Proteome Analysis of Human Metaphase Chromosomes

Uchiyama, Susumu; Kobayashi, Shogo; Takata, Hideaki; Ishihara, Takeshi; Hori, Naoto; Higashi, Taishi; Hayashihara, Kayoko; Sone, Takefumi; Higo, Daisuke; Nirasawa, Takashi; Takao, Toshifumi; Matsunaga, Sachihiro; Fukui, Kiichi

doi:10.1074/jbc.m412774200

Cited by 117 publications

(154 citation statements)

References 66 publications

(71 reference statements)

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“…In our recent proteome analysis, 107 proteins were identified in highly purified human metaphase chromosomes (Uchiyama et al, 2005). Furthermore, the observation that more than a quarter of the identified proteins were classified as chromosome peripheral proteins strongly suggests that these proteins are related to either chromosome organization or mitotic progression.…”

Section: Introductionmentioning

confidence: 99%

Nucleolin functions in nucleolus formation and chromosome congression

Matsunaga

Takata

et al. 2007

Journal of Cell Science

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A complex structure, designated the chromosome periphery, surrounds each chromosome during mitosis. Although several proteins have been shown to localize to the chromosome periphery, their functions during mitosis remain unclear. Here, we used a combination of highresolution microscopy and RNA-interference-mediated depletion to study the functions of nucleolin, a nucleolar protein localized at the chromosome periphery, in interphase and mitosis. During mitosis, nucleolin was localized in the peripheral region including the vicinity of the outer kinetochore of chromosomes. Staining with an antibody specific for nucleolin phosphorylated by CDC2 revealed that nucleolin was also associated with the spindle poles from prometaphase to anaphase. Nucleolin depletion resulted in disorganization of the nucleoli at interphase. Furthermore, nucleolin-depleted cells showed a prolonged cell cycle with misaligned chromosomes and defects in spindle organization. The misaligned chromosomes showed syntelic kinetochore-microtubule attachments with reduced centromere stretching. Taken together, our results indicate that nucleolin is required for nucleolus formation, and is also involved in chromosome congression and spindle formation. Journal of Cell Science 2092Nucleolin is an abundant nucleolar protein localized in the DFCs and GCs of nucleoli (Biqqioqera et al., 1990). Nucleolin plays essential roles in rDNA transcription, rRNA maturation, ribosome assembly and nucleocytoplasmic transport (Ginisty et al., 1999;Srivastava and Pollard, 1999). As a multifunctional protein, nucleolin has also been proposed to be a nuclear matrix-binding protein (Gotzmann et al., 1997), to interact with telomerase (Khurts et al., 2004) and to be involved in the regulation of apoptosis (Mi et al., 2003), intestinal cell differentiation (Turck et al., 2006) and remodeling of nucleosomes (Angelov et al., 2006). Although many functions of nucleolin have been clarified, previous studies have fallen short of demonstrating its functions during mitosis.Nucleolin is highly phosphorylated (Olson et al., 1975;Rao et al., 1982) and its phosphorylation is highly regulated during the cell cycle (Peter et al., 1990;Morimoto et al., 2005). Extensive phosphorylation by casein kinase 2 (CK2) occurs at interphase and by CDC2 during mitosis (Peter et al., 1990), and this regulated phosphorylation of nucleolin probably regulates nucleolin functions during the cell cycle (Ginisty et al., 1999). It has been demonstrated that phosphorylation of nucleolin at interphase is correlated with active rRNA transcription (Suzuki et al., 1987). Despite a previous study describing the localization of nucleolin phosphorylated by CDC2 (Dranovsky et al., 2001), no information about its functions during mitosis has been obtained to date.Here, we demonstrate that nucleolin localizes to the nucleoli, which are located in the peripheral region including the vicinity of the outer kinetochore of chromosomes, in interphase nuclei and during mitosis. Staining with an antibody specific for nucle...

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Section: Introductionmentioning

confidence: 99%

Nucleolin functions in nucleolus formation and chromosome congression

Matsunaga

Takata

et al. 2007

Journal of Cell Science

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120

124

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“…Two small proteins that are likely to be important to chromatin folding are high-mobility-group (HMG) proteins, and the barrier-to-integration-factor (BAF-1) protein (Margalit et al 2007), which are present in mitotic chromosomes in numbers comparable to those of histones (Uchiyama et al 2005). HMG proteins are divided into three families with different functions (Hock et al 2007): HMGA proteins bind AT-rich DNA, HMGB proteins bend DNA (Thomas & Travers 2001), and HMGN proteins reorganize nucleosomes.…”

Section: Chromatin Fibrementioning

confidence: 99%

Micromechanical studies of mitotic chromosomes

2008

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Mitotic chromosomes respond elastically to forces in the nanonewton range, a property important to transduction of stresses used as mechanical regulatory signals during cell division. In addition to being important biologically, chromosome elasticity can be used as a tool for investigating the folding of chromatin. This paper reviews experiments studying stretching and bending stiffness of mitotic chromosomes, plus experiments where changes in chromosome elasticity resulting from chemical and enzyme treatments were used to analyse connectivity of chromatin inside chromosomes. Experiments with nucleases indicate that non-DNA elements constraining mitotic chromatin must be isolated from one another, leading to the conclusion that mitotic chromosomes have a chromatin Fnetwork_ or Fgel_ organization, with stretches of chromatin strung between Fcrosslinking_ points. The as-yet unresolved questions of the identities of the putative chromatin crosslinkers and their organization inside mitotic chromosomes are discussed.

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“…Proteomic analyses under different conditions indicated that CPPs remain to be associated even with highly purified chromosomes. In contrast, most chromosomal coating proteins, including mitochondrial or endoplasmic reticulum (ER) proteins, are lost from chromosomes under the same conditions (17,18). This suggests that the chromosome periphery is maintained as a chromosomal structure, resisting isolation and purification procedures or mechanical treatments.…”

Section: What Is the Chromosome Periphery?mentioning

confidence: 92%

“…Immunostaining with antibodies against several CPPs and localization analyses of GFP-fused CPPs also demonstrated that the chromosome periphery is not an experimental artifact but a chromosome structure (7,17). Recent proteome analyses of metaphase chromosomes revealed that human chromosomes consist of four layers, including the chromosome periphery (17). Proteomic analyses under different conditions indicated that CPPs remain to be associated even with highly purified chromosomes.…”

Section: What Is the Chromosome Periphery?mentioning

confidence: 99%

“…Moreover, the chromosome periphery consists of closely packed dense granules which are often in direct contact with chromatin (16). Immunostaining with antibodies against several CPPs and localization analyses of GFP-fused CPPs also demonstrated that the chromosome periphery is not an experimental artifact but a chromosome structure (7,17). Recent proteome analyses of metaphase chromosomes revealed that human chromosomes consist of four layers, including the chromosome periphery (17).…”

Section: What Is the Chromosome Periphery?mentioning

confidence: 99%

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The chromosome peripheral proteins play an active role in chromosome dynamics

Matsunaga

Fukui

2010

BioMolecular Concepts

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The chromosome periphery is a chromosomal structure that covers the surface of mitotic chromosomes. The structure and function of the chromosome periphery has been poorly understood since its first description in 1882. It has, however, been proposed to be an insulator or barrier to protect chromosomes from subcellular substances and to act as a carrier of nuclear and nucleolar components to direct their equal distribution to daughter cells because most chromosome peripheral proteins (CPPs) are derived from the nucleolus or nucleus. Until now, more than 30 CPPs were identified in mammalians. Recent immunostaining analyses of CPPs have revealed that the chromosome periphery covers the centromeric region of mitotic chromosomes in addition to telomeres and regions between two sister chromatids. Knockdown analyses of CPPs using RNAi have revealed functions in chromosome dynamics, including cohesion of sister chromatids, kinetochore-microtubule attachments, spindle assembly and chromosome segregation. Because most CPPs are involved in various subcellular events in the nucleolus or nuclear at interphase, a temporal and spatialspecific knockdown method of CPPs in the chromosome periphery will be useful to understand the function of chromosome periphery in cell division.

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Proteome Analysis of Human Metaphase Chromosomes

Cited by 117 publications

References 66 publications

Nucleolin functions in nucleolus formation and chromosome congression

Nucleolin functions in nucleolus formation and chromosome congression

Micromechanical studies of mitotic chromosomes

The chromosome peripheral proteins play an active role in chromosome dynamics

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