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On the regulation, function, and localization of the DNA-dependent ATPase PICH.

Kaulich M, Cubizolles F, Nigg EA - Chromosoma (2012)

Bottom Line: The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1.This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity.Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Growth & Development, Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4065 Basel, Switzerland.

ABSTRACT
The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1. During mitosis PICH associates with centromeres and kinetochores and, most interestingly, constitutes a robust marker for ultrafine DNA bridges (UFBs) that connect separating chromatids in anaphase cells. The precise roles of PICH remain to be clarified. Here, we have used antibody microinjection and siRNA-rescue experiments to study PICH function and localization during M phase progression, with particular emphasis on the role of the predicted ATPase domain and the regulation of PICH localization by Plk1. We show that interference with PICH function results in chromatin bridge formation and micronucleation and that ATPase activity is critical for PICH function. Interestingly, an intact ATPase domain of PICH is required for prevention of chromatin bridge formation but not for UFB resolution, and quantitative analyses of UFB and chromatin bridge frequencies suggest that these structures are of different etiologies. We also show that the ATPase activity of PICH is required for temporal and spatial control of PICH localization to chromatin and that Plk1 likely controls PICH localization through phosphorylation of proteins distinct from PICH itself. This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity. Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.

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Anti-PICH antibody injection or siRNA-mediated PICH depletion causes anaphase chromatin bridges. a Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-GFP, after antibody microinjection (N.I. non-injected, C.I. control injected (buffer or Myc mAb), P.I. PICH injected (polyclonal or monoclonal antibodies)). TexasRed signal identifies microinjected cells. T = 0 was set at nuclear envelope breakdown (NEBD) and numbers indicate elapsed time (minutes). Arrows indicate chromatin bridge formation. bBox-and-whisker plot showing elapsed time (minutes) from NEBD to anaphase onset for individual microinjected cells. Analyses were performed on >120 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively. cBar graph showing the percentage of chromatin bridges after microinjection of the indicated antibodies. Analyses were performed on >120 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. d Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-mCherry after transfection with the Gl2 (control) and PICH-directed siRNA oligonucleotides. T = 0 was set at NEBD and numbers indicate elapsed time (minutes). Arrows indicate chromatin bridge formation. eBox-and-whisker plot showing elapsed time (minutes) from NEBD to anaphase onset for individual siRNA-transfected cells. Analyses were performed on >120 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively. fBar graph showing the percentage of chromatin bridges after the indicated siRNA transfections. Analyses were performed on >120 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. g Representative images of PTEMF fixed HeLaS3 anaphase cells, revealing typical chromatin bridges after PICH knockdown. Cells were stained for DNA with 4′-6-diamidino-2-phenylindole (DAPI). Scale bar represents 10 μm
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Fig1: Anti-PICH antibody injection or siRNA-mediated PICH depletion causes anaphase chromatin bridges. a Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-GFP, after antibody microinjection (N.I. non-injected, C.I. control injected (buffer or Myc mAb), P.I. PICH injected (polyclonal or monoclonal antibodies)). TexasRed signal identifies microinjected cells. T = 0 was set at nuclear envelope breakdown (NEBD) and numbers indicate elapsed time (minutes). Arrows indicate chromatin bridge formation. bBox-and-whisker plot showing elapsed time (minutes) from NEBD to anaphase onset for individual microinjected cells. Analyses were performed on >120 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively. cBar graph showing the percentage of chromatin bridges after microinjection of the indicated antibodies. Analyses were performed on >120 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. d Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-mCherry after transfection with the Gl2 (control) and PICH-directed siRNA oligonucleotides. T = 0 was set at NEBD and numbers indicate elapsed time (minutes). Arrows indicate chromatin bridge formation. eBox-and-whisker plot showing elapsed time (minutes) from NEBD to anaphase onset for individual siRNA-transfected cells. Analyses were performed on >120 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively. fBar graph showing the percentage of chromatin bridges after the indicated siRNA transfections. Analyses were performed on >120 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. g Representative images of PTEMF fixed HeLaS3 anaphase cells, revealing typical chromatin bridges after PICH knockdown. Cells were stained for DNA with 4′-6-diamidino-2-phenylindole (DAPI). Scale bar represents 10 μm

