The C protein of wild-type measles virus has the ability to shuttle between the nucleus and the cytoplasm

Measles virus (MV) C protein is a small and basic non-structural protein, but its function is not well understood. We have found that a FLAG-tagged wild-type MV C protein expressed from cDNA was accumulated exclusively in the nucleus. To analyze the amino acid sequence important for the nuclear localization of C protein, a plasmid expressing C protein fused to the enhanced green fluorescent protein (EGFP) was generated. Mutation analysis revealed that (41)PPARKRRQ(48), belonging to the classical nuclear localization signal was important for nuclear localization. Analysis of the amino acid sequence of C protein revealed that it has a nuclear export signal (NES)-like sequence, (76)LEKAMTTLKL(85). Addition of the putative NES to the EGFP resulted in the translocation of EGFP to the cytoplasm. The Rev(1.4)-EGFP nuclear export assay showed that this putative NES has a CRM1-dependent NES activity. C-EGFP accumulated in HeLa nuclei could be translocated to NIH3T3 nuclei in heterokaryon assays. In MV-infected cells, C-EGFP was accumulated in the nuclei in early phase but in the cytoplasm in late phase. These results indicate that the putative NES is functional and that C protein has the ability to shuttle between the nucleus and the cytoplasm.     

N‐glycosylation Does Not Affect the Catalytic Activity of Ricin A Chain but Stimulates Cytotoxicity by Promoting Its Transport Out of the Endoplasmic Reticulum

Ricin A chain (RTA) depurinates the α‐sarcin/ricin loop after it undergoes retrograde trafficking to the cytosol. The structural features of RTA involved in intracellular transport are not known. To explore this, we fused enhanced green fluorescent protein (EGFP) to precursor (preRTA‐EGFP), containing a 35‐residue leader, and mature RTA (matRTA‐EGFP). Both were enzymatically active and toxic in Saccharomyces cerevisiae. PreRTA‐EGFP was localized in the endoplasmic reticulum (ER) initially and was subsequently transported to the vacuole, whereas matRTA‐EGFP remained in the cytosol, indicating that ER localization is a prerequisite for vacuole transport. When the two glycosylation sites in RTA were mutated, the mature form was fully active and toxic, suggesting that the mutations do not affect catalytic activity. However, nonglycosylated preRTA‐EGFP had reduced toxicity, depurination and delayed vacuole transport, indicating that N‐glycosylation affects transport of RTA out of the ER. Point mutations in the C‐terminal hydrophobic region restricted RTA to the ER and eliminated toxicity and depurination, indicating that this sequence is critical for ER exit. These results demonstrate that N‐glycosylation and the C‐terminal hydrophobic region stimulate the toxicity of RTA by promoting ER export. The timing of depurination coincided with the timing of vacuole transport, suggesting that RTA may enter the cytosol during vacuole transport.     

Nuclear localization of enhanced green fluorescent protein homomultimers

The green fluorescent protein (GFP) and its variants are used in many studies to determine the subcellular localization of other proteins by analyzing fusion proteins. The main problem for nuclear localization studies is the fact that, to some extent, GFP translocates to the nucleus on its own. Because the nuclear import could be due to unspecific diffusion of the relatively small GFP through the nuclear pores, we analyzed the localization of multimers of a GFP variant, the enhanced GFP (EGFP). By detecting the fluorescence of the expressed proteins in gels after nonreducing SDS-PAGE, we demonstrate the integrity of the expressed proteins. Nevertheless, even EGFP homotetramers and homohexamers are found in the nuclei of the five analyzed mammalian cell lines. The use of fusion constructs of small proteins with multimeric EGFP alone, therefore, is not adequate to prove nuclear import processes. Fusion to tetrameric EGFP in combination with a careful quantification of the fluorescence intensities in the nucleus and cytoplasm might be sufficient in many cases to identify a significant difference between the fusion protein and tetrameric EGFP alone to deduce a nuclear localization signal.     

Nuclear localization of dengue virus nonstructural protein 5 through its importin alpha/beta-recognized nuclear localization sequences is integral to viral infection

Dengue virus nonstructural protein 5 (NS5) is a large multifunctional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type-2 (DENV-2) NS5 ('aNLS' and 'bNLS') that are recognized by the importin alpha/beta and importin beta1 nuclear transporters, respectively. Here, we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time and show that the aNLS is responsible. Site-specific mutations in the bipartite-type aNLS or bNLS region were introduced into a reporter plasmid encoding green fluorescent protein fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length complementary DNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K(387)K(388)K(389)); mutagenesis of both clusters abolished NS5 nuclear import and DENV-2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear-localizing NS5, as compared with wild-type virus expressing nuclear-localizing NS5, to reduce interleukin-8 production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.     

Identification of a nuclear targeting domain in the insertion between helices C and D in protease inhibitor-10

Protease inhibitor 10 (PI-10), an intracellular ovalbumin-serpin, contains a series of basic amino acids in the loop between helices C and D that exhibit homology to known nuclear targeting signals. Transfection of HeLa cells with plasmids encoding enhanced green fluorescent protein (EGFP) coupled to PI-10 revealed an intense fluorescence of the nucleus. Immunoblotting demonstrated a single Mr 80,000 EGFP.PI-10 complex in isolated nuclei. Mutation of four basic amino acids in the interhelical loop to alanines (i.e. K74A, K75A, R76A, K77A) resulted in the fluorescent complex being confined to the cytoplasm. Further evidence for a nuclear targeting signal in this region was provided by localization of the fluorescent label to the nucleus in cells transfected with a plasmid encoding EGFP fused to the 25 amino acids comprising the interhelical loop of PI-10 (i.e. Arg-63 to Glu-87), whereas a cytoplasmic distribution was noted for the construct encoding EGFP coupled to the mutated interhelical loop. These data raise the possibility that PI-10 may play a role in regulating protease activity within the nucleus, a property unique in the field of serpin biology.     

Molecular Control of Nuclear and Subnuclear Targeting of the Plant CDK Inhibitor ICK1 and ICK1-Mediated Nuclear Transport of CDKA

ICK1 is the first member of a family of plant cyclin-dependent kinase (CDK) inhibitors. It has been shown that ICK1 is localized in the nuclei of transgenic Arabidopsis plants. Since cellular localization is important for the functions of cell cycle regulators, a comprehensive analysis was undertaken to identify specific sequences regulating the cellular localization of ICK1. Deletion and site-specific mutants fused to the green fluorescent protein (GFP) were used in transgenic Arabidopsis plants and transfected tobacco cells. Surprisingly, three separate sequences in the N-terminal, central and C-terminal regions of ICK1 could independently confer nuclear localization of the GFP fusion proteins. The central nuclear localization signal NLS^ICK1 could transport the much larger GUS (β-glucuronidase)-GFP fusion protein into nuclei, while the other two sequences were unable to. These results suggest that NLS^ICK1 is a strong NLS that actively transports the fusion protein into nuclei, while the other two sequences are either a weaker NLS or confer the nuclear localization of GFP indirectly. It was further observed that the N-terminal sequence specifies a punctate pattern of subnuclear localization, while the C-terminal sequence suppresses it. Furthermore, co-expression of ICK1 and Arabidopsis CDKA, tagged with different GFP variants, showed that ICK1 could mediate the transport of CDKA into nuclei while a mutant ICK1^1–162 that does not interact with CDKA lost this ability. These results illustrate how the nuclear localization of ICK1 is regulated and also suggest a possible role of ICK1 in regulating the cellular distribution of CDKA.     

Analysis of localization and reorganization of germ plasm in Xenopus transgenic line with fluorescence‐labeled mitochondria

Germ plasm is found in germ‐line cells of Xenopus and thought to include the determinant of primordial germ cells (PGCs). As mitochondria is abundant in germ plasm, vital staining of mitochondria was used to analyze the movement and function of germ plasm; however, its application was limited in early cleavage embryos. We made transgenic Xenopus, harboring enhanced green fluorescent protein (EGFP) fused to the mitochondria transport signal (Dria‐line). Germ plasm with EGFP‐labeled mitochondria was clearly distinguishable from the other cytoplasm, and retained mostly during one generation of germ‐line cells in Dria‐line females. Using the Dria‐line, we show that germ plasm is reorganized from near the cell membrane to the perinuclear space at St. 9, dependent on the microtubule system.     

Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.     

Spatial regulation of the spindle assembly checkpoint and anaphase‐promoting complex in Aspergillus nidulans

The spindle assembly checkpoint (SAC) plays a critical role in preventing mitotic errors by inhibiting anaphase until all kinetochores are correctly attached to spindle microtubules. In spite of the economic and medical importance of filamentous fungi, relatively little is known about the behavior of SAC proteins in these organisms. In our efforts to understand the role of γ‐tubulin in cell cycle regulation, we have created functional fluorescent protein fusions of four SAC proteins in Aspergillus nidulans, the homologs of Mad2, Mps1, Bub1/BubR1 and Bub3. Time‐lapse imaging reveals that SAC proteins are in distinct compartments of the cell until early mitosis when they co‐localize at the spindle pole body. SAC activity is, thus, spatially regulated in A. nidulans. Likewise, Cdc20, an activator of the anaphase‐promoting complex/cyclosome, is excluded from interphase nuclei, but enters nuclei at mitotic onset and accumulates to a higher level in mitotic nuclei than in the surrounding nucleoplasm before leaving in anaphase/telophase. The activity of this critical cell cycle regulatory complex is likely regulated by the location of Cdc20. Finally, the γ‐tubulin mutation mipAD159 causes a nuclear‐specific failure of nuclear localization of Mps1 and Bub1/R1 but not of Cdc20, Bub3 or Mad2.     

In vivo visualization of the lymphatic vessels in pFLT4-EGFP transgenic medaka

Feline McDonough Sarcoma (FMS)-like tyrosine kinase 4 (FLT4) is a marker for lymphatic vessels and some high endothelial venules in human adult tissues. We generated a transgenic medaka fish in which the lymphatic vessels and some blood vessels are visible in vivo by transferring the promoter of medaka flt4 driving the expression of enhanced green fluorescent protein (EGFP) using a see-through medaka line. To do this, we identified and cloned medaka flt4 and generated a construct in which the promoter was the 4-kb region upstream of the translation initiation site. The fluorescent signal of EGFP could be observed with little background, and the expression pattern correlated well with that of flt4 determined by whole-mount RNA in situ hybridization. Because a see-through medaka line is transparent until adult, the model is useful for visualizing the lymphatic vessels not only in embryo and fry but also in adult. This model will be a useful tool for analyzing lymphatic development.     

oct4‐EGFP reporter gene expression marks the stem cells in embryonic development and in adult gonads of transgenic medaka

Maintenance of pluripotency in stem cells is tightly regulated among vertebrates. One of the key genes in this process is oct4, also referred to as pou5f1 in mammals and pou2 in teleosts. Pou5f1 evolved by duplication of pou2 early in the tetrapod lineage, but only monotremes and marsupials retained both genes. Either pou2 or pou5f1 was lost from the genomes of the other tetrapods that have been analyzed to date. Consequently, these two homologous genes are often designated oct4 in functional studies. In most vertebrates oct4 is expressed in pluripotent cells of the early embryo until the blastula stage, and later persist in germline stem cells until adulthood. The isolation and analysis of stem cells from embryo or adult individuals is hampered by the need for reliable markers that can identify and define the cell populations. Here, we report the faithful expression of EGFP under the control of endogenous pou2/oct4 promoters in transgenic medaka (Oryzias latipes). In vivo imaging in oct4‐EGFP transgenic medaka reveals the temporal and spatial expression of pou2 in embryos and adults alike. We describe the temporal and spatial patterns of endogenous pou2 and oct4‐EGFP expression in medaka with respect to germline and adult stem cells, and discuss applications of oct4‐EGFP transgenic medaka in reproductive and stem cell biology. Mol. Reprod. Dev. 80: 48–58, 2013. © 2012 Wiley Periodicals, Inc.     

A mouse reporter line to conditionally mark nuclei and cell membranes for in vivo live-imaging

Live-imaging is an essential tool to visualize live cells and monitor their behaviors during development. This technology demands a variety of mouse reporter lines, each uniquely expressing a fluorescent protein. Here, we developed an R26R-RG reporter mouse line that conditionally and simultaneously expresses mCherry and EGFP in nuclei and plasma membranes, respectively, from the Rosa26 locus. The intensity and resolution of mCherry nuclear localization and EGFP membrane localization were demonstrated to be sufficient for live-imaging with embryos that express RG (mCherry and EGFP) ubiquitously and specifically in fetal Sertoli cells. The conditional R26R-RG reporter mouse line should be a useful tool for labeling nuclei and membranes simultaneously in distinct cell populations.     

Quantitative and qualitative immunofluorescence studies of neoplastic cells transfected with a construct encoding p53-EGFP.

The p53 protein is a major regulator of cell cycle progression and apoptosis. We used a p53-enhanced green fluorescent protein (EGFP) construct for transfections into human breast cancer (MCF-7) cells. Cells expressing p53-EGFP showed an increased apoptotic index compared to cells transfected with EGFP alone. Interestingly, apoptotic cells showed localization of p53-EGFP to both nuclei and cytoplasm, whereas non-apoptotic cells usually only showed nuclear localization of p53-EGFP. This result is in agreement with the hypothesis that p53 induces apoptosis by interaction with both nuclear and cytoplasmic targets. Transfected p53-deficient osteosarcoma cells were used for immunofluorescence quantitation. The intensity of immunofluorescence for either p53 or EGFP showed excellent linear correlation to the EGFP autofluorescence, proving that measurements of immunofluorescence intensities can be used for determining endogenous protein levels.     

Redistribution of the kinesin-II subunit KAP from cilia to nuclei during the mitotic and ciliogenic cycles in sea urchin embryos

KAP is the non-motor subunit of the heteromeric plus-end directed microtubule (MT) motor protein kinesin-II essential for normal cilia formation. Studies in Chlamydomonas have demonstrated that kinesin-II drives the anterograde intraflagellar transport (IFT) of protein complexes along ciliary axonemes. We used a green fluorescent protein (GFP) chimera of KAP, KAP-GFP, to monitor movements of this kinesin-II subunit in cells of sea urchin blastulae where cilia are retracted and rebuilt with each mitosis. As expected if involved in IFT, KAP-GFP localized to apical cytoplasm, basal bodies, and cilia and became concentrated on basal bodies of newly forming cilia. Surprisingly, after ciliary retraction early in mitosis, KAP-GFP moved into nuclei before nuclear envelope breakdown, was again present in nuclei after nuclear envelope reformation, and only decreased in nuclei as ciliogenesis reinitiated. Nuclear transport of KAP-GFP could be due to a putative nuclear localization signal and nuclear export signals identified in the sea urchin KAP primary sequence. Our observation of a protein involved in IFT being imported into the nucleus after ciliary retraction and again after nuclear envelope reformation suggests KAP115 may serve as a signal to the nucleus to reinitiate cilia formation during sea urchin development.     

New approach to cell lineage analysis in mammals using the Cre-loxP system

The Cre-loxP site-specific recombination system was used for cell lineage analysis in mammals. We constructed an expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a portion of the rabbit beta-globin gene, loxP-flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E. coli beta-galactosidase (beta-gal). When circular pCETZ-17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were allowed to develop to two-cell stage, 62.8% (59/94) of the two-cell embryos exhibited distinct fluorescence in one or both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for beta-gal. When both circular plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, almost all (87.0%, 47/54) embryos exhibited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for beta-gal activity. This indicates that transient expression of the Cre recombinase gene removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence. We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only two-cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP-expressing two-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 1 day up to four-cell stage. When the developing four-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for beta-gal activity was observed in most (77.8%, 7/9) embryos. These findings were further confirmed using two-cell embryos derived from a transgenic mouse line carrying CETZ-17 transgene. Thus, our system, which is based on transient expression of the Cre recombinase gene directly introduced into nuclei of embryonic cells by microinjection, is a powerful means for cell lineage analysis in mammals.