Biomedical subjects

Tokuko Haraguchi

Publications and source records attributed to Tokuko Haraguchi.

27 records · Page 2Linked to original sources

PubMed2003

Overexpression of the human MNB/DYRK1A gene induces formation of multinucleate cells through overduplication of the centrosome.

BACKGROUND: Previously we cloned the human MNB/DYRK1A gene from the "Down syndrome critical region" on chromosome 21. This gene encodes a dual specificity protein kinase that catalyzes its autophosphorylation on serine/threonine and tyrosine residues. But, the functions of the MNB/DYRK1A gene in cellular processes are unknown. RESULTS: In this study, we examined HeLa cells transfected with cDNA encoding a green fluorescent protein (GFP)-MNB/DYRK1A fusion protein and found 2 patterns of expression: In one group of transfected cells, GFP-MNB/DYRK1A was localized as dots within the nucleus; and in the other group, it was overexpressed and had accumulated all over the nucleus. In the cells overexpressing GFP-MNB/DYRK1A, multinucleation was clearly observed; whereas in those with the nuclear dots, such aberrant nuclei were not found. Furthermore, in the latter cells, essential processes such as mitosis and cytokinesis occurred normally. Multinucleation was dependent on the kinase activity of MNB/DYRK1A, because it was not observed in cells overexpressing kinase activity-negative mutants, GFP-MNB/DYRK1A (K179R) and GFP-MNB/DYRK1A (Y310F/Y312F). Immunostaining of GFP-MNB/DYRK1A-overexpressing cells with specific antibodies against alpha- and gamma-tubulin revealed that multiple copies of centrosomes and aberrant multipolar spindles were generated in these cells. CONCLUSIONS: These results indicate that overexpression of MNB/DYRK1A induces multinucleation in HeLa cells through overduplication of the centrosome during interphase and production of aberrant spindles and missegregation of chromosomes during mitosis.

Cell Cycle↗

PubMed2003

Cell cycle behavior of human HP1 subtypes: distinct molecular domains of HP1 are required for their centromeric localization during interphase and metaphase.

Heterochromatin protein 1 (HP1) plays an important role in heterochromatin formation. Three subtypes of HP1, namely HP1alpha, beta, and gamma, have been identified in humans. In this study, using yellow fluorescent protein (YFP) fusion constructs, we examined the intracellular localization of human HP1 subtypes during the cell cycle. During interphase, all three HP1 subtypes were localized to centromeric heterochromatin and to promyelocytic leukemia (PML) nuclear bodies. Different preferences, however, were observed among the subtypes: during interphase HP1beta localized most preferentially to centromeric heterochromatin, whereas HP1alpha and gamma were more preferentially localized to PML nuclear bodies. During metaphase, only HP1alpha, was localized to the centromere. We thus determined which molecular domains of HP1 were necessary for their intracellular localization. Our results showed that the C-terminal fragment (amino acid residues 101-180) of HP1alpha was necessary for localization to the metaphase centromere and the N-terminal fragment (amino acid residues 1-76) of HP1beta was necessary for localization to the interphase centromere. Interestingly, simultaneous observations of residues 101-180 of HP1alpha and residues 1-76 of HP1beta in living HeLa cells revealed that during late prophase, the HP1beta fragment dissociated from centromeric regions and the HP1alpha fragment accumulated in centromeric regions. These results indicate that different specific regions of human HP1alpha and HP1beta mediate localization to metaphase and interphase centromeric regions resulting in association of different subtypes of HP1 with the centromere at different times during the cell cycle.

Amino Acid Sequence↗

PubMed2003

Dynamic behavior of Nuf2-Hec1 complex that localizes to the centrosome and centromere and is essential for mitotic progression in vertebrate cells.

Nuf2 and Hec1 are evolutionarily conserved centromere proteins. To clarify the functions of these proteins in vertebrate cells, we characterized them in chicken DT40 cells. We generated GFP fusion constructs of Nuf2 and Hec1 to examine in detail the localization of these proteins during the cell cycle. We found that Nuf2 is associated with Hec1 throughout the cell cycle and that this complex is localized to the centrosomes during G1 and S phases and then moves through the nuclear membrane to the centromere in G2 phase. During mitosis, this complex is localized to the centromere. We also created conditional loss-of-function mutants of Nuf2 and Hec1. In both mutants, the cell cycle arrested at prometaphase, suggesting that the Nuf2-Hec1 complex is essential for mitotic progression. The inner centromere proteins CENP-A, -C, and -H and checkpoint protein BubR1 were localized to chromosomes in the mutant cells arrested at prometaphase, but Mad2 localization was abolished. Furthermore, photobleaching experiments revealed that the Nuf2-Hec1 complex is stably associated with the centromere and that interaction of this complex with the centrosome is dynamic.

Animals↗

PubMed2002

CENP-I is essential for centromere function in vertebrate cells.

We identified a novel essential centromere protein, CENP-I, which shows sequence similarity with fission yeast Mis6 protein, and we showed that CENP-I is a constitutive component of the centromere that colocalizes with CENP-A, -C, and -H throughout the cell cycle in vertebrate cells. To determine the precise function of CENP-I, we examined its role in centromere function by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-I, cells arrested at prometaphase with misaligned chromosomes for long periods of time. Eventually, cells exited mitosis without undergoing cytokinesis. Immunocytochemical analysis of CENP-I-deficient cells demonstrated that both CENP-I and CENP-H are necessary for localization of CENP-C but not CENP-A to the centromere.

Animals↗

PubMed2002

Spectral imaging fluorescence microscopy.

The spectral resolution of fluorescence microscope images in living cells is achieved by using a confocal laser scanning microscope equipped with grating optics. This capability of temporal and spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; for example, those associated with fluorescence resonance energy transfer (FRET). Using the spectral imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescent dyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate (FITC) and green fluorescent protein (GFP).

Fluorescein-5-isothiocyanate↗

PubMed2002

Interaction of the chromatin compaction-inducing domain (LR domain) of Ki-67 antigen with HP1 proteins.

BACKGROUND: The LR domain of marsupial chmadrin is defined by its C-terminal amino acid sequence, which contains several pairs of leucine (L) and arginine (R) residues. The LR domain of chmadrin causes a significant compaction of chromatin over the entire length of chromosomes when it is overproduced. The possible human homologue of chmadrin, Ki-67 antigen (pKi-67), also has a stretch of LR pairs, but with no obvious overall similarity, at its C-terminus. RESULTS: The LR domain of human pKi-67 also induced chromatin compaction, both in human and marsupial cells. A yeast two-hybrid assay and an in vitro binding assay demonstrated that the human LR domain binds to heterochromatin protein 1 (HP1), a well-characterized molecule as a mediator of heterochromatin formation. In fixed cells stained with specific antibodies, the pKi-67 was found to be co-localized partially with HP1 at foci on chromosomes in an early G1 phase. Time-lapse observation in living cells co-expressing the fluorescently tagged proteins showed that the LR domain formed foci on chromosomes over a limited period of the cell cycle from the telophase to early G1 phase and that HP1 subsequently accumulated at these foci of the LR domain. CONCLUSIONS: Marsupial chmadrin and human pKi-67 induce chromatin compaction across species, possibly via the interaction of its LR domain with HP1.

Amino Acid Sequence↗

PubMed2002

Peroxisomes are formed from complex membrane structures in PEX6-deficient CHO cells upon genetic complementation.

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti-70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.

ATP-Binding Cassette Transporters↗

PubMed2002

Live cell imaging: approaches for studying protein dynamics in living cells.

In the last decade, the long-standing biologist's dream of seeing the molecular events within the living cell came true. This technological achievement is largely due to the development of fluorescence microscopy technologies and the advent of green fluorescent protein as a fluorescent probe. Such imaging technologies allowed us to determine the subcellular localization, mobility and transport pathways of specific proteins and even visualize protein-protein interactions of single molecules in living cells. Direct observation of such molecular dynamics can provide important information about cellular events that cannot be obtained by other methods. Thus, imaging of protein dynamics in living cells becomes an important tool for cell biology to study molecular and cellular functions. In this special issue of review articles, we review various imaging technologies of microscope hardware and fluorescent probes useful for cell biologists, with a focus on recent development of live cell imaging.

Animals↗

PubMed2002

Multispectral imaging fluorescence microscopy for living cells.

Multispectral imaging technologies have been widely used in fields of astronomy and remote sensing. Interdisciplinary approaches developed in, for example, the National Aeronautics and Space Administration (NASA, USA), the Jet Propulsion Laboratory (JPL, USA), or the Communications Research Laboratory (CRL, Japan) have extended the application areas of these technologies from planetary systems to cellular systems. Here we overview multispectral imaging systems that have been devised for microscope applications. We introduce these systems with particular interest in live cell imaging. Finally we demonstrate examples of spectral imaging of living cells using commercially available systems with no need for user engineering.

Animals↗