Mentions: Previous studies aimed at uncovering the function of PICH by siRNA had yielded inconsistent results (Baumann et al. 2007; Hubner et al. 2010; Kurasawa and Yu-Lee 2010; Leng et al. 2008). Thus, we resorted to antibody microinjection as a powerful, alternative approach for probing protein function. We raised and characterized a monoclonal antibody (mAb; clone 142-26-3) against human PICH, which recognized PICH in immunofluorescence microscopy, Western blotting, as well as immunoprecipitation (Fig. S1). Staining patterns matched those reported previously (Baumann et al. 2007; Wang et al. 2008), and antibody specificity was confirmed by siRNA-mediated knockdown of PICH (Fig. S1A, C). Furthermore, Plk1 was readily co-precipitated with PICH by mAb 142-26-3 (Fig. S1B), as expected (Baumann et al. 2007). To examine the consequences of introducing anti-PICH antibodies into living cells, HeLaS3 cells stably expressing histone 2B-GFP were synchronized in G1/S phase of the cell cycle (Sillje et al. 2006), before they were injected with mAb 142-26-3 or a previously characterized polyclonal anti-PICH Ab (Baumann et al. 2007). Control injections were performed using either injection buffer alone or a mAb specific for a Myc epitope (9E10; Evan et al. 1985). Using co-injected TexasRed for identification of injected cells, these were monitored by time-lapse video microscopy. None of the antibody injections detectably accelerated mitotic progression (Fig. 1a, b; videos 1–3), supporting the conclusion that PICH is not required for the establishment of a functional SAC (Hubner et al. 2010). To extend this conclusion, we also injected mitotically arrested HeLaS3 cells in which the SAC had been activated by a 5-h treatment with nocodazole. In this case, a function-neutralizing anti-Mad2 mAb served as a positive control (Fava et al. 2011). Cells were kept in culture for 3 h after injection, before they were fixed and analyzed by immunofluorescence microscopy. Whereas injection of the Myc mAb did not affect the SAC-induced mitotic arrest, injection of the anti-Mad2 mAb leads to the expected checkpoint override and premature mitotic exit (Fig. S2A, B). Importantly, neither mAb 142-26-3 nor the polyclonal anti-PICH Ab affected the nocodazole-induced SAC arrest (Fig. S2A, B), indicating that PICH is not required for maintenance of SAC activity.Fig. 1


On the regulation, function, and localization of the DNA-dependent ATPase PICH.

Kaulich M, Cubizolles F, Nigg EA - Chromosoma (2012)

Anti-PICH antibody injection or siRNA-mediated PICH depletion causes anaphase chromatin bridges. a Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-GFP, after antibody microinjection (N.I. non-injected, C.I. control injected (buffer or Myc mAb), P.I. PICH injected (polyclonal or monoclonal antibodies)). TexasRed signal identifies microinjected cells. T = 0 was set at nuclear envelope breakdown (NEBD) and numbers indicate elapsed time (minutes). Arrows indicate chromatin bridge formation. bBox-and-whisker plot showing elapsed time (minutes) from NEBD to anaphase onset for individual microinjected cells. Analyses were performed on >120 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively. cBar graph showing the percentage of chromatin bridges after microinjection of the indicated antibodies. Analyses were performed on >120 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. d Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-mCherry after transfection with the Gl2 (control) and PICH-directed siRNA oligonucleotides. T = 0 was set at NEBD and numbers indicate elapsed time (minutes). Arrows indicate chromatin bridge formation. eBox-and-whisker plot showing elapsed time (minutes) from NEBD to anaphase onset for individual siRNA-transfected cells. Analyses were performed on >120 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively. fBar graph showing the percentage of chromatin bridges after the indicated siRNA transfections. Analyses were performed on >120 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. g Representative images of PTEMF fixed HeLaS3 anaphase cells, revealing typical chromatin bridges after PICH knockdown. Cells were stained for DNA with 4′-6-diamidino-2-phenylindole (DAPI). Scale bar represents 10 μm
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Fig1: Anti-PICH antibody injection or siRNA-mediated PICH depletion causes anaphase chromatin bridges. a Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-GFP, after antibody microinjection (N.I. non-injected, C.I. control injected (buffer or Myc mAb), P.I. PICH injected (polyclonal or monoclonal antibodies)). TexasRed signal identifies microinjected cells. T = 0 was set at nuclear envelope breakdown (NEBD) and numbers indicate elapsed time (minutes). Arrows indicate chromatin bridge formation. bBox-and-whisker plot showing elapsed time (minutes) from NEBD to anaphase onset for individual microinjected cells. Analyses were performed on >120 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively. cBar graph showing the percentage of chromatin bridges after microinjection of the indicated antibodies. Analyses were performed on >120 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. d Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-mCherry after transfection with the Gl2 (control) and PICH-directed siRNA oligonucleotides. T = 0 was set at NEBD and numbers indicate elapsed time (minutes). Arrows indicate chromatin bridge formation. eBox-and-whisker plot showing elapsed time (minutes) from NEBD to anaphase onset for individual siRNA-transfected cells. Analyses were performed on >120 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively. fBar graph showing the percentage of chromatin bridges after the indicated siRNA transfections. Analyses were performed on >120 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. g Representative images of PTEMF fixed HeLaS3 anaphase cells, revealing typical chromatin bridges after PICH knockdown. Cells were stained for DNA with 4′-6-diamidino-2-phenylindole (DAPI). Scale bar represents 10 μm
Mentions: Previous studies aimed at uncovering the function of PICH by siRNA had yielded inconsistent results (Baumann et al. 2007; Hubner et al. 2010; Kurasawa and Yu-Lee 2010; Leng et al. 2008). Thus, we resorted to antibody microinjection as a powerful, alternative approach for probing protein function. We raised and characterized a monoclonal antibody (mAb; clone 142-26-3) against human PICH, which recognized PICH in immunofluorescence microscopy, Western blotting, as well as immunoprecipitation (Fig. S1). Staining patterns matched those reported previously (Baumann et al. 2007; Wang et al. 2008), and antibody specificity was confirmed by siRNA-mediated knockdown of PICH (Fig. S1A, C). Furthermore, Plk1 was readily co-precipitated with PICH by mAb 142-26-3 (Fig. S1B), as expected (Baumann et al. 2007). To examine the consequences of introducing anti-PICH antibodies into living cells, HeLaS3 cells stably expressing histone 2B-GFP were synchronized in G1/S phase of the cell cycle (Sillje et al. 2006), before they were injected with mAb 142-26-3 or a previously characterized polyclonal anti-PICH Ab (Baumann et al. 2007). Control injections were performed using either injection buffer alone or a mAb specific for a Myc epitope (9E10; Evan et al. 1985). Using co-injected TexasRed for identification of injected cells, these were monitored by time-lapse video microscopy. None of the antibody injections detectably accelerated mitotic progression (Fig. 1a, b; videos 1–3), supporting the conclusion that PICH is not required for the establishment of a functional SAC (Hubner et al. 2010). To extend this conclusion, we also injected mitotically arrested HeLaS3 cells in which the SAC had been activated by a 5-h treatment with nocodazole. In this case, a function-neutralizing anti-Mad2 mAb served as a positive control (Fava et al. 2011). Cells were kept in culture for 3 h after injection, before they were fixed and analyzed by immunofluorescence microscopy. Whereas injection of the Myc mAb did not affect the SAC-induced mitotic arrest, injection of the anti-Mad2 mAb leads to the expected checkpoint override and premature mitotic exit (Fig. S2A, B). Importantly, neither mAb 142-26-3 nor the polyclonal anti-PICH Ab affected the nocodazole-induced SAC arrest (Fig. S2A, B), indicating that PICH is not required for maintenance of SAC activity.Fig. 1

Bottom Line: The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1.This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity.Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Growth & Development, Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4065 Basel, Switzerland.

ABSTRACT
The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1. During mitosis PICH associates with centromeres and kinetochores and, most interestingly, constitutes a robust marker for ultrafine DNA bridges (UFBs) that connect separating chromatids in anaphase cells. The precise roles of PICH remain to be clarified. Here, we have used antibody microinjection and siRNA-rescue experiments to study PICH function and localization during M phase progression, with particular emphasis on the role of the predicted ATPase domain and the regulation of PICH localization by Plk1. We show that interference with PICH function results in chromatin bridge formation and micronucleation and that ATPase activity is critical for PICH function. Interestingly, an intact ATPase domain of PICH is required for prevention of chromatin bridge formation but not for UFB resolution, and quantitative analyses of UFB and chromatin bridge frequencies suggest that these structures are of different etiologies. We also show that the ATPase activity of PICH is required for temporal and spatial control of PICH localization to chromatin and that Plk1 likely controls PICH localization through phosphorylation of proteins distinct from PICH itself. This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity. Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.

Show MeSH
Related in: MedlinePlus