Journal: Journal of Virology

Article Title: Venezuelan Equine Encephalitis Virus Capsid Protein Forms a Tetrameric Complex with CRM1 and Importin ?/? That Obstructs Nuclear Pore Complex Function ▿

doi: 10.1128/JVI.02554-09

Figure Lengend Snippet: Identification of the NES in VEEV capsid protein. (A) Sequence alignment of H68 peptides derived from different New World alphaviruses with a consensus NES sequence (NES), functional NES of cyclic-AMP (c-AMP)-dependent kinase inhibitor (PKI) (51) and supraphysiological NES (S1) (13). The sequence alignment was performed using ClustalW. Conservative hydrophobic amino acids in the NES are shown in red. Amino acids that are identical between different members of the New World alphaviruses are shaded in green. Asterisks indicate identical residues; colons indicate conserved substitutions; periods indicate semiconserved substitutions. WEEV, western equine encephalitis virus. (B) BHK-21 cells were coinfected with packaged replicons expressing VEEV capsid-GFP and 4×Tomato-3×NLS and treated with leptomycin B at 2 h postinfection as described in Materials and Methods. The images were acquired after 4 h of leptomycin B treatment on a Zeiss LSM510 confocal microscope. Scale bars, 20 μm. (C) (Top) Amino acid alignments of mutated H68-GFP fusions. Conserved hydrophobic amino acids are indicated in red, and introduced point mutations are shown in blue. (Bottom) Representative confocal images of cells expressing 4×Tomato-3×NLS and mutant proteins. Scale bars, 20 μm. (D) Box plot demonstrating the nuclear/cytoplasmic distribution of 4×Tomato-3×NLS when expressed alone and the distribution of the same protein when coexpressed with mutant peptide-GFP. A small but statistically significant increase in nuclear-reporter accumulation was detected for single-amino-acid mutants. The double mutant no longer affected nuclear accumulation of 4×Tomato-3×NLS. (E) Box plot demonstrating the nuclear/cytoplasmic distributions of H68-GFP and its mutants. The increase in nuclear accumulation of the mutant-peptide-GFP fusions was correlated with their reduced efficiencies in nuclear import inhibition. The P values were calculated using the Mann-Whitney test (n = 30 for all experiments).

Article Snippet: The images were acquired after 4 h of leptomycin B treatment on a Zeiss LSM510 confocal microscope.

Techniques: Sequencing, Derivative Assay, Functional Assay, Western Blot, Expressing, Microscopy, Mutagenesis, Inhibition, MANN-WHITNEY

Journal: Cells

Article Title: Cyclin B Export to the Cytoplasm via the Nup62 Subcomplex and Subsequent Rapid Nuclear Import Are Required for the Initiation of Drosophila Male Meiosis

doi: 10.3390/cells12222611

Figure Lengend Snippet: Nuclear–cytoplasmic shuttling of cyclin B in spermatocytes during the growth phase. ( a – e ) Anti-Cyclin B (CycB) immunostaining of spermatocytes with anti-cyclin B (CycB) antibody from the growth phase (S3–S6 in ( a – d )) to the onset of meiosis (prophase I (Pro) in ( e )). Images show CycB immunofluorescence (red in ( a – e ), white in ( a ’– e ’)), Sa-GFP fluorescence for visualizing the nucleolus (green in ( a – e ), white in ( a ’’– e ’’)), and DNA staining with 4′,6-diamidino-2-phenylindole (DAPI) (blue in ( a – e ), white in ( a ’’’– e ’’’)). Scale bar: 10 μm. ( f ) Quantification of CycB immunofluorescence intensity in whole spermatocytes (black line) and nuclei relative to that in the cytoplasm of spermatocytes (gray line) at each stage represented in ( a – e ). Intensities of the immunofluorescence in spermatocytes at the S3 to ProI phases were individually measured in whole cell regions, the cytoplasm, and the nucleus. The mean intensity value in the whole region was calculated at each stage, displayed on the y -axis (arbitrary unit: AU), and drawn with a black line. The ratio of the intensity in the cytoplasm to that in the nucleus (N/C intensity) was displayed by a gray line. Data represent means ± 95% confidence intervals (CIs) (>16 cells). ( g , h ) Time-lapse observation of CycB in the growth phase (S5) of living spermatocytes expressing CycB–GFP and treated with leptomycin B (LMB) ( h ) and that of untreated cells ( g ). Ninety images were captured every minute. Four selected images of a live spermatocyte are presented. Scale bar: 10 μm. ( i ) Comparative quantification of nuclear GFP fluorescence intensity relative to cytoplasmic intensity in LMB-treated (orange line) and untreated (green line) spermatocytes expressing CycB–GFP. The intensities of CycB-GFP in the whole regions of the cells at the time indicated by t = 0 to 90 min were measured. The ratio of the intensity in the cytoplasm to that in the nucleus (N/C intensity) was displayed on the y -axis (with a green line (control cells at S5) and an orange line (the cells treated with LMB)). Data represent means ± 95% CIs (11 untreated and 8 LMB-treated spermatocytes). Significance was tested between control cells and LMB-treated cells at the last time point ( t = 90). *** p < 0.001 (Mann–Whitney test).

Article Snippet: To facilitate the observation of CycB–GFP nuclear import, we inhibited exportin with leptomycin B (LMB; Cayman Chemical, Ann Arbor, MI, USA).

Techniques: Immunostaining, Immunofluorescence, Fluorescence, Staining, Expressing, MANN-WHITNEY

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Ankle1 shuttles between nucleus and cytoplasm. a Schematic representation of Ankle1’s domain organization depicting predicted ankyrin repeats, the LEM domain and a GIY-YIG nuclease domain. Putative nuclear export sequences (NES1, NES2) and nuclear localization sequences (NLS1, NLS2), identified in silico are indicated. b Immuno-fluorescence analysis of ectopic Ankle1-V5 in U2OS cells without or following a 3 h treatment with 50 nM leptomycin B, an inhibitor of CRM1-mediated export. Cells were stained with antibodies to V5, and DNA with DAPI. Scale bar: 10 μm

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: In Silico, Fluorescence, Staining

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Localization of Ankle1 fragments containing different domains and export and import signals. Localization of GFP-tagged Ankle1 truncation constructs ectopically expressed in U2OS ( a ) and HeLa ( b ) cells was determined by confocal fluorescence microscopy. Molecular weights and schematic representations of domain organization of respective truncation protein constructs are indicated. Cells were fixed after 3 h of mock or leptomycin B treatment. DNA was counterstained with DAPI. Scale bar: 10 μm

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: Construct, Fluorescence, Microscopy

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Mutation analyses identify NES2 and NLS2 as the predominant sequences controlling nucleo-cytoplasmic shuttling of Ankle1. a , b , d , e U2OS cells were transiently transfected with Ankle1-V5 carrying point mutations in NLS or NES sequences and either mock-treated or treated with leptomycin B for 3 h and processed for confocal immunofluorescence analyses using antibodies to V5 and DAPI to detect DNA. Scale bars: 10 μm. c Mean fluorescence intensities in nuclei and cytoplasm of cells expressing wild-type Ankle1-V5, Ankle1-NES1mut-V5 or Ankle1-NES2mut-V5 were measured in original unprocessed digital images prior to contrast/brightness adjustment and nucleus to cytoplasm signal ratios were calculated. Data were obtained from three independent experiments and analyzed using Student’s t -test. Ankle1-NES1, P = 0.002; Ankle1-NES2, P = 5.4E-21; n = 50; 15–17 cells each from three independent experiments

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: Mutagenesis, Transfection, Immunofluorescence, Fluorescence, Expressing