Kazuaki Tokunaga

RAD18 promotes DNA double-strand break repair during G1 phase through chromatin retention of 53BP1

Recruitment of RAD18 to stalled replication forks facilitates monoubiquitination of PCNA during S-phase, promoting translesion synthesis at sites of UV irradiation-induced DNA damage. In this study, we show that RAD18 is also recruited to ionizing radiation (IR)-induced sites of DNA double-strand breaks (DSBs) forming foci which are co-localized with 53BP1, NBS1, phosphorylated ATM, BRCA1 and γ-H2AX. RAD18 associates with 53BP1 and is recruited to DSB sites in a 53BP1-dependent manner specifically during G1-phase, RAD18 monoubiquitinates KBD domain of 53BP1 at lysine 1268 in vitro. A monoubiquitination-resistant 53BP1 mutant harboring a substitution at lysine 1268 is not retained efficiently at the chromatin in the vicinity of DSBs. In Rad18-null cells, retention of 53BP1 foci, efficiency of DSB repair and post-irradiation viability are impaired compared with wild-type cells. Taken together, these results suggest that RAD18 promotes 53BP1-directed DSB repair by enhancing retention of 53BP1, possibly through an interaction between RAD18 and 53BP1 and the modification of 53BP1.

Nucleocytoplasmic transport of fluorescent mRNA in living mammalian cells: nuclear mRNA export is coupled to ongoing gene transcription

In eukaryotic cells, export of mRNA from the nucleus to the cytoplasm is one of the essential steps in gene expression. To examine mechanisms involved in the nucleocytoplasmic transport of mRNA, we microinjected fluorescently labeled fushi tarazu (ftz) pre‐mRNA into the nuclei of HeLa cells. The injected intron‐containing ftz pre‐mRNA was distributed to the SC35 speckles and exported to the cytoplasm after splicing by an energy‐requiring active process. In contrast, the injected intron‐less ftz mRNA was diffusely distributed in the nucleus and then presumably degraded. Interestingly, export of the ftz pre‐mRNA was inhibited by treatment with transcriptional inhibitors (actinomycin D, α‐amanitin or DRB). Cells treated with transcriptional inhibitor showed foci enriched with the injected mRNA, which localize side by side with SC35 speckles. Those nuclear foci, referred to as TIDRs (transcriptional‐inactivation dependent RNA domain), do not overlap with paraspeckles. In addition, in situ hybridization analysis revealed that the export of endogenous poly(A)+ mRNA is also affected by transcriptional inactivation. These results suggest that nuclear mRNA export is coupled to ongoing gene transcription in mammalian cells.

Computational image analysis of colony and nuclear morphology to evaluate human induced pluripotent stem cells

Non-invasive evaluation of cell reprogramming by advanced image analysis is required to maintain the quality of cells intended for regenerative medicine. Here, we constructed living and unlabelled colony image libraries of various human induced pluripotent stem cell (iPSC) lines for supervised machine learning pattern recognition to accurately distinguish bona fide iPSCs from improperly reprogrammed cells. Furthermore, we found that image features for efficient discrimination reside in cellular components. In fact, extensive analysis of nuclear morphologies revealed dynamic and characteristic signatures, including the linear form of the promyelocytic leukaemia (PML)-defined structure in iPSCs, which was reversed to a regular sphere upon differentiation. Our data revealed that iPSCs have a markedly different overall nuclear architecture that may contribute to highly accurate discrimination based on the cell reprogramming status.

Quantitative assessment of higher‐order chromatin structure of the INK4/ARF locus in human senescent cells

Somatic cells can be reset to oncogene‐induced senescent (OIS) cells or induced pluripotent stem (iPS) cells by expressing specified factors. The INK4/ARF locus encodes p15INK4b, ARF, and p16INK4a genes in human chromosome 9p21, the products of which are known as common key reprogramming regulators. Compared with growing fibroblasts, the CCCTC‐binding factor CTCF is remarkably up‐regulated in iPS cells with silencing of the three genes in the locus and is reversely down‐regulated in OIS cells with high expression of p15INK4b and p16INK4a genes. There are at least three CTCF‐enriched sites in the INK4/ARF locus, which possess chromatin loop‐forming activities. These CTCF‐enriched sites and the p16INK4a promoter associate to form compact chromatin loops in growing fibroblasts, while CTCF depletion disrupts the loop structure. Interestingly, the loose chromatin structure is found in OIS cells. In addition, the INK4/ARF locus has an intermediate type of chromatin compaction in iPS cells. These results suggest that senescent cells have distinct higher‐order chromatin signature in the INK4/ARF locus.

S1-1 nuclear domains: characterization and dynamics as a function of transcriptional activity

Background information. The RNA‐binding protein S1‐1, also called RBM10 (RNA‐binding motif 10), is a paralogue of putative tumour suppressor RBM5 and has been correlated with cancer proliferation and apoptosis. In the present study, we have investigated the cell biology of S1‐1.

PV1, a novel Plasmodium falciparum merozoite dense granule protein, interacts with exported protein in infected erythrocytes

Upon invasion, Plasmodium falciparum exports hundreds of proteins across its surrounding parasitophorous vacuole membrane (PVM) to remodel the infected erythrocyte. Although this phenomenon is crucial for the parasite growth and virulence, elucidation of precise steps in the export pathway is still required. A translocon protein complex, PTEX, is the only known pathway that mediates passage of exported proteins across the PVM. P. falciparum Parasitophorous Vacuolar protein 1 (PfPV1), a previously reported parasitophorous vacuole (PV) protein, is considered essential for parasite growth. In this study, we characterized PfPV1 as a novel merozoite dense granule protein. Structured illumination microscopy (SIM) analyses demonstrated that PfPV1 partially co-localized with EXP2, suggesting the protein could be a PTEX accessory molecule. Furthermore, PfPV1 and exported protein PTP5 co-immunoprecipitated with anti-PfPV1 antibody. Surface plasmon resonance (SPR) confirmed the proteins’ direct interaction. Additionally, we identified a PfPV1 High-affinity Region (PHR) at the C-terminal side of PTP5 where PfPV1 dominantly bound. SIM analysis demonstrated an export arrest of PTP5ΔPHR, a PTP5 mutant lacking PHR, suggesting PHR is essential for PTP5 export to the infected erythrocyte cytosol. The overall results suggest that PfPV1, a novel dense granule protein, plays an important role in protein export at PV.

Monitoring mRNA export.

Transport of mRNA from the nucleus to the cytoplasm is an essential process for gene expression in eukaryotic cells. In this unit, methods for monitoring nuclear mRNA export are described. Visualization of cellular mRNAs by fluorescence in situ hybridization with oligo(dT) probes is effectively applied to monitoring mRNA export from the nucleus in yeast and mammalian cells. In addition to the protocols for fluorescence in situ hybridization, this unit includes an alternate method that the authors have been developing for visual analysis of nuclear mRNA export in living mammalian cells by microinjection of fluorescently labeled pre‐mRNA into the nuclei. Curr. Protoc. Cell Biol. 41:22.13.1‐22.13.20.

Visual Screening for the Natural Compounds That Affect the Formation of Nuclear Structures

In eukaryotic cells, there are two major compartments separated by the nuclear membrane: the nucleus, where transcription and replication of DNA occur, and the cytoplasm, where translation of mRNAs to proteins occurs. Interestingly, the interphase nucleus is further divided into a dozen subnuclear compartments, such as nucleoli, speckles, Cajal bodies, paraspeckles, promyelocytic leukemia (PML) bodies, and gems (for gemini of Cajal bodies) (Fig. 1) [1].