Figure 2. A portion of DscI localizes to... | Rockefeller University Press

Figure 2.

$A portion of DscI localizes to the cortex, and Dsc is needed for CortI cortical distribution. (A) Representative images of immunofluorescence imaging of DscI (green) using anti-DscI antibody and nuclei (blue) labeled with Hoescht in WT and dsc1 cells. Images were collected using confocal microscopy. DscI signal was present in the cytoplasm and slightly enriched in the cortex of WT cells. This signal was absent in dsc1 null cells, confirming the antibody’s specificity for DscI. (B) Representative images of immunofluorescence imaging of DscI (green) using anti-DscI antibody and nuclei (blue) labeled with Hoescht in dividing WT cells. (C) Time series of a dsc1 null-complemented cell completing cytokinesis. DscIA was fused with GFP and images were collected with a GFP channel on an Olympus IX71 microscope, equipped with a 40× (NA 1.30) oil objective at room temperature using Zen software and analyzed with ImageJ. t = 0 is defined as the time when the video began. The video is provided in the Supplemental materials (Video 3). (D and F) Representative confocal immunofluorescence images of WT, dsc1 null, DscIA-overexpressed, vector control (Vec) and dscI null-complemented cells stained with anti-CortI (green) and Hoescht to label nuclei (blue). Images were collected using a Zeiss AxioObserver with 780-Quasar confocal module microscope, equipped with a C-Apochromat 40× (NA 1.2) water objective at room temperature using Zen software and analyzed with ImageJ. Vector encoding mCherry was used as the vector (Vec) control. (E) Western blot verifying overexpression of DscIA in WT background cells is shown. Dynacortin (Dyn) is provided as a loading control. (G) CortI cortical localization for D and F was quantified as the ratio of mean signal intensity at the cortex (Icort) to the mean signal intensity of the cytoplasm (Icyt). P values were calculated using the ANOVA followed by Fisher’s LSD test. Number of cells analyzed, n = 15–25 from two independent experiments. (H) Summary diagram shows that a fraction of intracellular DscI localizes to the cortex, and DscI helps with cortical distribution of CortI. Source data are available for this figure: SourceData F2.$

A portion of DscI localizes to the cortex, and Dsc is needed for CortI cortical distribution. (A) Representative images of immunofluorescence imaging of DscI (green) using anti-DscI antibody and nuclei (blue) labeled with Hoescht in WT and dsc1 cells. Images were collected using confocal microscopy. DscI signal was present in the cytoplasm and slightly enriched in the cortex of WT cells. This signal was absent in dsc1 null cells, confirming the antibody’s specificity for DscI. (B) Representative images of immunofluorescence imaging of DscI (green) using anti-DscI antibody and nuclei (blue) labeled with Hoescht in dividing WT cells. (C) Time series of a dsc1 null-complemented cell completing cytokinesis. DscIA was fused with GFP and images were collected with a GFP channel on an Olympus IX71 microscope, equipped with a 40× (NA 1.30) oil objective at room temperature using Zen software and analyzed with ImageJ. t = 0 is defined as the time when the video began. The video is provided in the Supplemental materials (Video 3). (D and F) Representative confocal immunofluorescence images of WT, dsc1 null, DscIA-overexpressed, vector control (Vec) and dscI null-complemented cells stained with anti-CortI (green) and Hoescht to label nuclei (blue). Images were collected using a Zeiss AxioObserver with 780-Quasar confocal module microscope, equipped with a C-Apochromat 40× (NA 1.2) water objective at room temperature using Zen software and analyzed with ImageJ. Vector encoding mCherry was used as the vector (Vec) control. (E) Western blot verifying overexpression of DscIA in WT background cells is shown. Dynacortin (Dyn) is provided as a loading control. (G) CortI cortical localization for D and F was quantified as the ratio of mean signal intensity at the cortex (I_cort) to the mean signal intensity of the cytoplasm (I_cyt). P values were calculated using the ANOVA followed by Fisher’s LSD test. Number of cells analyzed, n = 15–25 from two independent experiments. (H) Summary diagram shows that a fraction of intracellular DscI localizes to the cortex, and DscI helps with cortical distribution of CortI. Source data are available for this figure: SourceData F2.