Figure 1.
Figure 1. ER localization of INPP5K and ARL6IP1. (A) Domain organization of yeast INP54 and human INPP5K. Note the presence of an ER-anchoring tail at the C terminus of yeast INP54. Human INPP5K lacks such an anchoring ER and instead contains the SKICH domain. (B and C) Confocal images of COS-7 cells expressing EGFP-INPP5K alone (B) or coexpressing EGFP-INPP5K and the ER marker mCh-VAPB, an intrinsic membrane protein (C), showing the dual localization of EGFP-INPP5K in the cytosol (indicated by the diffuse fluorescence in B) and in the ER (shown by the colocalization with mCh-VAPB on tubular structures and the line scan analysis in C). Representative examples of several cells imaged in at least three independent experiments. All transfected cells exhibited the phenotype shown. Scale bars: 10 µm in B, 5 µm in C. (D and E) Confocal images of COS-7 cells expressing EGFP-ARL6IP1 (C) or coexpressing EGFP-ARL6IP1 and mCh-VAPB (D), demonstrating the localization of EGFP-ARL6IP1 in the ER (shown by the colocalization with mCh-VAPB on tubular structures and the line scan analysis in D). Representative examples of cells imaged in at least three independent experiments. All transfected cells exhibited the phenotype shown. Scale bars: 10 µm in D, 5 µm in E. (F) Representative confocal images and corresponding line-scan analysis of COS-7 cells showing that EGFP-INPP5K has a dual localization in the ER and cytosol in cells only expressing endogenous ARL6IP1, while it is primarily recruited to the ER when coexpressed with mCh-ARL6IP1 and loses its ER localization upon ARL6IP1 knockdown. Scale bars: 5 µm. The ratio of EGFP-INPP5K fluorescence intensity in the ER relative to the adjacent cytosol under the three conditions is plotted in the right panel. Data were acquired by line-scan analyses of multiple individual ER tubules from multiple cells (“Endogenous”: n = 39 ER tubules from 9 cells; “Overexpressed”: n = 39 ER tubules from 11 cells; “Depleted”: n = 33 ER tubules from 10 cells). ****, p < 0.0001 (two-tailed t test). (G and H) Representative confocal image of mouse cortical neurons grown 4 d in vitro and transfected with EGFP-INPP5K and mCh-ARL6IP1, showing their localization in the ER reticular network. Scale bars: 10 µm in G, 2 µm in H.

ER localization of INPP5K and ARL6IP1. (A) Domain organization of yeast INP54 and human INPP5K. Note the presence of an ER-anchoring tail at the C terminus of yeast INP54. Human INPP5K lacks such an anchoring ER and instead contains the SKICH domain. (B and C) Confocal images of COS-7 cells expressing EGFP-INPP5K alone (B) or coexpressing EGFP-INPP5K and the ER marker mCh-VAPB, an intrinsic membrane protein (C), showing the dual localization of EGFP-INPP5K in the cytosol (indicated by the diffuse fluorescence in B) and in the ER (shown by the colocalization with mCh-VAPB on tubular structures and the line scan analysis in C). Representative examples of several cells imaged in at least three independent experiments. All transfected cells exhibited the phenotype shown. Scale bars: 10 µm in B, 5 µm in C. (D and E) Confocal images of COS-7 cells expressing EGFP-ARL6IP1 (C) or coexpressing EGFP-ARL6IP1 and mCh-VAPB (D), demonstrating the localization of EGFP-ARL6IP1 in the ER (shown by the colocalization with mCh-VAPB on tubular structures and the line scan analysis in D). Representative examples of cells imaged in at least three independent experiments. All transfected cells exhibited the phenotype shown. Scale bars: 10 µm in D, 5 µm in E. (F) Representative confocal images and corresponding line-scan analysis of COS-7 cells showing that EGFP-INPP5K has a dual localization in the ER and cytosol in cells only expressing endogenous ARL6IP1, while it is primarily recruited to the ER when coexpressed with mCh-ARL6IP1 and loses its ER localization upon ARL6IP1 knockdown. Scale bars: 5 µm. The ratio of EGFP-INPP5K fluorescence intensity in the ER relative to the adjacent cytosol under the three conditions is plotted in the right panel. Data were acquired by line-scan analyses of multiple individual ER tubules from multiple cells (“Endogenous”: n = 39 ER tubules from 9 cells; “Overexpressed”: n = 39 ER tubules from 11 cells; “Depleted”: n = 33 ER tubules from 10 cells). ****, p < 0.0001 (two-tailed t test). (G and H) Representative confocal image of mouse cortical neurons grown 4 d in vitro and transfected with EGFP-INPP5K and mCh-ARL6IP1, showing their localization in the ER reticular network. Scale bars: 10 µm in G, 2 µm in H.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal