Journal: Cell reports

Article Title: G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration

doi: 10.1016/j.celrep.2025.115237

Figure Lengend Snippet: (A) Schematic of FA isolation protocol using untreated or RNase A-treated HDFs (1 mg/mL for 10 min). (B) Expression of G3BP1 with RNase A treatment from proteomics analysis of FA isolates from control or RNase A-treated HDFs (mean ± SD, two-way unpaired t test, p = 0.0003, n = 4 independent replicates). (C) Representative image of G3BP1 (yellow) and iRFP (cyan) within control or RNase A-treated cells (1 mg/mL for 10 min) co-stained with PXN (magenta) and DAPI (white). Dotted boxes in the left images represent the highly magnified region along the bottom. (D) Boxplots of FA-G3BP1 or FA-iRFP correlation coefficient from control or RNase A-treated cells. Paired measurements from G3BP1 and iRFP are from the same cells (two-way ANOVA with multiple comparisons, Con G3BP1 vs. Con iRFP p = 0.0088, vs. RNase G3BP1 p = 0.0005, and vs. RNase iRFP p = 0.0016; n = 25 cells). (E) Representative images of G3BP1 (yellow) and PXN (magenta) from cells treated with RNase A (1 mg/mL for 10 min), Blebb (25 μM for 30 min), or sodium arsenite (SA; 500 μM for 45 min). (F) Boxplots of FA-G3BP1 correlation coefficient from control or SA-treated cells (two-way unpaired t test, p < 0.0001, n = 14–21 cells). (G) Schematic of sample preparation for G3BP1 co-immunoprecipitation (coIP) for liquid chromatography-mass spectrometry (LC-MS). Cells were treated with RNase A (1 mg/mL for 10 min) or Blebb (25 μM for 30 min) before being lysed for coIP. (H) Heatmap of FA proteins lost or with decreased binding to G3BP1 following treatment ( n = 3 biological replicates). (I) Western blots of coIP samples of G3BP1, ACTN1, TLN1, or immunoglobulin (Ig)G controls for control or treated cells. Samples were treated under the same conditions as the LC-MS screen. See also and and .

Article Snippet: For G3BP1 re-expression, the ΔRBD and ΔNTF2L plasmids (a gift from Paul Taylor) were cloned into the lentiviral backbone and fused with an SNAP tag for visualization to match full length G3BP1 (Addgene, #119949).

Techniques: Isolation, Expressing, Control, Staining, Sample Prep, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Western Blot

Journal: Cell reports

Article Title: G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration

doi: 10.1016/j.celrep.2025.115237

Figure Lengend Snippet: (A) Schematic and confocal images of GFP-G3BP1 protein (10 μM, yellow) mixed with in - vitro -transcribed mRNAs (50 nM, cyan). Dotted lines represent the outlines of the G3BP1 RNP complexes. (B) High-resolution confocal images of HDFs after immunofluorescence staining of G3BP1 (yellow) and paxillin (PXN; magenta) and smFISH of endogenous mRNA ( TRAK2 , KIF1C , DCBLD2 , and IMPAD in cyan). Dotted lines represent outlines of proteins and mRNA. Dotted boxes in whole-cell images represent the region highly magnified along the bottom. (C) Normalized (to max intensity) profiles of G3BP1, PXN, and mRNA fluorescent intensities averaged across multiple FAs for mRNA directly co-localizing with FAs (mean ± SD, n = 20 mRNA/protein complexes, two-tailed correlation matrix for each mRNA vs. G3BP1 and PXN, where p < 0.0001 for all Pearson correlation coefficients where r = 0.849–0.947). (D) Composite MERFISH images for FA-mRNAs after all hybridization rounds with FAs marked by PXN (white arrows) in HDFs with a G3BP1 or control shRNA. (E and F) Boxplot (E) and histogram (F) of log2 of FA enrichment for FA-mRNAs determined by MERFISH in G3BP1 or control shRNA-treated HDFs ( n = 122 FA-mRNA species averaged across 3 or 4 independent experiments, p < 0.0001, two-way paired t test). See also and and .

Article Snippet: For G3BP1 re-expression, the ΔRBD and ΔNTF2L plasmids (a gift from Paul Taylor) were cloned into the lentiviral backbone and fused with an SNAP tag for visualization to match full length G3BP1 (Addgene, #119949).

Techniques: In Vitro, Immunofluorescence, Staining, Two Tailed Test, Hybridization, Control, shRNA

Journal: Cell reports

Article Title: G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration

doi: 10.1016/j.celrep.2025.115237

Figure Lengend Snippet: (A) Schematic of G3BP1 protein domains and mutants used. (B and C) Rose plots (B) and quantification of speed (μm/min), represented as boxplots, (C) from individually migrating cells from control or G3BP1 shRNA-treated cells with either no G3BP1, full-length (FL), ΔRBD, or ΔNTF2L re-expression (one-way ANOVA with multiple comparisons, p < 0.0001 for Con or FL vs. shRNA alone, ΔRBD, or ΔNTF2L, and p = 0.0445 for ΔRBD vs. ΔNTF2L; n = 247–346 cells per group). (D) Boxplots of FA-G3BP1 correlation coefficient from control or re-expression cells in E (one-way ANOVA with multiple comparisons, p < 0.0001 for G3BP1 shRNA only vs. all other comparisons, for Con shRNA vs. ΔRBD or ΔNTF2L, or FL vs. ΔNTF2L; p = 0.0115 for FL vs. ΔRBD; n = 15 for all groups, but n = 7 for G3BP1 shRNA alone). (E) Representative images of G3BP1 (yellow) and PXN (magenta) in control or G3BP1 shRNA-treated cells with G3BP1 re-expression. Dotted lines represent outlines of proteins. Dotted boxes in the whole-cell image represent the region highly magnified along the bottom below. (F) Schematic of MS2-MCP-GFP-PXN system. TRAK2-MS2-tagged mRNA was co-expressed with an MCP-GFP-PXN fusion protein to directly link RNA to FAs. (G) Representative images of G3BP1 RNP complexes (yellow) and TRAK2-MS2 (cyan) in HDFs co-expressing MCP-GFP-PXN, which is incorporated into the FAs (magenta). Dotted lines represent outlines of protein/mRNA. Dotted boxes in images represent the highly magnified regions below. (H) Quantification of smFISH images taken of cells expressing MCP-GFP-PXN and either MS2 alone or TRAK2-MS2. Boxplots represent the log2 of FA enrichment for mRNA foci directly on top of FA (co-localization) (two-way unpaired t test, p < 0.0001, n = 21 cells). (I) Boxplots of FA-G3BP1 correlation coefficient from MS2-only- or TRAK2-MS2-expressing cells (two-way unpaired t test, p = 0.0472, n = 25 cells). (J and K) Rose plots (microns) (J) and quantification of speed (μm/min), represented as boxplots, (K) from individually migrating cells from control or G3BP1 shRNA-treated cells with either MS2 only or TRAK2-MS2 (one-way ANOVA with multiple comparisons, p < 0.0001 for MS2 only vs. TRAK2-MS2 Con shRNA and MS2 only or TRAK2-MS2 Con shRNA vs. both G3BP1 shRNA groups; n = 498–755 cells/group). See also and , , , , , , , , and .

Article Snippet: For G3BP1 re-expression, the ΔRBD and ΔNTF2L plasmids (a gift from Paul Taylor) were cloned into the lentiviral backbone and fused with an SNAP tag for visualization to match full length G3BP1 (Addgene, #119949).

Techniques: Control, shRNA, Expressing

Journal: Cell reports

Article Title: G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration

doi: 10.1016/j.celrep.2025.115237

Figure Lengend Snippet: (A and B) Western blot images and quantification of puromycin incorporation after 5 min of puromycin treatment (5 μM) in (A) whole cells or (B) isolated FAs from control or G3BP1 shRNA-treated cells. Puromycin intensity was normalized to total protein visualized via ponceau S staining (see and ) (mean ± SD, two-way unpaired t test, p = 0.4024 with n = 3 replicates for whole cells, p = 0.6837 with n = 2 replicates [4 pooled dishes for each replicate] for isolated FAs). (C) Western blot images and quantification for FA-mRNA encoding proteins (TRAK2, KIF1C, LPAR1, PPFIBP1, IQGAP1, NET1, RAI14, and CTNNB1), FA/cytoskeletal proteins (ACTB, VCL, PXN, TLN1, and ACTN1), or G3BP1 in Con shRNA or G3BP1 shRNA-treated cells. Protein intensity was normalized to GAPDH (mean ± SD, two-way unpaired t tests for all, n = 3 replicates, p > 0.05 for all comparisons except for G3BP1, where p = 0.0014). (D and E) Rose plots (D) and quantification of speed (μm/min), represented as boxplots, (E) from individually migrating cells from control or G3BP1 shRNA-treated cells with either DMSO or CHX (100 μg/mL added at the start of imaging and continued for the 2-h video acquisition) (one-way ANOVA with multiple comparisons, p < 0.0001 for all comparisons except for Con shRNA CHX vs. G3BP1 shRNA DMSO [ p = 0.0023] and Con shRNA CHX vs. G3BP1 shRNA CHX [ p = 0.0008]; n = 284–382 cells per group). See also and , , , and .

Article Snippet: For G3BP1 re-expression, the ΔRBD and ΔNTF2L plasmids (a gift from Paul Taylor) were cloned into the lentiviral backbone and fused with an SNAP tag for visualization to match full length G3BP1 (Addgene, #119949).

Techniques: Western Blot, Isolation, Control, shRNA, Staining, Imaging

Journal: Cell reports

Article Title: G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration

doi: 10.1016/j.celrep.2025.115237

Figure Lengend Snippet: (A and B) Representative images (A) and quantification (B) of FA size in HDF expressing a control or a G3BP1-targeting shRNA and treated with either DMSO or CHX (100 μg/mL for 1 h). Quantification is represented as boxplots (two-way ANOVA with multiple comparisons, Con shRNA [DMSO or CHX] vs. G3BP1 shRNA [DMSO or CHX] p < 0.0001 and Con shRNA DMSO vs. Con shRNA CHX p = 0.0316; n = 999–2,362 FAs per group). (C and D) Representative images (C) and quantification (D) of FRAP of PXN+ FAs in cells expressing a control or a G3BP1-targeting shRNA and treated with either DMSO or CHX (100 μg/mL for 1 h). PXN was ectopically expressed as PXN-iRFP. Quantification is represented as boxplots (two-way ANOVA with multiple comparisons, p < 0.0001 for all comparisons shown and p > 0.05 for all other comparisons; n = 47–49 FA FRAPs per group). (E and F) Representative images (E) and quantification (F) of FA size in control or G3BP1 shRNA-treated cells with either no G3BP1, full-length (FL), ΔRBD, or ΔNTF2L re-expression. Quantification is represented as boxplots (one-way ANOVA with multiple comparisons, p < 0.0001 for Con shRNA or FL vs. all other groups, n = 292–480 FA per group). (G and H) Representative images (G) and quantification of (H) FRAP of PXN+ FAs in cells expressing a control or a G3BP1-targeting shRNA with G3BP1 re-expression. PXN was ectopically expressed as PXN-mCherry. Quantification is represented as boxplots (one-way ANOVA with multiple comparisons, p < 0.0001 for Con shRNA or FL vs. all other groups, n = 46–55 FA FRAPs per group). See also and , , , , , , and .

Article Snippet: For G3BP1 re-expression, the ΔRBD and ΔNTF2L plasmids (a gift from Paul Taylor) were cloned into the lentiviral backbone and fused with an SNAP tag for visualization to match full length G3BP1 (Addgene, #119949).

Techniques: Expressing, Control, shRNA

Journal: Cell reports

Article Title: G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration

doi: 10.1016/j.celrep.2025.115237

Figure Lengend Snippet: We demonstrate that focal adhesions (FAs) are enriched with mRNAs that have longer coding and untranslated regions, exhibit lower translation levels, and contain AU-rich elements, forming G3BP1 ribonucleoprotein (RNP) complexes essential for cell migration. In normal cells, G3BP1 localizes at FAs, and FA protein mobility is enhanced, leading to increased FA size and faster cell migration. Removing G3BP1 inhibits FA protein mobility and halts cell migration without affecting FA proteins or general protein translation. Our findings indicate that G3BP1 RNPs regulate cell migration by modulating FA protein dynamics.

Article Snippet: For G3BP1 re-expression, the ΔRBD and ΔNTF2L plasmids (a gift from Paul Taylor) were cloned into the lentiviral backbone and fused with an SNAP tag for visualization to match full length G3BP1 (Addgene, #119949).

Techniques: Migration

Journal: Cell reports

Article Title: G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration

doi: 10.1016/j.celrep.2025.115237

Figure Lengend Snippet:

Article Snippet: For G3BP1 re-expression, the ΔRBD and ΔNTF2L plasmids (a gift from Paul Taylor) were cloned into the lentiviral backbone and fused with an SNAP tag for visualization to match full length G3BP1 (Addgene, #119949).

Techniques: Control, Recombinant, SYBR Green Assay, Cell Culture, Protease Inhibitor, Mutagenesis, Isolation, Sequencing, shRNA, Plasmid Preparation, Software

Journal: eLife

Article Title: Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1α

doi: 10.7554/eLife.75580

Figure Lengend Snippet: ( A ) Reporter construct design and illustration of the method: XBP1u translation reporters feature a 9× GCN4 array (green) inserted into the opening reading frame downstream of the ER intron and in frame with the XBP1u protein. Upon translation of GCN4-XBP1u, emerging GCN4 peptide repeats are recognized by GFP-labeled single-chain antibodies (scAB-GFP), which allow detection of translating ribosomes together with mRNA transcripts. Upon splicing, the reading frame is changed and GCN4 expression is lost. ( B ) qPCR-based splicing assay to test functionality of XBP1u translation reporter (green) as compared to a non-GCN4-tagged control (gray). Shown is the splicing ratio (XBP1s/XBP1u) in response to induction of ER stress with 100 nM thapsigargin (TG). Graph represents the average ± SD (n=3). Statistical test Kruskal-Wallis and Dunn's multiple comparison test. No significant differences were observed.( C ) Western blot against XBP1 proteins. Spliced XBP1 appearance is dependent on reporter expression (Dox) and induction of ER stress with 100 nM TG. Black arrows: XBP1 protein products expressed upon TG and Dox treatment. White arrows: unspecific bands present irrespective of reporter expression (Dox) in response to TG. Asterisk: unspecific bands present in all samples. ( D ) Representative live-cell image of XBP1u translation sites (green diffraction limited spots) in a HeLa cell expressing scAB-GFP and a fluorescent ER marker (gray). ( E ) Boxplot showing ER association of XBP1u translation sites (green) as compared to secreted protein encoding Gaussia mRNAs (gray) that serve as an ER-associated positive control. Statistical test: unpaired t-test, p-value = 0.49. ( F ) Combined single-molecule fluorescence in-situ hybridization (smFISH) and immunofluorescence (IF) analysis for colocalization of XBP1 mRNA (magenta) and translation site signal (green) in fixed HeLa cells (DAPI = blue). The majority of translation site spots disappear upon induction of ER stress with 5 µg/ml tunicamycin (TM) for 2 hours. ( G ) Quantification of data shown in ( F ). Individual dots represent per-cell averages. Black bars show mean ± SD. All scale bars = 5 µm. For raw data see . Figure 2—source data 1. Source data containing raw gel images for .

Article Snippet: Single-chain antibodies fused to GFP (scAB-GFP, Addgene #104,998) were used for imaging translation sites through nascent polypeptide labeling.

Techniques: Construct, Labeling, Expressing, Splicing Assay, Control, Comparison, Western Blot, Marker, Positive Control, Fluorescence, In Situ Hybridization, Immunofluorescence

Journal: eLife

Article Title: Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1α

doi: 10.7554/eLife.75580

Figure Lengend Snippet: ( A ) Correlated diffusion and endoplasmic reticulum (ER) colocalization analysis for individual XBP1u translation site tracks (green) and Gaussia mRNA transcripts (gray). Dots are single particles that were tracked for at least 30 frames. Y axis: instantaneous diffusion coefficients . X axis: cumulative ER localization index. Positive values indicate ER colocalization. XBP1u translation site tracks scatter similar to Gaussia mRNA tracks. ( B ) Representative live-cell image of XBP1u translation reporter mRNA (magenta) and translation site (green) spots in a HeLa cell stably expressing NLS-stdMCP-stdHalo and scAB-GFP. In the absence of stress, the majority of mRNA transcripts are translated. ( C ) Same as ( B ) but acquired upon addition of puromycin (PUR) (100 µg/ml). Translation site spots (green) vanish upon PUR-mediated translation inhibition. All scale bars = 5 µm. ( D ) Histogram plot showing distribution of mean spot intensities in exemplary fixed cell experiment (smFISH-IF, replicate 2). Intensities of mRNA spots that did not colocalize with scAB-GFP signal are shown in dark magenta, while intensities of those mRNA spots that did colocalize with scAB-GFP ( = translation sites) are depicted in light magenta.

Article Snippet: Single-chain antibodies fused to GFP (scAB-GFP, Addgene #104,998) were used for imaging translation sites through nascent polypeptide labeling.

Techniques: Diffusion-based Assay, Stable Transfection, Expressing, Inhibition

Journal: eLife

Article Title: Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1α

doi: 10.7554/eLife.75580

Figure Lengend Snippet: ( A ) Schematic illustration of experimental design: GCN4-SM translation reporters either encoding Renilla luciferase (left) or XBP1u (right) were stably expressed in the HeLa imaging cell lines introduced above. To test if scAB-GFP could unspecifically associate with MCP (MS2 coat protein)-Halo or any other part of the XBP1 mRNA, a plasmid encoding the XBP1 wild-type (WT) reporter transcript was transfected into cells stably expressing the GCN4-SM-Renilla reporter (Eichenberger et al., in preparation). Expression of all reporter mRNAs was induced through addition of 0.2 ug/ml doxycycline for 1–2 hours. Cells were fixed, processed for single-molecule fluorescence in-situ hybridization (smFISH) against XBP1 mRNA and immunofluorescence (IF) against scAB-GFP, and the degree of colocalization of mRNA and scAB-GFP signal was quantified for both conditions. ( B ) Combined smFISH and IF experiment to assess colocalization of XBP1 mRNA (magenta) and translation site signal (green) in fixed HeLa cells (DAPI = blue). XBP1 mRNA and scAB-GFP translation site spots only colocalize if expressed from the same mRNA transcript. ( C ) Quantification of data shown in ( B ). Individual dots represent per-cell averages. Black bars show mean ± SD (XBP1u only = 0.50 ± 0.17; XBP1 wt + Renilla Luciferase = 0.01 ± 0.03). All scale bars = 5 µm.

Article Snippet: Single-chain antibodies fused to GFP (scAB-GFP, Addgene #104,998) were used for imaging translation sites through nascent polypeptide labeling.

