Figure 3.
Figure 3. Preferential localization of ARL6IP1 and INPP5K, relative to other ER proteins, in peripheral ER tubules. (A) COS-7 cell coexpressing the ER membrane marker EGFP-Sec61β, mCh-INPP5K, and Myc-ARL6IP1 imaged by confocal fluorescence microscopy. Both EGFP-Sec61β and mCh-INPP5K fluorescence are present throughout the ER tubular network, but mCh-INPP5K is enriched over EGFP-Sec61β in the peripheral tubular ER (as marked by arrowheads) and is nearly undetectable in ER sheets. Scale bars: 5 µm. Insets at the bottom left corners show the nuclear envelope of a different cell from the same field, demonstrating that mCh-INPP5K fluorescence is absent from the nuclear envelope marked by EGFP-Sec61β fluorescence. Inset scale bars: 2 µm. Representative examples of several cells imaged in at least three independent experiments. (B) COS-7 cell coexpressing EGFP-Sec61β and mCh-ARL6IP1, showing the relative enrichment of mCh-ARL6IP1 over EGFP-Sec61β in the peripheral tubular ER (as marked by arrowheads) and the presence of EGFP-Sec61β, but not mCh-ARL6IP1, on the nuclear envelope. Scale bars: 5 µm. Representative examples of several cells imaged in at least three independent experiments. (C and D) Representative confocal images of COS-7 cells coexpressing mCh-ARL6IP1 and EGFP-Sec61β showing regions including the nuclear envelope (C) or peripheral ER sheets (D), respectively. Graphs show a representative example of the quantification of the normalized fluorescence intensity measured along the dashed lines as delineated in the merged fields. Note the lack of mCh-ARL6IP1 signal from the nuclear envelope (B) and from the peripheral ER sheets, except for their edges (C), while EGFP-Sec61β labels these structures (arrowheads). Scale bars: 2 µm. (E–I) Representative confocal images and respective line-scan analysis of the periphery of COS-7 cells coexpressing mCh-ARL6IP1 and other GFP-tagged proteins as indicated. While ARL6IP1 and INPP5K are homogenously present on all the ER tubules and precisely localized (E), the fluorescence of ER membrane proteins Sec61β, VAPB, and Sac1 and of the luminal ER marker ss-GFPox-KDEL declines toward the ends of the most distal tubules (F–I, arrowheads). Scale bars: 5 µm. Representative examples of several cells imaged in at least three independent experiments. (J) Quantification of normalized fluorescent intensity of mCh-ARL6IP1 and ss-GFPox-KDEL along peripheral ER tubules based on line scans as exemplified on the top. Note that the dashed line elongates beyond the ends of the ER tubules into the background. Data are represented as mean ± SD (n = 13 ER tubules from six cells). The bar graphs at bottom right show the average fluorescence intensity along 1-µm segments at the proximal and distal end, respectively. ****, p < 0.0001; n.s., not significant (two-tailed t test).

Preferential localization of ARL6IP1 and INPP5K, relative to other ER proteins, in peripheral ER tubules. (A) COS-7 cell coexpressing the ER membrane marker EGFP-Sec61β, mCh-INPP5K, and Myc-ARL6IP1 imaged by confocal fluorescence microscopy. Both EGFP-Sec61β and mCh-INPP5K fluorescence are present throughout the ER tubular network, but mCh-INPP5K is enriched over EGFP-Sec61β in the peripheral tubular ER (as marked by arrowheads) and is nearly undetectable in ER sheets. Scale bars: 5 µm. Insets at the bottom left corners show the nuclear envelope of a different cell from the same field, demonstrating that mCh-INPP5K fluorescence is absent from the nuclear envelope marked by EGFP-Sec61β fluorescence. Inset scale bars: 2 µm. Representative examples of several cells imaged in at least three independent experiments. (B) COS-7 cell coexpressing EGFP-Sec61β and mCh-ARL6IP1, showing the relative enrichment of mCh-ARL6IP1 over EGFP-Sec61β in the peripheral tubular ER (as marked by arrowheads) and the presence of EGFP-Sec61β, but not mCh-ARL6IP1, on the nuclear envelope. Scale bars: 5 µm. Representative examples of several cells imaged in at least three independent experiments. (C and D) Representative confocal images of COS-7 cells coexpressing mCh-ARL6IP1 and EGFP-Sec61β showing regions including the nuclear envelope (C) or peripheral ER sheets (D), respectively. Graphs show a representative example of the quantification of the normalized fluorescence intensity measured along the dashed lines as delineated in the merged fields. Note the lack of mCh-ARL6IP1 signal from the nuclear envelope (B) and from the peripheral ER sheets, except for their edges (C), while EGFP-Sec61β labels these structures (arrowheads). Scale bars: 2 µm. (E–I) Representative confocal images and respective line-scan analysis of the periphery of COS-7 cells coexpressing mCh-ARL6IP1 and other GFP-tagged proteins as indicated. While ARL6IP1 and INPP5K are homogenously present on all the ER tubules and precisely localized (E), the fluorescence of ER membrane proteins Sec61β, VAPB, and Sac1 and of the luminal ER marker ss-GFPox-KDEL declines toward the ends of the most distal tubules (F–I, arrowheads). Scale bars: 5 µm. Representative examples of several cells imaged in at least three independent experiments. (J) Quantification of normalized fluorescent intensity of mCh-ARL6IP1 and ss-GFPox-KDEL along peripheral ER tubules based on line scans as exemplified on the top. Note that the dashed line elongates beyond the ends of the ER tubules into the background. Data are represented as mean ± SD (n = 13 ER tubules from six cells). The bar graphs at bottom right show the average fluorescence intensity along 1-µm segments at the proximal and distal end, respectively. ****, p < 0.0001; n.s., not significant (two-tailed t test).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal