Poly(ADP-ribose) is required for spindle assembly and structure

Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nature (Impact Factor: 41.46). 01/2005; 432(7017):645-9. DOI: 10.1038/nature03061
Source: PubMed


The mitotic spindle is typically thought of as an array of microtubules, microtubule-associated proteins and motors that self-organizes to align and segregate chromosomes. The major spindle components consist of proteins and DNA, the primary structural elements of the spindle. Other macromolecules including RNA and lipids also associate with spindles, but their spindle function, if any, is unknown. Poly(ADP-ribose) (PAR) is a large, branched, negatively charged polymeric macromolecule whose polymerization onto acceptor proteins is catalysed by a family of poly(ADP-ribose) polymerases (PARPs). Several PARPs localize to the spindle in vertebrate cells, suggesting that PARPs and/or PAR have a role in spindle function. Here we show that PAR is enriched in the spindle and is required for spindle function--PAR hydrolysis or perturbation leads to rapid disruption of spindle structure, and hydrolysis during spindle assembly blocks the formation of bipolar spindles. PAR exhibits localization dynamics that differ from known spindle proteins and are consistent with a low rate of turnover in the spindle. Thus, PAR is a non-proteinaceous, non-chromosomal component of the spindle required for bipolar spindle assembly and function.

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Available from: Paul Chang, Jun 26, 2014
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    • "A conserved protein, BuGZ, which was identified as part of the lamin-B (LB) spindle matrix in Xenopus (Tsai et al., 2006; Ma et al., 2009), has recently been shown to facilitate chromosome alignment by controlling both stability and kinetochore loading of the SAC component Bub3 (Jiang et al., 2014; Toledo et al., 2014). Additionally, LB (Tsai et al., 2006) and poly ADP-ribose (Chang et al., 2004), along with other spindle assembly factors (SAFs), such as dynein, Nudel, NuMA, and kinesin Eg5 (Civelekoglu-Scholey et al., 2010; Goodman et al., 2010; Ma et al., 2009; Tsai et al., 2006), may regulate spindle morphogenesis . Despite these studies, the structural nature of the spindle matrix remains undefined and whether it constitutes a cohesive functional unit is unclear. "
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    ABSTRACT: Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks.
    Cell 09/2015; 163:1-15. DOI:10.1016/j.cell.2015.08.010 · 32.24 Impact Factor
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    • "As a result, scaffolds of protein-PAR::protein interactions are formed (reviewed in [18]). Such scaffolding property is observed in the recruitment of DNA repair proteins at the site of DNA damage [21], and for the assembly of spindle poles [22] and RNA organelles such as stress granules [14]. PARylation can be reversed mainly through two classes of degradation enzymes—one that can break the ribose–ribose bonds within the PAR chain and the other that breaks the covalent bonds between the proximal ADP-ribose units and the modified proteins. "
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    ABSTRACT: ADP-ribosylation refers to the addition of one or more ADP-ribose units onto protein substrates and this protein modification has been implicated in various cellular processes including DNA damage repair, RNA metabolism, transcription and cell cycle regulation. This review focuses on a compilation of large-scale proteomics studies that identify ADP-ribosylated proteins and their associated proteins by mass spectrometry using a variety of enrichment strategies. Some methods, such as the use of a poly(ADP-ribose)-specific antibody and boronate affinity chromatography and NAD+ analogues, have been employed for decades while others, such as the use of protein microarrays and recombinant proteins that bind ADP-ribose moieties (such as macrodomains), have only recently been developed. The advantages and disadvantages of each method and whether these methods are specific for identifying mono(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins will be discussed. Lastly, since poly(ADP-ribose) is heterogeneous in length, it has been difficult to attain a mass signature associated with the modification sites. Several strategies on how to reduce polymer chain length heterogeneity for site identification will be reviewed.This article is protected by copyright. All rights reserved
    Proteomics 09/2014; 15(2-3). DOI:10.1002/pmic.201400217 · 3.81 Impact Factor
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    • "Although they are best known for their role in DNA repair, particularly Base Excision Repair and other types of ssDNA repair, PARPs are reported to function in other cellular processes, such as mitosis, regulating chromatin state, and transcription [34]–[39]. In addition, PARPs are important for cellular responses to many types of environmental assaults, including heat, genotoxic, metabolic, and oxidative stresses [40]–[43]. "
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    ABSTRACT: Maintaining the length of the telomere tract at chromosome ends is a complex process vital to normal cell division. Telomere length is controlled through the action of telomerase as well as a cadre of telomere-associated proteins that facilitate replication of the chromosome end and protect it from eliciting a DNA damage response. In vertebrates, multiple poly(ADP-ribose) polymerases (PARPs) have been implicated in the regulation of telomere length, telomerase activity and chromosome end protection. Here we investigate the role of PARPs in plant telomere biology. We analyzed Arabidopsis thaliana mutants null for PARP1 and PARP2 as well as plants treated with the PARP competitive inhibitor 3-AB. Plants deficient in PARP were hypersensitive to genotoxic stress, and expression of PARP1 and PARP2 mRNA was elevated in response to MMS or zeocin treatment or by the loss of telomerase. Additionally, PARP1 mRNA was induced in parp2 mutants, and conversely, PARP2 mRNA was induced in parp1 mutants. PARP3 mRNA, by contrast, was elevated in both parp1 and parp2 mutants, but not in seedlings treated with 3-AB or zeocin. PARP mutants and 3-AB treated plants displayed robust telomerase activity, no significant changes in telomere length, and no end-to-end chromosome fusions. Although there remains a possibility that PARPs play a role in Arabidopsis telomere biology, these findings argue that the contribution is a minor one.
    PLoS ONE 02/2014; 9(2):e88872. DOI:10.1371/journal.pone.0088872 · 3.23 Impact Factor
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