti-separase and aliquots of the immunoprecipitates were analysed by western blotting. (F) Absence of MASTL impairs the activation of separase in mitosis. HeLa cells transfected with indicated siRNAs were synchronised in G2 and M phase by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the separase cleavage product signal was quantified for each condition, starting in mitosis, and corrected for the signal of the same product present in mitotic lysates. The resulting amount was corrected for the total level of separase (full length plus cleavage product). Finally, these values were normalised to 100% at the onset (30 min G1 condition) of mitotic exit (n = 3; mean±s.e.m.). (G) Schematic working model of cyclin B1-mediated separase inhibition. The activation of APC/CCdc20 results in the destruction of cyclin B1 and securin (indicated by the ubiquitin chains), thereby liberating separase from its inhibitors. Subsequently, activated separase triggers the induction of anaphase by cleaving centromeric cohesin (red dots). When cyclin B1 degradation is impaired, its binding to separase will prevent proper sister chromatid separation, leading to the DNA bridges observed in MASTL RNAi cells.
Mentions: In a different line of investigation, when we compared the effects of either CDC20 or MASTL RNAi by immunofluorescence, we had observed that only depletion of MASTL increased the incidence of DNA bridges, as detected by DAPI-positive threads connecting the G1 daughter cells (Fig. 5A). Partial CDC20 depletion increased the amount of multinucleated cells, possibly due to a delay in metaphase, leading to premature sister chromatid separation as a consequence of cohesion fatigue (Daum et al., 2011; Stevens et al., 2011). However, neither siCDC20, nor siCDH1 resulted in any detectable formation of DNA bridges (Fig. 5A; data not shown). These mitotic defects that arise upon MASTL RNAi are indicative of a premature exit from mitosis, most likely due to an increase in PP2A activity. We therefore tested whether co-depletion of MASTL and PPP2CA could restore the formation of DNA bridges between G1 daughter cells (Fig. 5B,C). Indeed, the effect of MASTL depletion was dependent on the presence of PP2A.

Bottom Line: However, we found that cyclin B1, through Cdk1 and Cks, is targeted to the phosphorylated APC/C(Cdc20) at the start of prometaphase, when the spindle checkpoint is still active.Here, we show that MASTL is essential for cyclin B1 recruitment to the mitotic APC/C and that this occurs entirely independently of Cdc20.Importantly, MASTL-directed binding of cyclin B1 to spindle checkpoint-inhibited APC/C(Cdc20) critically supports efficient cyclin B1 destruction after checkpoint release.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology I (B5) and Division of Molecular Carcinogenesis (B7), The Netherlands Cancer Institute (NKI-AvL), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

No MeSH data available.


Related in: MedlinePlus