Chie Mori

Two distinct repeat sequences of Nup98 nucleoporins characterize dual nuclei in the binucleated ciliate tetrahymena.

Ciliated protozoa have two functionally distinct nuclei, a micronucleus (MIC) and a macronucleus (MAC) [1]. These two nuclei are distinct in size, transcriptional activity, and division cycle control, proceeding with cycles of DNA replication and nuclear division at different times within the same cell [2, 3]. The structural basis generating functionally distinct nuclei remains unknown. Here, we show that, in Tetrahymena thermophila, the nuclear pore complexes (NPCs) of MIC and MAC are composed of different sets of nucleoporins. Among the 13 nucleoporins identified, Nup98 homologs were of interest because two out of the four homologs were localized exclusively in the MAC and the other two were localized exclusively in the MIC. The two MAC-localizing Nup98s contain repeats of GLFG [4]. In contrast, the two MIC-localizing Nup98s lack the GLFG repeats and instead contain a novel repeat signature of NIFN. Ectopic expression of a chimeric MIC-localizing Nup98 homolog bearing GLFG repeats obstructed the nuclear accumulation of MIC-specific nuclear proteins, and expression of a chimeric MAC-localizing Nup98 homolog bearing NIFN repeats obstructed the nuclear accumulation of MAC-specific nuclear proteins. These results suggest that Nup98s act as a barrier to misdirected localization of nucleus-specific proteins. Our findings provide the first evidence that the NPC contributes to nucleus-selective transport in ciliates.

Nuclear localization of barrier-to-autointegration factor is correlated with progression of S phase in human cells

Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.g. TIG-1, WI-38 and IMR-90 cells) BAF localization depended on the age of the cell, localizing primarily in the nucleus of >90% of young proliferating cells but only 20-25% of aged senescent cells. In immortal cell lines (e.g. HeLa, SiHa and HT1080 cells) BAF showed heterogeneous localization between the nucleus and cytoplasm. This heterogeneity was lost when the cells were synchronized in S phase. In S-phase-synchronized populations, the percentage of cells with predominantly nuclear BAF increased from 30% (asynchronous controls) to ∼80%. In HeLa cells, RNAi-induced downregulation of BAF significantly increased the proportion of early S-phase cells that retained high levels of cyclin D3 and cyclin E expression and slowed progression through early S phase. BAF downregulation also caused lamin A to mislocalize away from the nuclear envelope. These results indicate that BAF is required for the integrity of the nuclear lamina and normal progression of S phase in human cells.

A non-inheritable maternal Cas9-based multiple-gene editing system in mice.

The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection–based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create “Cas9 transgene-free” gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice.

Biased assembly of the nuclear pore complex is required for somatic and germline nuclear differentiation in Tetrahymena

Ciliates have two functionally distinct nuclei, a somatic macronucleus (MAC) and a germline micronucleus (MIC) that develop from daughter nuclei of the last postzygotic division (PZD) during the sexual process of conjugation. Understanding this nuclear dimorphism is a central issue in ciliate biology. We show, by live‐cell imaging of Tetrahymena, that biased assembly of the nuclear pore complex (NPC) occurs immediately after the last PZD, which generates anterior‐posterior polarized nuclei: MAC‐specific NPCs assemble in anterior presumptive MACs but not in posterior presumptive MICs. MAC‐specific NPC assembly in the anterior nuclei occurs much earlier than transport of Twi1p, which is required for MAC genome rearrangement. Correlative light‐electron microscopy shows that addition of new nuclear envelope (NE) precursors occurs through the formation of domains of redundant NE, where the outer double membrane contains the newly assembled NPCs. Nocodazole inhibition of the second PZD results in assembly of MAC‐specific NPCs in the division‐failed zygotic nuclei, leading to failure of MIC differentiation. Our findings demonstrate that NPC type switching has a crucial role in the establishment of nuclear differentiation in ciliates.

Inner nuclear membrane protein Lem2 augments heterochromatin formation in response to nutritional conditions

Inner nuclear membrane proteins interact with chromosomes in the nucleus and are important for chromosome activity. Lem2 and Man1 are conserved members of the LEM‐domain nuclear membrane protein family. Mutations of LEM‐domain proteins are associated with laminopathy, but their cellular functions remain unclear. Here, we report that Lem2 maintains genome stability in the fission yeast Schizosaccharomyces pombe. S. pombe cells disrupted for the lem2+ gene (lem2∆) showed slow growth and increased rate of the minichromosome loss. These phenotypes were prominent in the rich culture medium, but not in the minimum medium. Centromeric heterochromatin formation was augmented upon transfer to the rich medium in wild‐type cells. This augmentation of heterochromatin formation was impaired in lem2∆ cells. Notably, lem2∆ cells occasionally exhibited spontaneous duplication of genome sequences flanked by the long‐terminal repeats of retrotransposons. The resulting duplication of the lnp1+ gene, which encodes an endoplasmic reticulum membrane protein, suppressed lem2∆ phenotypes, whereas the lem2∆ lnp1∆ double mutant showed a severe growth defect. A combination of mutations in Lem2 and Bqt4, which encodes a nuclear membrane protein that anchors telomeres to the nuclear membrane, caused synthetic lethality. These genetic interactions imply that Lem2 cooperates with the nuclear membrane protein network to regulate genome stability.

BAF is a cytosolic DNA sensor that leads to exogenous DNA avoiding autophagy.

Significance Rapid detection of invasion of exogenous materials and subsequent responses are important for living organisms to survive hazards, such as pathogen infection. Understanding cellular responses against exogenous DNA provides clues not only for controlling pathogen infections that bring exogenous DNA into host cells, but also for designing efficient DNA delivery vectors for transgene expression. Here, by monitoring the invasion of exogenous DNA-coated polystyrene beads into living cells, we show that barrier-to-autointegration factor detects exogenous DNA immediately after its appearance at endosome breakdown and plays a role in DNA avoiding autophagy. These findings provide new insights into the mechanisms by which a cell detects and responds to exogenous double-stranded DNA. Knowledge of the mechanisms by which a cell detects exogenous DNA is important for controlling pathogen infection, because most pathogens entail the presence of exogenous DNA in the cytosol, as well as for understanding the cell’s response to artificially transfected DNA. The cellular response to pathogen invasion has been well studied. However, spatiotemporal information of the cellular response immediately after exogenous double-stranded DNA (dsDNA) appears in the cytosol is lacking, in part because of difficulties in monitoring when exogenous dsDNA enters the cytosol of the cell. We have recently developed a method to monitor endosome breakdown around exogenous materials using transfection reagent-coated polystyrene beads incorporated into living human cells as the objective for microscopic observations. In the present study, using dsDNA-coated polystyrene beads (DNA-beads) incorporated into living cells, we show that barrier-to-autointegration factor (BAF) bound to exogenous dsDNA immediately after its appearance in the cytosol at endosome breakdown. The BAF+ DNA-beads then assembled a nuclear envelope (NE)-like membrane and avoided autophagy that targeted the remnants of the endosome membranes. Knockdown of BAF caused a significant decrease in the assembly of NE-like membranes and increased the formation of autophagic membranes around the DNA-beads, suggesting that BAF-mediated assembly of NE-like membranes was required for the DNA-beads to evade autophagy. Importantly, BAF-bound beads without dsDNA also assembled NE-like membranes and avoided autophagy. We propose a new role for BAF: remodeling intracellular membranes upon detection of dsDNA in mammalian cells.

Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena.

A puromycin-N-acetyltransferase gene (pac) is widely used as a selection marker for eukaryotic gene manipulation. However, it has never been utilized for molecular studies in the ciliate Tetrahymena thermophila, in spite of the limited number of selection markers available for this organism. To utilize pac as a maker gene for T. thermophila, the nucleotide sequence of the pac gene was altered to accord with the most preferred codon-usage in T. thermophila. This codon-optimized pac gene expressed in T. thermophila conferred a resistance to transformed cells against 2,000 μg/ml of puromycin dihydrochloride, whereas the growth of wild-type cells was completely inhibited by 200 μg/ml. Furthermore, an expression cassette constructed with the codon-optimized pac and an MTT1 promoter was effectively utilized for experiments to tag endogenous proteins of interest by fusing the cassette into the target gene locus. These results indicate that pac can be used as a selection marker in molecular studies of T. thermophila.

Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in the ciliate Tetrahymena

ABSTRACT The nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of ∼30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Tetrahymena thermophila, each cell contains both a transcriptionally active macronucleus (MAC) and a germline micronucleus (MIC). By combining in silico analysis, mass spectrometry analysis for immuno-isolated proteins and subcellular localization analysis of GFP-fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107–Nup160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either the MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on the compositional and structural specificity of NPCs. Summary: There are compositional and structural differences in the nuclear pore complexes present in the functionally differentiated macronucleus and micronucleus within the single cytoplasm of ciliated protozoa.

Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells

Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here, we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62‐knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA‐mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery.

Uncleavable Nup98–Nup96 is functional in the fission yeast Schizosaccharomyces pombe

Essential nucleoporins Nup98 and Nup96 are coded by a single open reading frame, and produced by autopeptidase cleavage. The autocleavage site of Nup98–Nup96 is highly conserved in a wide range of organisms. To understand the importance of autocleavage, we examined a mutant that produces the Nup98–Nup96 joint molecule as a sole protein product of thenup189 + gene in the fission yeastSchizosaccharomyces pombe. Cells expressing only the joint molecule were found to be viable. This result indicates that autocleavage of Nup98–Nup96 is dispensable for cell growth, at least under normal culture conditions inS. pombe.