Journal: Cell

Article Title: Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

doi: 10.1016/j.cell.2017.03.043

Figure Lengend Snippet: B cells were harvested from the spleen of an Exosc10COIN/LacZ mouse and fixed and prepared for 3D-STORM after 72 hrs of treatment with (1) stimulation cocktail and (2) 4OHT+stimulation cocktail. Reconstructed two color 3D STORM (super-Aresolution) image from a data set of 50,000 frames with Atto488 labeled AID, AlexaFluor647 labeled Mtr4 and DAPI labeled nucleus. Three dimensional views of the boxed region show spatial distribution of AID and Mtr4 molecules inside the nuclei of B cells isolated from (A) wild type cells and (D) Exosc10 knockout cells. Histogram of the distribution of interactions of AID and Mtr4 calculated in the B cell nucleus of (B) wild type & (E) Exosc10 knockout cells and in the corresponding cytoplasms (C) & (F), by using Matlab (2014b, MathWorks) software. (G) Comparison of the distribution of paired interaction of AID and Mtr4 in the nucleus versus cytoplasm by one way ANOVA (Tukey-Kramer test) method in Matlab (2014b, MathWorks) software. Comparison of the distribution of paired interaction of AID and Mtr4 were calculated in the cytoplasm versus nucleus of (H) wild type & (I) Exosc10 knockout B cells using a Student’s t-test in Matlab (2014b, MathWorks) software and P values are noted in the graph. (J) Comparison of the distribution of paired interaction of AID and Mtr4 in the nuclear center versus displaced from center versus cytoplasm by one way ANOVA (Tukey-Kramer test) method in Matlab (2014b, MathWorks) software. (K) Reconstructed two color 3D STORM (super-resolution) image with Alexa488 labeled AID, AlexaFluor647 labeled Mtr4 and DAPI labeled nucleus of wild type Exosc10 cells. Histogram of the distribution of interactions of AID and Mtr4 calculated in (L) nucleus center and (M) displaced from center of wild type cells, by using Matlab (2014b, MathWorks) software. All of the 3D STORM imaging was performed in three different B cells (from independent experiments) and repeated three or more times. 3D STORM super resolution image magnification is ×100. Scale bar: 1μm(K). Error bars indicate S.D. (P values: ** <0.01, *** <0.001)

Article Snippet: Alexa488 (Against-Goat) , Jackson Immuno Research , 805-547-008.

Techniques: Labeling, Isolation, Knock-Out, Software, Comparison, Imaging

Journal: Cell

Article Title: Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

doi: 10.1016/j.cell.2017.03.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Alexa488 (Against-Goat) , Jackson Immuno Research , 805-547-008.

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Modification, Electron Microscopy, Shear, Transgenic Assay, Sequencing, Subcloning, Software

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 2. Amino acids 1-63 of AE1-4 can direct a cytoplasmic tailless mutant of the Fc receptor to the basolateral membrane of MDCK cells. Confluent MDCK cells stably expressing Fc– (A), Fc1-63 (B), Fc1-63Y44A (C), Fc1-63Y47A (D) or Fc1-63Y44A,Y47A (E) were fixed and incubated with the rat monoclonal antibody specific for the Fc receptor and phalloidin-FITC. The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine, and the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is near the center (B, C and D) or near the apical surface (A and E) of the cells. Regions that are yellow indicate significant overlap in the distribution of the chimera and actin. The black arrowhead next to each panel marks the position of the basal membrane in the xz image of the transfected cells. The bar in each xy image is 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Mutagenesis, Membrane, Stable Transfection, Expressing, Incubation, Labeling, Microscopy, Transfection

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 3. Amino acids 1-37 and 38-63 of AE1-4 target a cytoplasmic tailless Fc receptor to distinct intracellular membrane compartments. MDCK cells stably expressing Fc1-37 (A-C) or Fc38-63 (D-F) were fixed and incubated with rat monoclonal antibodies specific for the Fc receptor (A and D), a mouse monoclonal antibody specific for the mannose 6-phosphate receptor (B) or a rabbit polyclonal antibody specific for furin (E). The cells were washed and incubated with donkey anti-rat IgG conjugated to lissamine (A and D), goat anti- mouse IgG conjugated to FITC (B) or donkey anti-rabbit IgG conjugated to FITC (E). The localization of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The merged images illustrate significant overlap in the distribution of Fc 1-37 and the mannose 6-phosphate receptor (C) and Fc38-63 and furin (F). Bar in A and D, 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Membrane, Stable Transfection, Expressing, Incubation, Bioprocessing, Labeling, Microscopy

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 4. Fc1-37 recycles from the plasma membrane to a membrane compartment that overlaps the distribution of the mannose 6-P receptor. MDCK cells stably expressing Fc1-37 were incubated with the anti-Fc receptor antibody for 1 hour at 4°C. Following washing with cold DMEM, the cells were incubated for 15 minutes (A,C,E) or 45 minutes (B,D,F) at 37°C. At each time point, the cells were fixed, permeabilized and incubated with a mouse monoclonal directed against the mannose 6-phosphate receptor (M-6-P). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine and goat anti-mouse IgG conjugated to FITC. Following washing, the localization of Fc1-37 (A,B) and the mannose 6-phosphate receptor (C,D) was visualized on a Zeiss LSM 510 laser-scanning microscope. The merged images showing the overlap of Fc1-37 and the mannose 6-phosphate receptor are shown in E and F. The arrows in A indicate the Fc1-37 chimeras that still reside on the cell surface following a 15 minute incubation at 37°C. Bar (A,B), 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Clinical Proteomics, Membrane, Stable Transfection, Expressing, Incubation, Laser-Scanning Microscopy

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 5. Fc38-63 recycles from the plasma membrane to a membrane compartment that overlaps the distribution of furin. MDCK cells stably expressing Fc38-63 were incubated with the anti-Fc receptor antibody for 1 hour at 4°C. Following washing with cold DMEM, the cells were incubated for 15 minutes (A,C,E) or 45 minutes (B,D,F) at 37°C. At each time point, the cells were fixed, permeabilized and incubated with a rabbit polyclonal directed against furin. The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine and donkey anti-rabbit IgG conjugated to FITC. Following washing, the localization of Fc38-63 (A,B) and furin (C,D) was visualized on a Zeiss LSM 510 laser scanning microscope. The merged images showing the overlap of Fc38-63 and furin are shown in E and F. Bar (A,B), 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Clinical Proteomics, Membrane, Stable Transfection, Expressing, Incubation, Laser-Scanning Microscopy

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 6. The surface distribution and steady-state localization of wild- type and mutant Fc38-63 constructs in polarized MDCK cells. Polarized MDCK cells stably expressing Fc38-63 (A,B) or Fc38- 63Y47A (C) or transiently expressing Fc38-63Y47L (D) or Fc38- 63L50A (E) were grown on Transwell filters. The intact cells were either incubated with the Fc-receptor-specific antibody for 1 hour at 4°C, washed and fixed in 3% paraformaldehyde (A) or the cells were fixed in 3% paraformaldehyde, permeabilized by incubation in PBST and incubated with the Fc-receptor-specific antibody (B-E). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine (A-E) and phalloidin conjugated to FITC (B-E). Following washing, the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is near the center of the cells. Regions that are yellow in B-E indicate significant overlap in the distribution of the chimera and actin. The black arrowhead next to each panel marks the position of the basal membrane in the xz image. Bars, 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Mutagenesis, Construct, Stable Transfection, Expressing, Incubation, Labeling, Microscopy, Membrane

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 7. The surface distribution and steady-state localization of wild- type and mutant Fc1-37 constructs in polarized MDCK cells. Polarized MDCK cells transiently (A,C) or stably (B) expressing Fc1- 37 (A and B) or Fc1-37S25A (C) were grown on Transwell filters. The intact cells were either incubated with the Fc-receptor-specific antibody for 1 hour at 4°C, washed and fixed in 3% paraformaldehyde (A) or the cells were fixed in 3% paraformaldehyde, permeabilized by incubation in PBST and incubated with the Fc-receptor-specific antibody (B,C). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine (A-C) and phalloidin conjugated to FITC (B and C). Following washing, the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is either near the center (A,B) or near the apical surface (C) of the cells. The surface population of Fc1-37 could be detected in a longer exposure of the image in B. The black arrowhead next to each panel marks the position of the basal membrane in the xz image. Bars, 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Mutagenesis, Construct, Stable Transfection, Expressing, Incubation, Labeling, Microscopy, Membrane

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 8. Fc1-63 and Fc38-63Y47A colocalize with phalloidin-stained stress fibers in subconfluent MDCK cells. Subconfluent MDCK cells stably expressing Fc1-63 (A-C) or Fc38-63Y47A (D-F) were fixed, permeabilized and incubated with the Fc-receptor-specific antibody (A,D) and phalloidin conjugated to FITC (B,E). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine. Following washing, the localization of fluorescently labeled polypeptides was visualized on a Zeiss Axiophot microscope. The merged images showing the overlap of these chimeras with phalloidin-stained microfilaments are shown in C and F. Bars, 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Staining, Stable Transfection, Expressing, Incubation, Labeling, Microscopy