Figure 4.
Figure 4. ER tubules populated by ARL6IP1 and INPP5K undergo rapid motion. (A–D) Analysis of the motility of ER proteins. (A and C) Representative confocal images of COS-7 cells coexpressing EGFP-ARL6IP1 and mCh-VAPB (top fields of A) or mCh-ARL6IP1 and EGFP-Sec61β (top fields of C). In the images shown, three time points were color-coded and merged, so that the stationary ER elements staying on the same sets of pixels appear white, while the motile ER elements occupying alternate pixels appear in colors. Note the abundance of the colored ER tubules in ARL6IP1 images, relative to the VAPB and Sec61β (arrowheads). Bottom: Graphic display of motility from the same field shown above during a 5-min recording. Differences of fluorescence intensity at each pixel between subsequent time-lapse images were calculated, and these values are added up and pseudocolored (see Fig. S3 for methods). Scale bars: 5 µm. (B and D) Plots of motility index (see Fig. S3 for methods) where cumulative values of motility in several cells, calculated as in A and B, were normalized to the initial fluorescence intensity. n = 21 cells expressing EGFP-ARL6IP1 and mCh-VAPB (C), and n = 24 cells for cells expressing mCh-ARL6IP1 and EGFP-Sec61β (D). Data are represented as scattered dots with the solid black bar as mean (two-tailed t test). (E) Histograms of transport distances and velocity of the tips of motile ER tubules in cells overexpressing EGFP-ARL6IP1 (out of 81 events from five cells). All the events shown represent ER tubule movement that originated and ended during the recorded time. (F) Example of a growing ER tubule sliding along a preexisting ER tubule (arrows point to the tips of a ER tubule). Scale bar: 1 µm. (G) ER tubule extension events were captured during live-cell imaging of cells expressing YFP-α-tubulin and mCh-ARL6IP1 at the times indicated. Arrows depict the movement of ARL6IP1-postive ER tubules along microtubules. Scale bar: 2 µm. (H) Frequency of ARL6IP1-positive ER tubules that grow along microtubules marked by YFP-α-Tubulin (data from 14 cells). (I) Live-cell images of a COS-7 cell expressing YFP-α-Tubulin and mCh-ARL6IP1 upon 5 µM nocodazole treatment. Note the depolymerization of microtubules, the collapse of tubular ER network and the accumulation of bright foci containing mCh-ARL6IP1. Scale bar: 2 µm. Images are representative of three independent experiments. (J) Time-lapse images of cells expressing mCh-ARL6IP1 and GFP-CLIP170, a microtubule plus end-tracking protein. Arrowheads point to the tip of an elongating ARL6IP1-positive ER tubule. Note this tip lacks CLIP170 fluorescence. Scale bar: 2 µm. (K) Frequency of ARL6IP1-positive ER tubules tips adjacent (<1 µm) to CLIP170 puncta (data from 10 cells).

ER tubules populated by ARL6IP1 and INPP5K undergo rapid motion. (A–D) Analysis of the motility of ER proteins. (A and C) Representative confocal images of COS-7 cells coexpressing EGFP-ARL6IP1 and mCh-VAPB (top fields of A) or mCh-ARL6IP1 and EGFP-Sec61β (top fields of C). In the images shown, three time points were color-coded and merged, so that the stationary ER elements staying on the same sets of pixels appear white, while the motile ER elements occupying alternate pixels appear in colors. Note the abundance of the colored ER tubules in ARL6IP1 images, relative to the VAPB and Sec61β (arrowheads). Bottom: Graphic display of motility from the same field shown above during a 5-min recording. Differences of fluorescence intensity at each pixel between subsequent time-lapse images were calculated, and these values are added up and pseudocolored (see Fig. S3 for methods). Scale bars: 5 µm. (B and D) Plots of motility index (see Fig. S3 for methods) where cumulative values of motility in several cells, calculated as in A and B, were normalized to the initial fluorescence intensity. n = 21 cells expressing EGFP-ARL6IP1 and mCh-VAPB (C), and n = 24 cells for cells expressing mCh-ARL6IP1 and EGFP-Sec61β (D). Data are represented as scattered dots with the solid black bar as mean (two-tailed t test). (E) Histograms of transport distances and velocity of the tips of motile ER tubules in cells overexpressing EGFP-ARL6IP1 (out of 81 events from five cells). All the events shown represent ER tubule movement that originated and ended during the recorded time. (F) Example of a growing ER tubule sliding along a preexisting ER tubule (arrows point to the tips of a ER tubule). Scale bar: 1 µm. (G) ER tubule extension events were captured during live-cell imaging of cells expressing YFP-α-tubulin and mCh-ARL6IP1 at the times indicated. Arrows depict the movement of ARL6IP1-postive ER tubules along microtubules. Scale bar: 2 µm. (H) Frequency of ARL6IP1-positive ER tubules that grow along microtubules marked by YFP-α-Tubulin (data from 14 cells). (I) Live-cell images of a COS-7 cell expressing YFP-α-Tubulin and mCh-ARL6IP1 upon 5 µM nocodazole treatment. Note the depolymerization of microtubules, the collapse of tubular ER network and the accumulation of bright foci containing mCh-ARL6IP1. Scale bar: 2 µm. Images are representative of three independent experiments. (J) Time-lapse images of cells expressing mCh-ARL6IP1 and GFP-CLIP170, a microtubule plus end-tracking protein. Arrowheads point to the tip of an elongating ARL6IP1-positive ER tubule. Note this tip lacks CLIP170 fluorescence. Scale bar: 2 µm. (K) Frequency of ARL6IP1-positive ER tubules tips adjacent (<1 µm) to CLIP170 puncta (data from 10 cells).

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