XB-IMG-49186
Xenbase Image ID: 49186
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Figure 3. Importin α2 and Ntf2 Regulate Nuclear Size and Import(A) Nuclei were assembled in X. tropicalis extract, and at 40 min, importin α-E was added at the indicated concentrations in addition to GFP-NLS. At 80 min, images for at least 50 nuclei per condition were acquired with the same exposure time, and NE surface area was quantified, averaged, and normalized to the buffer control. Error bars represent standard error (SE). Scale bar, 20 μm.(B) Experiments were performed as in (A) with a fixed concentration (0.8 μM) of added importin α-E or a mutant version lacking the importin β-binding domain (δIBB). Average fold change from the buffer control and SD are shown (n = 4 extracts). The δIBB mutant did not have a strong dominant-negative effect on import because it was added at a concentration below the endogenous importin α level.(C) Nuclei were assembled in X. laevis extract mock and partially immunodepleted of importin α2 (0.5–1 μM depleted). Kinetics of nuclear assembly were similar in the two extracts. At 40 min, indicated proteins were added at 1 μM as well as GFP-NLS. At 80 min, images for at least 50 nuclei per condition were acquired with the same exposure time, and NE surface area and nuclear GFP-NLS fluorescence intensity were quantified. Average fold change from the mock depletion and SD are shown (n = 4 extracts).(D) Recombinant Ntf2 was titrated into X. laevis extract prior to nuclear assembly. Initial kinetics of nuclear assembly were not altered by supplemental Ntf2. GFP-NLS or IBB-coated Qdots were added at 30 min. At 80 min, nuclei were processed for immunofluorescence with an antibody against Ran, and images for at least 50 nuclei per condition were acquired with the same exposure time. NE surface area was quantified from Ran-stained nuclei, averaged, and normalized to the buffer control. Nuclear fluorescence intensities for Qdots, GFP-NLS, and Ran were similarly processed. Error bars represent SE. One representative experiment of three is shown. For each parameter, the difference between 0 and 1.6 μM added Ntf2 was statistically significant by Student's t test (p < 0.005).(E) Experiments similar to (D) were performed with a fixed Ntf2 concentration (1.6 μM) and over time. Nuclear Qdot or GFP-NLS fluorescence intensities for at least 50 nuclei per time point were averaged and normalized to 1.0 (arbitrary units). Error bars represent SE. At 95 min, the difference in Q dot import between 0 and 1.6 μM added Ntf2 was statistically significant by Student's t test (p < 0.001).(F) Nuclei were assembled in X. tropicalis extract supplemented with anti-Ntf2 or IgG antibodies (0.1 mg/ml). At 30 min, nuclear assembly was similar in the two conditions, and Qdots or GFP-NLS was added. At 80 min, immunofluorescence for Ran was performed, and nuclear parameters were quantified as in (D). Average fold change from the IgG control and SD are shown (n = 6 extracts).See also Figure S3. Image published in: Levy DL and Heald R (2010) Copyright © 2010. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |