memerald er3  (Addgene inc)

Journal: Science (New York, N.Y.)

Article Title: Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER

doi: 10.1126/science.aaf3928

Figure Lengend Snippet: (A) Tubular ER in the periphery of a COS-7 cell expressing Sec61β imaged live at 40 Hz using GI-SIM microscopy. Scale bar, 2 mm. (B) ER tubules within the boxed region in (A), identified using a skeletonization algorithm. Left: The midline of each tubule (green) is mapped onto the fluorescence (magenta). Right: Positions of the midlines are plotted as kymographs against time for each of the three locations shown in cyan at left; scale bars, 200 nm and 0.5 s. (C and D) Amplitudes (C) and frequencies (D) of tubular ER oscillations in COS-7 cells expressing Sec61β treated with deoxyglucose plus sodium azide (DOG + NaN3), AlF, nocodazole (NZ), blebbistatin (Bleb), puromycin (Puro), and cycloheximide (CHX). Untreated controls using a luminal ER marker (ER3) and results for a different cell line (U-2 OS–Sec61β) are also shown. (E) Plot of frequency versus amplitude for tubular oscillations in treated and untreated cells. Error bars represent SEM. (F) Locations of three-way junctions derived from skeletonized data (white). Original fluorescence is shown in magenta; example tracks of junctions (cyan) over 2.5 s are indicated in green. Scale bar, 2 mm. (G) MSD scaling exponent (α values) for treated and control cells. Box plots indicate the mean and SD in (C), (D), and (G); range is indicated by outer tick marks. See tables S1 and S2 for a detailed list of means and test statistics.

Article Snippet: Lifeact-mApple has been previously described ( 27 ), and mEmerald-ER-3 (ER3) was a gift from Michael Davidson (Addgene plasmid # 54082).

Techniques: Expressing, Microscopy, Fluorescence, Marker, Derivative Assay, Control

Journal: Science (New York, N.Y.)

Article Title: Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER

doi: 10.1126/science.aaf3928

Figure Lengend Snippet: (A) COS-7 cell expressing Sec61β imaged live by GI at 40 Hz exhibits many peripheral sheet-like structures. (B) GI-SIM of the boxed region in (A) shows many discrete spaces throughout the structure. Colored lines at left correspond to the locations of the kymographs shown at right. Voids in intensity within the structure can be seen appearing and disappearing over time. (C) Single-particle tracking (SPT) of dark spaces within the structure. The fluorescence image (i) was inverted and spaces were tracked using SPT algorithms. Tracks overlaid onto the inverted image are shown in (ii), with trajectories shown in different colors. (D) Each track length corresponds to the lifetime of the space; distance across the space (i.e., distance between tubules) is also quantified. The box plot indicates the SD and mean; range is indicated by outer tick marks. The asterisks denote significant difference between means, detailed in table S4. (E and F) Temporal intensity derivative analysis (see materials and methods) of representative peripheral sheet-like structures in a COS-7 cell expressing Sec61β, with a luminal ER marker (ER3) and another cell line (U-2 OS–Sec61β) as controls. (E) Original fluorescence images. (F) Each consecutive frame over a 250-ms time period is color-coded, with intensity corresponding to the magnitude of fluorescence change. Scale bars, 2 μm. See tables S3 and S4 for a detailed list of means and test statistics.

Article Snippet: Lifeact-mApple has been previously described ( 27 ), and mEmerald-ER-3 (ER3) was a gift from Michael Davidson (Addgene plasmid # 54082).

Techniques: Expressing, Single-particle Tracking, Fluorescence, Marker