Targeting Plk1 to chromosome arms and regulating chromosome compaction by the PICH ATPase

Department of Biochemistry & Molecular Biology, Baylor College of Medicine, Houston, Texas, United States
Cell cycle (Georgetown, Tex.) (Impact Factor: 4.57). 06/2008; 7(10):1480-9. DOI: 10.4161/cc.7.10.5951
Source: PubMed


During mitosis, chromosomes undergo dynamic structural changes that include condensation of chromosomes-the formation of individual compact chromosomes necessary for faithful segregation of sister chromatids in anaphase. Polo-like kinase 1 (Plk1) regulates multiple mitotic events by binding to targeting factors at different mitotic structures in a phosphorylation dependent manner. In this study, we report the identification of a putative ATPase that targets Plk1 to chromosome arms during mitosis. PICH (Plk1-interacting checkpoint "helicase") displays a temporal localization on chromosome arms and kinetochores during early mitosis. Interaction with PICH recruits Plk1 to chromosome arms and disruption of this interaction abolishes Plk1 localization on chromosome arms. Moreover, depletion of PICH or overexpression of PICH mutant that is defective in Plk1 binding or ATP binding causes defects in mitotic chromosome compaction, formation of anaphase bridge and cytokinesis failure. We provide data to show that both PICH phosphorylation and its ATPase activity are required for mitotic chromosome compaction. Our study provides a mechanism for targeting Plk1 to chromosome arms and suggests that the PICH ATPase activity is important for the regulation of mitotic chromosome architecture.

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Available from: Sung Jung, Oct 13, 2014
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    • "We observed defective chromosomal segregation and mitotic nuclei with supernumerary chromosomes at high frequencies during mitosis in the syncytial endosperm, which was considered to be the cause of the endospermless phenotype of enl1. This conclusion was supported by the molecular identification of ENL1 as the ortholog of human PICH, whose knockdown results in increased frequencies of missegregation and the deformed architectures of mitotic chromosomes (Baumann et al., 2007; Leng et al., 2008; Kurasawa and Yu-Lee, 2010; Ke et al., 2011). Our results shed light on the chromosome dynamics in relation to the highly specialized manners of rice (or cereal) endosperm development and those that are related to the conserved functions of the PICH family proteins among eukaryotes. "
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    ABSTRACT: The endosperm of cereal grains represents the most important source of human nutrition. In addition, the endosperm provides many investigatory opportunities for biologists because of the unique processes that occur during its ontogeny, including syncytial development at early stages. Rice endospermless 1 (enl1) develops seeds lacking an endosperm but carrying a functional embryo. The enl1 endosperm produces strikingly enlarged amoeboid nuclei. These abnormal nuclei result from a malfunction in mitotic chromosomal segregation during syncytial endosperm development. The molecular identification of the causal gene revealed that ENL1 encodes an SNF2 helicase family protein that is orthologous to human PICH, which has been implicated in the resolution of persistent DNA catenation during anaphase. ENL1-Venus (enhanced YFP) localizes to the cytoplasm during interphase but moves to the chromosome arms during mitosis. ENL1-Venus is also detected on a thread-like structure that connects separating sister chromosomes. These observations indicate the functional conservation between PICH and ENL1 and confirm the proposed role of PICH. Although ENL1 dysfunction also affects karyokinesis in the root meristem, enl1 plants can grow in a field and set seeds, indicating that its indispensability is tissue-dependent. Notably, despite the wide conservation of ENL1/PICH among eukaryotes, the loss of function of the ENL1 ortholog in Arabidopsis (CHR24) has only marginal effects on endosperm nuclei and results in normal plant development. Our results suggest that ENL1 is endowed with an indispensable role to secure the extremely rapid nuclear cycle during syncytial endosperm development in rice.This article is protected by copyright. All rights reserved.
    The Plant Journal 10/2014; 81(1). DOI:10.1111/tpj.12705 · 5.97 Impact Factor
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    • "There have been several phenotypes reported for mammalian cells depleted of PICH. These include effects on mitotic checkpoints and metaphase chromosome architecture, but many of these phenotypes were not seen in all studies, probably due to off-target effects of the siRNAs (Baumann et al., 2007; Kurasawa and Yu-Lee, 2010; Leng et al., 2008). Recent data, derived from the use of more specific siRNAs or the microinjection of anti-PICH antibodies, indicate that impairment of PICH function leads to an elevation in the frequency of bulky (DAPIpositive ) anaphase bridges (Hubner et al., 2010). "
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    ABSTRACT: The Plk1-interacting checkpoint helicase (PICH) protein localizes to ultrafine anaphase bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including the Bloom's syndrome protein (BLM). However, very little is known about the function of PICH or how it is recruited to UFBs. Using a combination of microfluidics, fluorescence microscopy, and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to duplex DNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding DNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a model in which PICH recognizes and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids.
    Molecular cell 08/2013; 51(5). DOI:10.1016/j.molcel.2013.07.016 · 14.02 Impact Factor
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    • "Plk1 closely followed the relocalization of the binding-competent PICH-WAB from kinetochores to chromosome arms, but no such re-localization was observed in the case of the binding-incompetent PICH-WAB- TA (Fig. 3a). These results demonstrate that the ATPase activity is critical for determining PICH localization, and they confirm that PICH is a major interaction partner of Plk1 on mitotic chromosomes (Baumann et al. 2007; Hutchins et al. 2010; Leng et al. 2008; Santamaria et al. 2007). Interestingly, expression of PICH-TA in the PICH siRNA background reduced the frequency of chromatin bridges to a similar extent as expression of PICH WT (video 8 and data not shown), indicating that Plk1 binding to PICH is not required to suppress chromatin bridge formation. "
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    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. Electronic supplementary material The online version of this article (doi:10.1007/s00412-012-0370-0) contains supplementary material, which is available to authorized users.
    Chromosoma 04/2012; 121(4):395-408. DOI:10.1007/s00412-012-0370-0 · 4.60 Impact Factor
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