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 visualizes the protein and lipid context of cells. (A) Workflow of land-ExM. (B) Schematic of NHS-biotin-MA linker. (C) Schematic of mCLING. (D) land-ExM image of U2OS cells incubated with NHS-biotin-MA linker. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4. (E) ExM image of U2OS cells incubated with NHS-MA linker and stained with Alexa Fluor 488 NHS ester dye. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (F) ExM image of U2OS cells incubated with GMA linker and stained with SYPRO Orange. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (G) Bar chart comparing signal-to-noise ratios of protein context images obtained with different ExM methods shown in D–F. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 10 cells. (H–J) Different views of land-ExM images of a breast cancer cell, UCI082014, stained with mCLING for lipid content. The orange dashed lines in H show where the orthogonal views (I and J) align. Scale bar: 5 µm (H), 2 µm (I and J) in pre-expansion unit. Linear expansion factor: 3.8. (K) Magnified images of H. (L) Magnified images of I. The orange dashed line in K shows where the orthogonal view (L) aligns. Scale bar: 0.5 µm in pre-expansion unit. Linear expansion factor: 3.8. All images were taken with an Airyscan microscope. Images D–F were adjusted to the same contrast. Image in D is also shown in .

Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

Techniques: Incubation, 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: mCLING optimization for lipid staining of cells. (A–D) Airyscan images of U2OS cells stained with different batches of mCLING at different dilution factors. Scale bars: 20 µm. Red arrowheads indicate lipid structures in the cytoplasm. All images were taken with an Airyscan microscope.

Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

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: Alternative land-ExM workflow to avoid cross talk between NHS-biotin-MA and mCLING. (A) Alternative workflow of land-ExM. (B) i and ii: land-ExM images of U2OS cells stained first with NHS-biotin-MA and then mCLING. iii to v: Magnified images of boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). (C) i and ii: land-ExM images of U2OS cells stained first with mCLING and then NHS-biotin-MA. iii to v: Magnified images of orange boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). All images were taken with an Airyscan microscope.

Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

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: land-ExM using proteinase K digestion. (A) Workflow of land-ExM using proteinase K digestion to homogenize cells instead of heat denaturation. (B) land-ExM protein image of U2OS cells with proteinase K digestion (proK). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (C) land-ExM protein image of U2OS cells with heat denaturation (heat). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM lipid image of U2OS cell with proteinase K digestion (proK). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (E) land-ExM lipid image of U2OS cells with heat denaturation (heat). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. All images were taken with an Airyscan microscope. Image in C is also shown in .

Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

Techniques: 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 labeling and anchoring strategies improve the signal of TREx and pan-ExM. (A) Workflow of land-pan-ExM, which only replaces the labeling strategy of pan-ExM with the labeling strategy of land-ExM. (B) land-TREx protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (C) TREx protein channel of U2OS cells, where proteins were anchored with acryloyl-X SE and stained with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (D) Bar chart comparing the signal-to-noise ratio of the protein channel in land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (E) land-TREx lipid channel of U2OS cells, where lipids were labeled by mCLING and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (F) TREx lipid channel of U2OS cells, where lipids were anchored with acryloyl-X SE and stained with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (G) Bar chart comparing the signal-to-noise ratio of the lipid channel of land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (H) land-pan-ExM protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (I) Pan-ExM protein channel of U2OS cells labeled with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (J) Bar chart comparing the signal-to-noise ratio of the protein channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (K) land-pan-ExM lipid channel of U2OS cells, where lipids were stained following the workflow (A). Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (L) Pan-ExM lipid channel of U2OS cells labeled with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (M) Bar chart comparing the signal-to-noise ratio of the lipid (mCLING) channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. All images were taken with an Airyscan microscope.

Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

Techniques: Labeling, 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: land-ExM visualizes phase-separated and membrane organelles. (A–G) land-ExM protein images of membraneless phase separation structures. The proteins were labeled with NHS-biotin-MS and after gelation stained with streptavidin-Alexa Fluor 488. (A) land-ExM protein image of nucleoli in a U2OS cell. Red arrowheads indicate the fibrillar center (FC) or dense fibrillar component (DFC) of the nucleolus. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (B) land-ExM protein image of nuclear bodies of breast cancer cell, UCI082014. Red arrowheads indicate the nuclear bodies. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (C) land-ExM protein image of SGs of a U2OS cell treated with NaAsO 2 for 20 min. The red arrowhead indicates a SG. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM protein image of chromatin of a breast cancer cell. Scale bar: 500 nm in pre-expansion unit. Linear expansion factor: 4.2. (E) land-ExM protein image of NPCs of a breast cancer cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (F and G) land-ExM protein images of mitochondria and cytoskeleton of a U2OS cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (H–P) land-ExM lipid images of membrane structures. The lipids were labeled with mCLING-Atto647N. (H) land-ExM lipid image of breast cancer cell. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (I–M) magnified images of H showing different membrane structures: lipid vesicles (I), mitochondria (J), filopodia (K), nuclear invagination (L), and Golgi apparatus (M). Scale bar: 1 µm (I–M) in pre-expansion unit. (N) 3D land-ExM lipid image of a breast cancer cell after maximum intensity projection, showing the cell membrane. Color-coded by the z-dimension slices from bottom to top. Color bar: purple to white: 0–6 µm in pre-expansion unit. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (O and P) magnified images of N showing detailed structures of the cell membrane. Scale bar: 1 µm in pre-expansion unit. All images were taken with an Airyscan microscope.

Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

Techniques: Membrane, Labeling, 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: land-ExM coupled with immunostaining LR-ExM for lipid vesicle identification. (A–C) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Lamp2 antibodies (yellow). The anti-Lamp2 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 lysosomes. Scale bar: 500 nm in pre-expansion unit. (H) Intensity profile along the gray line across the lysosome in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-clathrin antibodies (yellow). The anti-clathrin 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 images of I–K showing details of clathrin-coated pits. Scale bar: 500 nm in pre-expansion unit. (P) Intensity profile along the gray line across the clathrin-coated pit in image (L). All images were taken with an Airyscan microscope.

Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

Techniques: Immunostaining, 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 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: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

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: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

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: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

Techniques: Microscopy

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A) MTORC1 and AMPK activity is influenced by amino acids and glucose, respectively, and regulate ULK1 activity, which induces autophagy. (B-E) U2OS cells were treated with full media (FM), FM lacking glucose (-Glc), or FM lacking amino acids (-aa) for the indicated times and cell lysates analyzed by immunoblot for ACC, total and phosphorylated at S79 (B, D) , and S6K1 phosphorylated at T389 and actin as a loading control (C, E) . Immunoblots were quantified and signal normalized to total ACC (for D) or actin loading control (for E) plotted. Symbols represent mean of 3 independent experiments and bars are s.e.m. (F-I) Monoclonal U2OS cell lines expressing DFCP1 (F) , WIPI2B (G) , WIPI1 (H) , or ATG5 ( I ) were treated with FM (blue), -Glc (green), or -aa media (red) for 6 hours and subjected to live-cell fluorescent imaging. GFP-puncta (objects) for each reporter were quantified from single cells. Trajectories include mean objects per cell (symbols); bars represent 95% CI. (J-K) GFP-LC3B objects were quantified from cells treated with FM, -aa, or -Glc in the presence of BafA1 (to prevent lysosome degradation) or a vehicle control in 2 hour increments (J) . The number of GFP-LC3 puncta synthesized (solid symbols) and degraded (open symbols) from time 0 min was calculated and plotted. The dashed lines demarcate where individual datasets were collected and data stitched together. Trajectories include mean puncta per cell (symbols); bars represent standard deviation.

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Activity Assay, Western Blot, Control, Expressing, Imaging, Synthesized, Standard Deviation

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A-B) U2OS cells were treated for 6 hours with FM (blue), indicated as 100% aa (the concentration found in RPMI-1640), or 10% (green), 5% (orange), or 0% (red) of that amino acid concentration. Cells were lysed and ULK1 phosphorylated at S758 quantified (relative to actin loading control and normalized to time 0 controls) (A) . Bars represent means of 3 biological replicates. The data in (A) was fit to a sigmoidal dose-response curve (dashed line) to generate an EC 50 of 6% aa (B) . (C-D) Cells were treated with the medias described in A and imaged live from hours 4-6 in the presence of BafA1 (as in ). GFP-LC3 puncta were quantified from cells and sum intensity plotted (this is the sum of the intensity of all GFP-positive pixels, an output used to avoid potential issues with aggregated vesicles). Trajectories include mean objects per cell (symbols); bars represent s.e.m.; black lines represent simple linear regression (C) . The GFP-LC3 synthesis rates from the linear regression lines in (C) across amino acid concentrations were fit to a sigmoidal dose-response curve (dashed line) to generate an EC 50 of 7% aa (D) . (E) The rate of GFP-LC3 synthesis (derived from linear regression analysis, shown in (C) and the relative level of pULK1-S758 (from A) plotted to show a negative, linear association (dashed line, r 2 = 0.815).

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Concentration Assay, Control, Derivative Assay

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A) GFP-WIPI1 (green, left Y-axis) and GFP-WIPI2B (purple, right Y-axis) object counts over the 6 hour -aa treatment were overlaid. The gray region indicates the immediate starvation period (0-1 hours), and the yellow highlights the period of delayed autophagy under sustained starvation (3-6 hours). (B) EGFP-2xFYVE puncta (PI(3)P-positive cell membranes) were quantified from cells under FM (blue) or -aa (red) treatment. Note a lack of substantial puncta increase in the immediate (0-1 hour) period (gray shading). (C) The fraction of cells containing at least 1 GFP-WIPI2B puncta is plotted with time of -aa starvation. The dashed line represents 50% of the cell population. (D) Representative EGFP-2xFYVE puncta in U2OS cells treated with a VPS34 inhibitor (1 μM compound 31, lower panels) or vehicle control (upper panels). Blue = Hoechst nuclear stain; green = EGFP-2xFYVE; captured with a 60x oil objective. Scale bars in left panels are 20 μm and scale bars in right panels (insets) are 5 μm. (E) GFP-LC3 synthesis with BafA1 in the presence of compound 31 (1 μM) or vehicle control. BafA1 was added for 1 hour during either the first hour of -aa starvation (“0-1 hr” bars) or after 4 hours of -aa starvation (“4-5 hr” bars). Data shown represent GFP-LC3 puncta synthesis relative to vehicle control. Symbols represent mean and bars are s.e.m. **** = adjusted p < 0.0001, one-way ANOVA.

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Control, Staining

Journal: PLOS One

Article Title: Quantitative and temporal analysis of autophagy: Differential Response to amino acid and glucose starvation

doi: 10.1371/journal.pone.0340957

Figure Lengend Snippet: (A) Cells were cultured with or without amino acids for 6 hours prior to a restimulation phase of 60 min with FM (containing amino acids). (B-C) Representative images of GFP-DFCP1 (B) or GFP-WIPI2B (C) puncta in U2OS cells that were starved of amino acids for 6 hours and subject to aa-restimulation for 0 min (left), 10 min (middle) or 20 min (right). Insets show 2x magnification of indicated region to highlight disappearance of puncta. (D-G) DFCP1 (D) , WIPI2B (E) , WIPI1 (F) , and LC3B (G) quantified from cells during the restimulation period following -aa (red) or FM (blue) treatments. Symbols are mean GFP-positive puncta per cell and bars are s.e.m. Solid lines are non-linear regression models (one phase exponential decay). Gray shaded area emphasizes restoration to FM levels within 20 min of aa restimulation.

Article Snippet: The human osteosarcoma cell line U2OS (HTB-96) was purchased from American Type Culture Collection, and cells maintained in RPMI-1640 medium (Gibco, 11-875-119) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Cell Culture

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: The RUSH system applied to integrin α5. (A) Principles of the RUSH-α5 integrin. In all experiments, SBP-EGFP-ITGA5 (RUSH-α5) is co-expressed with streptavidin-KDEL (ER-hook). In the absence of biotin, this combined complex is retained within the ER. Biotin addition displaces the ER-hook and releases RUSH-α5 into the cytoplasm. (B) The number of contacts between EGFP and FN during simulations of the coarse-grained model. Left: simulation of EGFP being pulled towards the FN-binding site, starting when the C-terminus of the EGFP and the N-terminus of the integrin α5 are <1 nm apart, the linker included, leading to the formation of contacts . Right: simulation of a fully stretched EGFP, initially in close proximity to the FN-binding site, that is allowed to relax without a biasing force resulting in a spontaneous and rapid loss of contacts (<100 ns; ). The pulling process spanned 8 nm and 80 ns. The relaxation spanned 3200 ns. Contacts were calculated between EGFP and FN with a cutoff of 0.6 nm. (C and D) RUSH-α5 is expressed on the cell surface and forms a functional heterodimer with integrin β1. (C) Representative flow cytometry analysis of cell surface RUSH-α5 levels (detected with the anti-GFP-AF647 antibody) in RUSH-α5–expressing U2OS cells ± biotin. (D) Representative immunoblots of GFP pulldowns performed in RUSH-α5 or control transfected cells ± biotin treatment for the indicated times and probed for endogenous integrin β1. The faster migrating band of immature integrin β1 is indicated by a green arrow and box and the slower migrating band of mature integrin β1 with a magenta arrow and box. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Binding Assay, Functional Assay, Flow Cytometry, Expressing, Western Blot, Control, Transfection

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: RUSH-α5 delivery to the plasma membrane is spatially regulated by the ECM. (A) Model of RUSH-α5 (EGFP-integrin α5)–integrin-β1 heterodimer based on the structure of human α5β1-integrin bound to FN (PDB: 7NWL ) (see also ). (B) Representative immunofluorescence timelapse images of a U2OS cell expressing RUSH-α5 (SBP-EGFP-integrin α5) and plated on FN ± biotin treatment (single grayscale images for the indicated time points are shown; see ). (C) Representative immunofluorescence images of RUSH-α5 and active integrin α5β1 (SNAKA51 antibody) in RUSH-α5–expressing U2OS cells plated on FN or collagen + biotin (60 min). Grayscale single-channel images and merged images (white, colocalization; blue, nuclei [DAPI]) are shown. (D) Representative images of RUSH-α5 and RUSH-CD59 release in U2OS cells co-expressing both constructs and plated on dual-coated micropatterns (alternating FN coating (cyan) and collagen-peptide (GFOGER) (non-fluorescent) lines). Nuclei (blue) are co-labeled. Intensity line profiles generated across the yellow line are displayed relative to the position of the FN-coated micropattern lines. White insets represent regions of interest (ROIs) that are magnified for each channel. FN, fibronectin.

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Expressing, Construct, Labeling, Generated

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse spinning-disk confocal imaging of RUSH-α5–expressing U2OS cell plated on FN (10 µg/ml), biotin added after acquisition of time point 0 min. One frame per minute. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: RUSH-α5 recruitment to adhesions is ligand-dependent. (A) Representative immunoblot of GFP pulldowns performed in RUSH-α5 or control transfected cells plated on FN or collagen and probed for endogenous integrin β1 and for GFP. Mature (magenta arrow) and immature (green arrow) integrin β1 are indicated. (B) Quantification of the relative fraction of mature to immature integrin β1 interacting with RUSH-α5 ± biotin treatment for the indicated times. N = 6 independent experiments; data are mean ± SD, One-way ANOVA, Dunn’s multiple comparison test, no significant difference between FN and collagen at all time points. (C) Representative images (see ) of U2OS cells co-expressing RUSH-α5 and RUSH-CD59 and plated on FN (top) or collagen (bottom) ± biotin treatment for the indicated times. Insets represent ROIs that are magnified. Scale bars: 20 µm. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Western Blot, Control, Transfection, Comparison, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse spinning-disk confocal imaging of U2OS cells co-expressing RUSH-α5 (green) and RUSH-CD59 (magenta) and plated on FN (left, 10 µg/ml) or collagen (right, 10 µg/ml), biotin added after acquisition of time point 0 min. One frame per 30 s. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: RUSH-α5 delivery to FAs. (A) Representative immunofluorescence images of U2OS cells co-expressing RUSH-α5 and pmKate2-Paxillin plated on FN or collagen ± biotin treatment for the indicated times. Insets represent ROIs that are magnified and show paxillin-segmented adhesions (red outlines). (B) Quantification of the relative mean intensity of RUSH-α5 in segmented adhesions/cell ± biotin treatment for the indicated times. Data are mean ± SD; n = 64 cells on collagen, 50 cells on FN, pooled from three independent experiments; One-way ANOVA, Holm-Šídák’s multiple comparison test; data distribution was assumed to be normal but this was not formally tested. (C) TIRF imaging of U2OS cells expressing RUSH-α5-pHluorin on a FN-coated surface after biotin release at T = 0. The arrows indicate exocytosis events. Exocytosis events were detected by performing a ratiometric analysis, which consisted of dividing each frame by the previous. All detected events before 19 min of release are indicated in red. The graph indicates the distance between the exocytosis events and the nearest FA segmented on the last frame of (37 min after release), compared to the distance of random dots to FAs, showing that the localization to FAs is not random. Individual measurements and the mean ± SD are represented. Unpaired T test. RUSH-α5-pHluorin spots n = 336 spots, random spots n = 116 spots from one experiment.

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Immunofluorescence, Expressing, Comparison, Imaging

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: RUSH-α5 delivery and localization following release. (A) RUSH-α5-pHluorin released in U2OS co-expressing Paxillin-mScarlet on FN- and anti-GFP antibody-coated surfaces. The intensity of RUSH-α5-pHluorin signal was quantified in and outside adhesions (paxillin positive, represented in the insets). Data are mean ± SD, N = 12 cells ( N = 6 cells from 1 experiment for T = 45 min), pooled from 2 independent experiments. Ordinary one-way Anova with Holm-Šídák’s multiple comparisons test; data distribution was assumed to be normal but this was not formally tested. Scale bars: 10 µm (main and insets). (B) High resolution imaging of RUSH-α5 after 15 min of release in U2OS. PDI (ER marker) or GM130 (Golgi marker) are co-stained. Arrows in the insets indicate RUSH-α5 positive vesicles. Scale bar: 10 µm (main), 5 µm (insets). (C and D) High-mannose integrin-α5 is delivered to the cell surface. (C) Flow cytometry analysis of high-mannose proteins at the cell surface detected with the fluorescent lectin PFL647 in U2OS cells expressing RUSH-α5, without release and 1 h after release. The left panel shows histograms of one experiment, the right panel shows the geometric fluorescence mean of the PFL647 signal for individual experiments ( N = 2 independent experiments). (D) U2OS expressing RUSH-α5 were labeled at their surface after 1 h release with a lectibody specifically recognizing high-mannose proteins. The lectibody was then pulled down by protein G beads. This Western blot shows GFP detection in the pull-down, indicating the presence of high-mannose RUSH-α5 at the cell surface after release. Representative of N = 3 independent experiments. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Expressing, Imaging, Marker, Staining, Flow Cytometry, Fluorescence, Labeling, Western Blot

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse TIRF imaging of U2OS expressing RUSH-α5-pHluorin plated on FN-coated surface. Left: RUSH-α5-pHluorin. Right: ratiometric analysis, the exocytosis spots appear in yellow. Scale bar: 20 µm. One frame per 10 s. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Polarized delivery of newly synthesized integrin to the cell protruding edge. (A–C) RUSH-α5 intensity in U2OS cells plated on 9 µm-wide micropatterns coated with FN and anti-GFP or collagen and anti-GFP ± biotin treatment for the indicated times was analyzed at both cell edges (the predominantly protruding edge was denoted ROI1 and the other edge ROI2; and ). Representative intensity coded images (A) and quantification of RUSH-α5 release on FN (B; ) and collagen (C; ) (normalized first to the total intensity of the cell and then to 0 min biotin) are shown. Data are mean ± SEM. (D) Representative images and spatiotemporal track maps of cell edge contours over time in U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Red insets represent protruding ROIs that are magnified. Blue insets represent retracting ROIs that are magnified. Spatiotemporal track maps: blue colors represent early time points and magenta colors represent late time points in the time-lapse series. (E) Quantifications of RUSH-α5 intensity in ROIs (retracting or protruding areas determined from spatiotemporal track maps). Data are mean ± SD. (B and C) N = 33 cells on FN and 38 cells on collagen, pooled from three independent experiments, two-way ANOVA, Holm-Šídák’s multiple comparison test. (E) N = 53 cells on collagen, 49 cells on FN, pooled from three independent experiments; one-way ANOVA, Holm-Šídák’s multiple comparisons test; data distribution was assumed to be normal but this was not formally tested.

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Synthesized, Expressing, Comparison

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse spinning-disk confocal imaging of U2OS expressing RUSH-α5 plated on 9 µm-wide FN and anti-GFP-coated micropattern lines. Biotin added after acquisition of time point 0 min. One frame per minute. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse imaging of spinning-disk confocal U2OS expressing RUSH-α5 plated on 9 µm-wide collagen and anti-GFP-coated micropattern lines. Biotin added after acquisition of time point 0 min. One frame per minute. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Early delivery of RUSH-α5 to the cell surface. (A) Representative immunofluorescence images of U2OS cells co-expressing RUSH-α5 (green) and the ER marker ERoxBFP (magenta) plated on FN (10 µg/ml) ± biotin treatment for the indicated times. Arrows indicate rapidly budding RUSH-α5–positive vesicles adjacent to cell protrusions (≤15 min after release) (see also ). (B) TIRF imaging of RUSH-α5 after release (0 min). The polarized delivery to the cell surface at the protruding area can be observed from 15 min after release (see also ). (C) Representative images of RUSH-α5 (green) and RUSH-CD59 (magenta) release in U2OS cells co-expressing both constructs and plated on dual-coated micropatterns (alternating FN coating (cyan) and collagen-peptide (GFOGER) (non-fluorescent) lines). Nuclei (blue) are co-labeled. White insets represent ROIs that are magnified for each channel. FN, fibronectin. (D) Flow cytometry analysis of cell surface RUSH-α5 levels (detected with the anti-GFP-AF647 antibody) in RUSH-α5–expressing U2OS cells ± biotin. Representative histograms and quantification from two independent experiments of cell surface GFP (ratio of the geometric means of the surface signal divided by the total GFP signal, normalized by subtracting the 0 min value) are shown.

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Immunofluorescence, Expressing, Marker, Imaging, Construct, Labeling, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse spinning-disk confocal imaging of U2OS cells co-expressing RUSH-α5 (green) and the ER marker ERoxBFP (magenta) plated on FN (10 µg/ml), biotin added after acquisition of time point 0 min. One frame per 30 s. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing, Marker

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Time lapse imaging of U2OS cells expressing RUSH-α5 plated on FN (10 µg/ml), biotin added after acquisition of time point 0 min, imaged by TIRF microscopy. One frame per 30 s. Related to .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Imaging, Expressing, Microscopy

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Golgi bypass early delivery of RUSH-α5 requires the integrin-α5 PDZ-binding motif. (A and B) Quantification of relative RUSH-α5 recruitment to protruding areas (A) or adhesions (B) in U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times with or without Golgicide A (10 µM). (C) Representative immunoblot of GFP pulldowns from RUSH-α5 or GFP control transfected cells plated on FN and probed for GFP and endogenous GRASP65. N = 3 independent experiments. (D) Amino acid sequence of the integrin α5 tail highlighting the canonical PDZ-binding motif (SDA) and the two proline residues critical for the formation of the non-canonical PDZ-binding motif. The mutations of these sites used in our experiments are indicated below. (E) Representative streptavidin pulldowns of the indicated biotinylated recombinant integrin peptides incubated with cell lysates collected from CHO cells overexpressing GFP-GRASP65. A representative immunoblot probed for GRASP65 (note, two bands are present in the lysate: upper, GFP-GRASP65; lower, endogenous GRASP65; GFP-GRASP65 is apparent in the pulldown). N = 3 independent experiments. (F) Quantification of RUSH-α5 or RUSH-α5 ΔSDA recruitment to adhesions in U2OS cells ± biotin treatment for the indicated times. All data are mean ± SD. (A and B) One-way ANOVA with Tukey’s multiple comparison test for comparing time points, one-way ANOVA with Holm-Šídák’s multiple comparisons test for comparing untreated and Golgicide A, data distribution was assumed to be normal but this was not formally tested. (A) N = 26 cells RUSH-α5, N = 22 cells RUSH-α5 Golgicide A, pooled from three independent experiments. (B) N = 24 cells RUSH-α5, N = 27 cells RUSH-α5 Golgicide A, pooled from three independent experiments. (F) One sample t test to compare time points with T = 0, ordinary one-way ANOVA with Holm-Šídák’s multiple comparisons test to compare RUSH-α5 and RUSH-α5 ΔSDA, data distribution was assumed to be normal but this was not formally tested. N = 23 cells RUSH-α5, N = 23 cells RUSH-α5 ΔSDA, pooled from two independent experiments. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Binding Assay, Expressing, Western Blot, Control, Transfection, Sequencing, Recombinant, Incubation, Comparison

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Early release of RUSH-α5 is adhesion dependent and polarized recruitment to protrusions is supported by endogenous integrin α5. (A–D) Validation of ITGA5 CRISPR-Cas9 KO U2OS cells. (A) Western blot analysis of WT and ITGA5 KO single cell clones showing the efficiency of the CRISPR-Cas9 ITGA5 KO in U2OS cells. (B) Genome sequence alignment of U2OS WT and ITGA5 KO clones with the ITGA5 WT sequence. The targeted exon and the gRNA used for CRISPR KO positions are indicated. (C) Representative flow cytometry analysis of cell surface integrin α5 in U2OS WT and ITGA5 KO clones. (D) Images of WT and ITGA5 KO U2OS clones stained for active integrin α5 (SNAKA51) and paxillin. Scale bar: 20 µm. (E) Flow cytometry analysis of cell surface RUSH-α5 levels (detected with the anti-GFP-AF647 antibody) in WT and ITGA5 KO U2OS cells transfected with RUSH-α5 ± biotin treatment for the indicated times. Representative histograms and quantification of cell surface GFP (ratio of the geometric means of the surface signal divided by the total GFP signal, normalized by subtracting the 0 min value) are shown. Data are mean ± SD; N = 3 independent experiments. The two-tailed paired t test showed no significant differences between WT and ITGA5 KO. Data distribution was assumed to be normal but this was not formally tested. (F) Flow cytometry analysis of cell surface RUSH-α5 levels in adherent versus suspension U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Representative histograms and quantification of cell surface GFP analyzed as in E are shown. Data are mean ± SD; N = 3 independent experiments. The two-tailed paired t test, data distribution was assumed to be normal but this was not formally tested. (G) Quantifications of RUSH-α5 intensity in ROIs (retracting or protruding areas) in WT and ITGA5 KO U2OS cells ± biotin treatment for the indicated times. One-way ANOVA, Holm-Šídák’s multiple comparison test, data distribution was assumed to be normal but this was not formally tested. Data are mean ± SD; N = 59 WT cells, 53 ITGA5 KO cells, pooled from three independent experiments. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Biomarker Discovery, CRISPR, Western Blot, Clone Assay, Sequencing, Flow Cytometry, Staining, Transfection, Two Tailed Test, Suspension, Expressing, Comparison

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: Early release of RUSH-α5 is sensitive to GRASP silencing. (A) Immunoblot of GST pull-downs of recombinant integrin α5 WT or mutant (ΔSDA or PPAA) peptides incubated with GST alone or recombinant GST-GRASP65. No enrichment of GST-GRASP65 signal over background (no peptide beads) is detected with integrin α5 WT peptide, indicating that the integrin α5 peptides do not interact with recombinant purified GST-GRASP65. (B) ELISA assay detecting biotinylated recombinant integrin α5 WT or ΔSDA or PPAA mutant with HRP-streptavidin incubated on wells coated with GST alone or GST-GRASP65. No direct interaction between GRASP65 and integrin α5 WT peptide was detected. (C) Immunoblot of lysates collected from control-silenced or GRASP65 and GRASP55-silenced U2OS cells used in D, E, probed for GRASP65 and GRASP55. β-actin was probed as a loading control. (D) Representative immunofluorescence images of control-silenced or GRASP65 and GRASP55-silenced U2OS cells expressing RUSH-α5 and plated on dual-coated micropatterns (magenta dots, FN; non-fluorescent regions, collagen peptide GFOGER). (E) Relative recruitment of RUSH-α5 in control- or GRASP65- and GRASP55-silenced U2OS cells to FN dots within the cell boundary. Data are mean ± SD; n = 9 siCTRL cells, 11 siGRASP cells (36 and 44 dots, respectively) from one experiment. (F and G) Quantification of RUSH-α5 intensity in the four areas relative to signal intensity in the respective area 2.5 min prior to RUSH-α5 appearance in (F) new adhesions or (G) already existing adhesions (determined from the time-lapse images) on FN- and anti-GFP antibody-coated surfaces. Adhesions close to the cell edge and with a minimum lifetime of 15 min were analyzed and changes of RUSH-α5 intensity were plotted over time in the indicated areas ranging from distal to proximal to the cell body. Data are mean ± SD; One independent experiment 9 adhesions from 6 cells on 2 coverslips (F) and one independent experiment 9 adhesions from 5 cells on 2 coverslips (G). (H and I) Representative images (H) and (I) track maps related to . Red arrows indicate direction of adhesion growth. Scale bars: 20 µm. Source data are available for this figure: .

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Western Blot, Recombinant, Mutagenesis, Incubation, Purification, Enzyme-linked Immunosorbent Assay, Control, Immunofluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway

doi: 10.1083/jcb.202508155

Figure Lengend Snippet: RUSH-α5 is delivered to the tip of adhesions and mediates adhesion growth. (A and B) Representative immunofluorescence image of U2OS cells expressing RUSH-α5 and pmKate2-Paxillin (white, colocalization) plated on FN (10 µg/ml) and anti-GFP (2.5 µg/ml; to trap cell surface RUSH-α5 at the point of delivery). Insets represent ROIs that are magnified. ROI2 is a FA demarcated into four equal areas for analysis and is further magnified in B. Scale bars 20 µm (whole cell image), 5 µm (ROI1), and 0.5 µm (ROI2 and B). (C) Representative image of an already established FA where RUSH-α5 delivery was quantified. Scale bar 0.5 µm. (D) Cartoon showing clutch model elements. Myosin motors pull on actin filaments with a speed v . This applies force to a substrate via integrins and adapter proteins (talin). The effect of force regulates the unbinding rates from integrins to the substrate ( k off ) and the folding/unfolding rates of talin ( k fold / k unfold ). When talin unfolds, adhesion reinforcement is assumed to happen, which is modeled by an increase in integrin density with value d add . Changes in integrin availability are modeled by changing the parameter d add . (E) Model prediction of adhesion growth with time for conditions in which integrin availability is low ( d add = 0.005 integrins/μm 2 ) or high ( d add = 0.01 integrins/μm 2 ). Adhesion growth (y-axis) is modeled through integrin density, which is plotted normalized to the starting value. (F) Quantification of adhesion growth in U2OS cells expressing RUSH-α5 and plated on FN or collagen ± biotin treatment for the indicated times. Shown are the relative sums of segmented adhesion area/cell. Data are mean ± SD. (G) Quantification of adhesion growth in WT and ITGA5 KO U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Shown are the relative sums of segmented adhesion area/cell. Data are mean ± SD. (H) Quantification of cell spreading in WT and ITGA5 KO cells expressing RUSH-α5 ± biotin treatment for the indicated times. Data are mean ± SEM. (I) Quantification of the length of the longest protrusion (extending furthest from the initial plasma membrane localization during imaging) formed per cell after 45 min of biotin. Data are mean ± SEM. (J) Schematic depiction of the regulation of cell dynamics by transport of integrins through the biosynthetic pathway. Adhesion and cell spreading-dependent delivery of integrin from the ER is detected rapidly after release in cell protrusions. Canonical Golgi-dependent delivery is also polarized to cell protruding areas in an ECM-specific manner and contributes to FA growth and cell protrusion. (F–H) One-way ANOVA, Holm-Šídák’s multiple comparison test, data distribution was assumed to be normal, but this was not formally tested. (F) N = 64 cells on collagen, 50 cells on FN, pooled from three independent experiments. (G) 57 WT cells and 52 ITGA5 KO cells, (H) 59 WT cells and 55 ITGA5 KO cells, pooled from three independent experiments. (I) Mann–Whitney test, N = 55 cells on FN, 66 cells on collagen, pooled from three independent experiments.

Article Snippet: U2OS cells (DSMZ; ACC 785) were grown in Dulbecco’s Modified Eagle’s medium (D5796; DMEM, Sigma-Aldrich) supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin (P0781; Sigma-Aldrich).

Techniques: Immunofluorescence, Expressing, Clinical Proteomics, Membrane, Imaging, Comparison, MANN-WHITNEY