Journal: The Journal of Cell Biology

Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

doi: 10.1083/jcb.202502035

Figure Lengend Snippet: land-ExM coupled with immunostaining LR-ExM for membrane-bound organelle visualization. (A–C) land-ExM total lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Tom20 antibodies (yellow). The anti-Tom20 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of mitochondria. Scale bar: 1 µm in pre-expansion unit. (H) Intensity profile along the cyan line across the mitochondria in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Sec61b antibodies (yellow). The anti-Sec61b antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified in images of I–K showing details of ER. Scale bar: 1 µm in pre-expansion unit. (P) Intensity profile along the cyan line across the ER in image (L). All images were taken with an Airyscan microscope.

Article Snippet: Sec61b (D5Q1W) Rabbit mAb , Cell Signaling Technology , 14648.

Techniques: Immunostaining, Membrane, Labeling, Microscopy

Journal: The Journal of Cell Biology

Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

doi: 10.1083/jcb.202502035

Figure Lengend Snippet: land-ExM reveals SGs at different locations of cells. (A–C) land-ExM images of U2OS cells untreated or treated with NaAsO2 for 20 or 60 min, then immunostained with anti-G3BP1 antibody. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–F) land-ExM images of U2OS cells stained with mCLING (magenta) and NHS ester dye (cyan) and immunostained with anti-G3BP1 (yellow) and anti-Sec61b (white) antibodies. Cells were untreated or treated with NaAsO2 for 20 min or 60 min. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (G) Magnified images of E showing SGs formed adjacent to ER (orange arrowheads). Scale bar: 1 µm in pre-expansion unit. (H) Analysis of the number of nuclear tunnels per cell with or without 60 min NaAsO2 treatment. Each bar represents the mean ± standard error of more than 18 cells. The ns indicates P > 0.05 by Welch’s t test. (I) Analysis of the diameter of nuclear tunnels in cells with or without 60 min NaAsO2 treatment. Each bar represents the mean ± standard error of more than 20 cells. ns indicates P > 0.05 by Welch’s t test. All images were taken with an Airyscan microscope. The cell shown in F is also shown in .

Article Snippet: Sec61b (D5Q1W) Rabbit mAb , Cell Signaling Technology , 14648.

Techniques: Staining, Microscopy

Journal: The Journal of Cell Biology

Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

doi: 10.1083/jcb.202502035

Figure Lengend Snippet: The nuclear tunnel forms a triple-organellar contact site that includes the SG, the nucleolus, and itself. (A) land-ExM protein (gray) image of U2OS cells immunostained with anti-G3BP1 (red) antibody. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (B–D) Different views of SG in the white dashed box of A. Scale bar: 1 µm in pre-expansion unit. (E) 3D rendering of SG in the white dashed box of A. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (F) land-ExM protein (gray) and lipid (blue) image of U2OS cells immunostained with anti-G3BP1 (red) antibody. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (G–I) Different views of SG in the white dashed box 1 of F. Scale bar: 1 µm in pre-expansion unit. (J) 3D rendering of SG in the white dashed box 1 of F. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in the pre-expansion unit. (K) 3D rendering of SGs in the white dashed box 1–4 of F. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in the pre-expansion unit. (L) land-ExM protein (gray) image of U2OS cells immunostained with anti-G3BP1 (red) and anti-Sec61b (yellow) antibodies. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (M–O) Different views of SG in the white dashed box 1 of L. Scale bar: 1 µm in pre-expansion unit. (P) 3D rendering of SG in the white dashed box 1 of L. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (Q) 3D rendering of SGs in the white dashed box 1–4 of L. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (R) Pie chart of nuclear tunnels with or without SGs. Total tunnels analyzed: 114. (S) Pie chart of SG-filled nuclear tunnels that contact nucleoli versus those that do not. Total tunnel analyzed: 83. All images were taken with an Airyscan microscope. The cell shown in A, F, and L is also shown in .

Article Snippet: Sec61b (D5Q1W) Rabbit mAb , Cell Signaling Technology , 14648.

Techniques: Microscopy

Journal: bioRxiv

Article Title: Derlin-mediated ERAD of lipid regulator ORMDL3 safeguards mitochondrial function

doi: 10.64898/2026.02.27.708653

Figure Lengend Snippet: (A) Post-nuclear supernatant (PNS), mitochondria, endoplasmic reticulum (ER), and MERCs fractions were isolated from WT, Derlin-2 KO, and Derlin-3 KO cells expressing V5-tagged ORMDL1, ORMDL2, or ORMDL3. Immunoblot analysis was performed using anti-V5 antibodies to detect ORMDL proteins across subcellular fractions. Endogenous Derlin-1 and Derlin-2 were probed, and fraction purity and enrichment were validated using Sec61β as an ER marker, TOMM20 as a mitochondrial marker, and Sigma-1 receptor (Sigma1R) as a MERC marker. (B) MERC enrichment of V5-tagged ORMDL1, ORMDL2, and ORMDL3 was quantified from subcellular fractionation experiments in (A) ORMDL abundance in the MERC fraction was normalized to the ER marker Sec61B (ORMDL/Sec61) to account for ER content within contact sites. Data are shown as mean ± SEM from independent experiments, with individual data points representing biological replicates. Statistical significance was determined by one-way ANOVA with post-hoc multiple-comparison testing. *P < 0.05; ***P < 0.001. (C-D) MERC fractionation and quantification were performed as in (A) and (B), except that ORMDL distribution was analyzed in Derlin-2 KO cells with empty vector and Derlin-2 KO with ectopic expression of Derlin-2-Myc. (E) Images of in situ PLA (indicated in red) monitoring VDAC1-ORMDL3-V5 interaction in WT, Derlin-2 KO, and Derlin-2 KO with ectopic Derlin-2 cells. Scale bar, 10 uM. (F) Quantitative analysis of VDAC1-ORMDL3-V5 signals in WT, Derlin-2 KO, and Derlin-2 KO with ectopic Derlin-2 cells. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparison test. *P < 0.05, **P < 0.01, ****P < 0.0001; ns, not significant.

Article Snippet: F-10 sc-17764), Sec61B (ER; Proteintech 51020-2-AP), and SIGMA1R (MERCS; Santa Cruz Biotech.

Techniques: Isolation, Expressing, Western Blot, Marker, Fractionation, Comparison, Plasmid Preparation, In Situ

Journal: Nature Communications

Article Title: FidlTrack: high-fidelity structure-aware single particle tracking resolves intracellular molecular motion in organelles sensing APP processing

doi: 10.1038/s41467-026-69067-y

Figure Lengend Snippet: a Time-averaged TIRF images of a Hela cell expressing a SEC61B::GFP ER marker (grayscale) with knock-in SEC13::SNAP (stained with cpSNAP-JF549) ER exit site marker (cyan). b Reconstructed Halo ER (ER-targeted HaloTag with KDEL retention signal, stained with PA-JF646) trajectories (individually colour-coded) reconstructed without (left) or with FidlTrack (right). c Percentage of ambiguous displacements for the trajectories reconstructed without or with FidlTrack. d Trajectories (individually colour-coded) visiting an ERES (spending at least 10 frames in ERES) found without (left) or with FidlTrack (right) overlaid on top of the averaged ER structure (grayscale) and ERES positions (cyan). e Blow up on the two regions highlighted in ( d ) showing how ERES act as attractors to local trajectories without FidlTrack (left), a problem mostly corrected when using FidlTrack (right). f Number of trajectories at ERES (spending at least 2 points close to an ERES) without and with FidlTrack. g , Average amount of time spent by trajectories close to ERES versus close to random ERES-like sites (see Fig. ). h Amount of the time spent in ERES by trajectories spending at least 5 frames at an ERES with a cutoff at 500 ms. The red line corresponds to a fit of the distribution to a biexponential function of parameters \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$a=156$$\end{document} a = 156 , \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau 1=10$$\end{document} τ 1 = 10 ms, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$b=29$$\end{document} b = 29 , \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau 2=56$$\end{document} τ 2 = 56 ms and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}^{2}=0.997$$\end{document} R 2 = 0.997 . i Example of a trajectory flying-by (passing without stopping) an ERES (left) and quantification of its distance to the ERES (right, top) and instantaneous displacement length (right, bottom) showing no sign of association with the ERES. j Same as ( i ) but for a trajectory visiting for some frames and then exiting an ERES. k Same as ( j ) but for a trajectory dwelling for a long time in an ERES (until bleaching). l Ratio of visiting to flyby trajectories per exit site. m Exit sites colour-coded by their visiting to flyby trajectory ratio as presented in ( l ). n Example of two exit sites exhibiting different behaviours, the left one is “sticky” with a high ratio of visiting trajectories while the right one has mostly flyby trajectories. Source data are provided as a Source Data file.

Article Snippet: 1014 , pcDNA3.1_GFP-SEC61B , SEC61B:: GFP , SEC61B fused to GFP , Fig. , Addgene #121159.

Techniques: Expressing, Marker, Knock-In, Staining

Journal: Nature Communications

Article Title: FidlTrack: high-fidelity structure-aware single particle tracking resolves intracellular molecular motion in organelles sensing APP processing

doi: 10.1038/s41467-026-69067-y

Figure Lengend Snippet: a TIRF images of a frame from simultaneously acquired single-molecules (left) and the associated organelle geometries (right) for a mitochondria matrix targeted HaloTag probe and mNeonGreen marker (top), the mitochondria membrane protein TOMM20 and mitochondria matrix targeted mEmerald (middle), and the ER membrane protein SEC61B and ER lumen targeted mEmerald (bottom) in COS-7 cells. b Masks extracted via AI-assisted image processing from the first and last frame of the recordings for the mitochondria matrix targeted probe (top), TOMM20 (middle) and SEC61B (bottom) recordings presented in ( a ). c Stability maps, representing for each pixel the percentage of total frames in which it is part of a mask, for the mitochondria matrix targeted probe (top), TOMM20 (middle) and SEC61B (bottom) recordings presented in ( a ). d Bar plot presenting the fraction of recovered spots relative to the quantity recovered from the entire stack of masks when using the mask of either the first or last frame to perform structure-aware tracking in the different recordings presented in ( a ). e Reconstructed trajectories (individually colour-coded) from the datasets presented in ( a ) using conventional tracking (not structure-aware, top, left), structure-aware tracking using the mask from the first frame (top, right) or the whole stack of masks (bottom, left). f Plot of the displacement distributions extracted from the trajectories presented in ( e ) fitted to a mixture of two Rayleigh distributions (dashed red lines, see Methods section “Fitting of displacement lengths distributions”). g Plot of the pooled displacement distributions for structure-aware ambiguity-removed tracking of SEC61B::Halotag in COS-7 cells after 4 h of BSA or Oleic Acid treatment at 400 µM. Pooling was done over n =7/6 recordings for BSA and oleic acid, respectively, the reported statistics correspond to a two-tailed Kolmogorov–Smirnov test p < 2.2251e-308. h Bar plot of the Kolmogorov–Smirnov statistics from ( g ) for either conventional or structure-aware tracking and with or without ambiguity removal. i Temporally colour-coded mitochondria positions over 700 frames (left), trajectory of the centre of the pointed mitochondria (right, top) and the raw (black) and mitochondrion motion-corrected trajectory (red) of a TOMM20 receptor moving at the surface of the pointed mitochondrion (right, bottom). Source data are provided as a Source Data file.

Article Snippet: 1014 , pcDNA3.1_GFP-SEC61B , SEC61B:: GFP , SEC61B fused to GFP , Fig. , Addgene #121159.

Techniques: Marker, Membrane, Two Tailed Test

Journal: Nature Communications

Article Title: FidlTrack: high-fidelity structure-aware single particle tracking resolves intracellular molecular motion in organelles sensing APP processing

doi: 10.1038/s41467-026-69067-y

Figure Lengend Snippet: a Time-averaged TIRF images of a Hela cell expressing a SEC61B::GFP ER marker (grayscale) with knock-in SEC13::SNAP (stained with cpSNAP-JF549) ER exit site marker (cyan). b Reconstructed Halo ER (ER-targeted HaloTag with KDEL retention signal, stained with PA-JF646) trajectories (individually colour-coded) reconstructed without (left) or with FidlTrack (right). c Percentage of ambiguous displacements for the trajectories reconstructed without or with FidlTrack. d Trajectories (individually colour-coded) visiting an ERES (spending at least 10 frames in ERES) found without (left) or with FidlTrack (right) overlaid on top of the averaged ER structure (grayscale) and ERES positions (cyan). e Blow up on the two regions highlighted in ( d ) showing how ERES act as attractors to local trajectories without FidlTrack (left), a problem mostly corrected when using FidlTrack (right). f Number of trajectories at ERES (spending at least 2 points close to an ERES) without and with FidlTrack. g , Average amount of time spent by trajectories close to ERES versus close to random ERES-like sites (see Fig. ). h Amount of the time spent in ERES by trajectories spending at least 5 frames at an ERES with a cutoff at 500 ms. The red line corresponds to a fit of the distribution to a biexponential function of parameters \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$a=156$$\end{document} a = 156 , \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau 1=10$$\end{document} τ 1 = 10 ms, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$b=29$$\end{document} b = 29 , \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau 2=56$$\end{document} τ 2 = 56 ms and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}^{2}=0.997$$\end{document} R 2 = 0.997 . i Example of a trajectory flying-by (passing without stopping) an ERES (left) and quantification of its distance to the ERES (right, top) and instantaneous displacement length (right, bottom) showing no sign of association with the ERES. j Same as ( i ) but for a trajectory visiting for some frames and then exiting an ERES. k Same as ( j ) but for a trajectory dwelling for a long time in an ERES (until bleaching). l Ratio of visiting to flyby trajectories per exit site. m Exit sites colour-coded by their visiting to flyby trajectory ratio as presented in ( l ). n Example of two exit sites exhibiting different behaviours, the left one is “sticky” with a high ratio of visiting trajectories while the right one has mostly flyby trajectories. Source data are provided as a Source Data file.

Article Snippet: 1014 , pcDNA3.1_GFP-SEC61B , SEC61B:: GFP , SEC61B fused to GFP , Fig. , Addgene #121159.

Techniques: Expressing, Marker, Knock-In, Staining