Characterization of nucleolar localization and exclusion signals in terminal deoxynucleotidyltransferase interacting factor 2/estrogen receptor α-binding protein.

Terminal deoxynucleotidyltransferase interacting factor 2/estrogen receptor α-binding protein (TdIF2/ERBP) is a multifunctional nucleolar protein. The nucleolar localization of TdIF2/ERBP is important for its functions because it promotes ribosomal RNA transcription. However, signal sequences that direct TdIF2/ERBP to the nucleolus are not well characterized. We examined the TdIF2/ERBP sequence using truncation and mutation analyses to determine whether the nucleosome binding and C-terminal domains of TdIF2/ERBP possess nucleolar localization signals (NoLSs). In these domains, four NoLSs that could direct the mCherry protein to the nucleolus were detected. In addition, a short stretch of hydrophobic residues (VLLVL) in the center of TdIF2/ERBP acted as a nucleolar exclusion signal, which reduced the nucleolar accumulation of mCherry-NoLS fusion proteins. These results would contribute to improving the prediction of NoLSs from protein sequences. The short, transferrable localization signals would be valuable tools for understanding the association between localization and functions of nucleolar proteins. Abbreviations TdIF2: terminal deoxynucleotidyltransferase interacting factor 2; ERBP: estrogen receptor α-binding protein; EGFP: enhanced green fluorescent protein; NLS: nuclear localization signal; NoLS: nucleolar localization signal; NoES: nucleolar exclusion signal; DAPI: 4',6-diamidino-2-phenylindole.

A rapid and simple method of evaluating the dimeric tendency of fluorescent proteins in living cells using a truncated protein of importin α as fusion tag.

Enhanced green fluorescent protein (EGFP) and its yellow variant (Venus) are weakly dimeric under physiological conditions. We designed a simple method to evaluate the dimeric tendency of fluorescent proteins in living mammalian cells. A novel single mutation, A206L, interfering with the hydrophobic interactions of the dimer interface in Venus, contributed to its monomerization, and was as effective as the A206K mutation in this assay.

Live cell imaging of micronucleus formation and development.

The micronucleus (MN) test is widely used to biomonitor humans exposed to clastogens and aneugens, but little is known about MN development. Here we used confocal time-lapse imaging and a fluorescent human lymphoblastoid cell line (T105GTCH), in which histone H3 and α-tubulin stained differentially, to record the emergence and behavior of micronuclei (MNi) in cells exposed to MN-inducing agents. In mitomycin C (MMC)-treated cells, MNi originated in early anaphase from lagging chromosome fragments just after chromosome segregation. In γ-ray-treated cells showing multipolar cell division, MN originated in late anaphase from lagging chromosome fragments generated by the abnormal cell division associated with supernumerary centrosomes. In vincristine(VC)-treated cells, MN formation was similar to that in MMC-treated cells, but MNi were also derived from whole chromosomes that did not align properly on the metaphase plate. Thus, the MN formation process induced by MMC, γ-rays, and VC, were strikingly different, suggesting that different mechanisms were involved. MN stability, however, was similar regardless of the treatment and unrelated to MN formation mechanisms. MNi were stable in daughter cells, and MN-harboring cells tended to die during cell cycle progression with greater frequency than cells without MN. Because of their persistence, MN may have significant impact on cells, causing genomic instability and abnormally transcribed genes.

Visualization of aberrant perinuclear microtubule aster organization by microtubule-destabilizing agents.

To analyze aberrant spindle formation by microtubule-targeting drugs, live cell imaging was performed using multi-fluorescent human MDA-MB-435 cells in which several spindle components were visualized. Time-lapse images revealed that nocodazole and vinblastine induced additional perinuclear asters at the onset of mitosis. These results imply that these drugs stimulate the microtubule-organizing activity, despite their microtubule-destabilizing properties.

Construction of three quadruple-fluorescent MDA435 cell lines that enable monitoring of the whole chromosome segregation process in the living state.

Mitotic events from prophase to telophase are defined by morphology or movement of chromatin, nuclear envelope, centrosomes and spindles. Live-cell imaging is useful for characterizing the whole chromosome segregation process in the living state. In this study, we constructed three quadruple-fluorescent MDA435 cell lines in which chromatin, kinetochores, nuclear envelope and either inner centromere, microtubules or centrosomes/spindles were differentially visualized with cyan, green, orange and red fluorescent proteins (ECFP, EGFP, mKO and DsRed). Each mitotic stage of the individual cells could be identified by capturing live-cell images without the requirement of fixing or staining steps. In addition, we obtained four-color time-lapse images of one cell line, MDA-Auro/imp/H3/AF, from prophase to metaphase and from early anaphase to telophase. These quadruple-fluorescent cell lines will be useful for precisely analyzing the mitotic events from prophase through to telophase in single cells in the future.

Differential responses of mitotic spindle pole formation to microtubule-stabilizing agents epothilones A and B at low concentrations.

We previously reported that the microtubule-stabilizing agent docetaxel induced formation of fragile acentrosomal spindle poles but that structurally related paclitaxel did not. In the present study, we examined whether the microtubule-stabilizing agents epothilones A and B, which are structurally similar, affect the centrosome/spindle pole architecture.We investigated mitotic processes in epothilone A or B-treated human MDA-MB-435 cells, in which the centrosomes, spindle poles and mitotic micro-tubules were simultaneously visualized by GFP-Aurora A kinase. Fluorescence microscopy of metaphase cells indicated that several chromosomes were misaligned away from the metaphase plate when treated with IC(50) concentrations of epothilone A or B (4.5 or 2 nM, respectively), suggesting that microtubule dynamics was impaired. Interestingly, epothilone B induced formation of acentro-somal spindle poles, but this effect was not observed for epothilone A. Live-cell imaging showed that aster-like structures ectopically arose around the nuclear envelope at the onset of mitosis in epothilone B-treated cells and that one of these asters became an acentrosomal spindle pole. Aster-like structures also arose in the presence of epothilone A, but they were merged into centro-some-derived spindle poles during prometaphase and completely disappeared until metaphase. These results indicate that the centro-some/spindle pole integrity is strongly affected by epothilone B but is not greatly affected by epothilone A. Our findings show that the two epothilones cause different cellular responses at equipotent concentrations and suggest that they have different mechanisms of activity in cells.

A multi-fluorescent MDA435 cell line for mitosis inhibitor studies: simultaneous visualization of chromatin, microtubules, and nuclear envelope in living cells.

As a part of our efforts to create a pre-made multi-fluorescent cell library for various cytological assays, we made a triple-fluorescent cell line in which microtubules, chromosomes, and nuclear envelopes were visualized for simultaneous observation of spindle structure and chromosome distribution in living cells. Pilot experiments with microtubule-disturbing drugs showed the advantages of this cell line in mitosis inhibitor studies.

Dynamic behavior of FCHO1 revealed by live-cell imaging microscopy: its possible involvement in clathrin-coated vesicle formation.

The intracellular behavior of human FCHO1 protein was investigated by live-cell imaging microscopy. The fluorescence intensity of green fluorescent protein (GFP)-FCHO1 fluctuated periodically in a perinuclear region approximately every 100 s, reminding us of the periodic fluctuations of clathrin reported in our recent work. The periodicity of FCHO1 was temporally correlated with that of clathrin, suggesting that FCHO1 is involved in clathrin-coated vesicle formation.

Rhythmic cycle of clathrin-coated pit formation at the trans-golgi network in human MDA-MB-435 cells.

We studied the in vivo dynamics of enhanced green fluorescent protein-tagged clathrin light chain a (GFP-CLCa) at the trans-Golgi network (TGN) in MDA-MB-435 cells. The intensity of fluorescence signals of GFP-CLCa periodically increased and decreased at the TGN approximately every 100 s. This suggests that the formation of clathrin-coated pits occurs synchronously and periodically at the TGN.