Techniques: Luciferase, Stable Transfection, Imaging, Plasmid Preparation, Transfection, Expressing, Fluorescence, In Situ Hybridization, Immunofluorescence

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 1. COPII-mediated cargo transport continues in the absence of Sar1 (A) Cartoon depicting the human SAR1A and SAR1B genomic loci. The positions of the gRNAs used during CRISPR-Cas9 editing (red lines) are highlighted, and the sizes of exons (shown as green boxes) are 1/50 that of introns (shown as black lines). (B) Representative immunoblots of extracts generated from a CRISPR-modified cell line lack- ing Sar1a and subjected to siRNA-mediated treatments as shown, using antibodies directed against Sar1 and actin. Extracts were generated at the time point indicated following siRNA treat- ment. (C) Quantification of the percentage of Sar1 re- maining at different time points following Sar1b siRNA treatment (relative to mock siRNA treat- ment). Error bars represent mean ± SEM (n = 4 biological replicates). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test. (D) Spinning disk confocal microscopy was used to image control RPE1 cells and cells lacking Sar1a either in the presence or in the absence of Sar1b, each expressing ss-DsRed following treatment with SLF (50 mM) to induce cargo disaggregation and release from the ER. Repre- sentative time-lapse images are shown (n = 15 cells, each condition; at least three biological replicates). Scale bar, 5 mm. (E and F) Quantification of cargo (E, ss-DsRed; F, ManII-SBP-GFP) accumulation within the peri- nuclear region (GM130 positive) in the various mutant backgrounds indicated. Error bars repre- sent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test (relative to the 0 min time point). See also Figures S1–S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: CRISPR, Western Blot, Generated, Confocal Microscopy, Control, Expressing, Mutagenesis

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 2. Loss of Sar1 alters the distribution of COPII coat subunits but fails to block their ability to co-assemble (A) Genome-edited cells expressing HaloTag- Sec23a and lacking Sar1a (mock transfected) and those depleted of Sar1b for 48 h were imaged live using spinning disk confocal microscopy following labeling with JFX646-HaloTag ligand. Scale bar, 5 mm; inset bar, 2 mm. (B and C) Quantification of the number of Sec23a- positive sites in cells lacking Sar1a in the presence and absence of Sar1b is shown at various time points following Sar1b depletion. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test, relative to mock treatment (B) or the 48 h time point (C). (D) Cells lacking Sar1a, depleted of Sar1b, and co- expressing HaloTag-Sec23a and YFP-Sec31a were imaged live using spinning disk confocal microscopy following labeling using JFX646- HaloTag ligand. Representative zoomed images are shown (n = 10 cells; at least three biological replicates each). Scale bar, 2 mm. (E) The volume distribution of Sec23a-positive structures in the absence of Sar1a is shown following 48 h of mock treatment or Sar1b deple- tion. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test (relative to mock treatment). (F) Cells lacking Sar1a (mock transfected) or depleted of Sar1b for 72 h were immunostained using antibodies directed against Sec24a and TFG (shown only in insets). Representative confocal images (maximum intensity projections) are shown, and an arrow indicates co-localization of Sec24a and TFG. Scale bar, 5 mm; inset bar, 2 mm. (G–I) HaloTag-Sec23a in cells lacking Sar1a, either in the presence or in the absence of Sar1b, were labeled with JFX646-HaloTag ligand and sub- jected to photobleaching. Fluorescence recovery is depicted for various-sized HaloTag-Sec23a structures (n = 15 cells each; at least three bio- logical replicates each). See also Figures S4–S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: Blocking Assay, Expressing, Transfection, Confocal Microscopy, Labeling, Fluorescence

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 3. COPII condensates associate with secretory cargoes that leave the ER in the absence of Sar1 (A) Cells expressing ss-DsRed and lacking both Sar1 isoforms were immunostained using antibodies directed against Sec24a and GM130 following treatment with SLF (50 mM) and imaged using STED microscopy. Representative images are shown (n = 15 cells; at least three biological replicates). Scale bar, 2 mm. (B) Cells expressing EGFP-Sec61b and HaloTag-Sec23a in the absence of Sar1 were transfected with a construct encoding ss-DsRed and imaged live using spinning disk confocal microscopy following cargo release and labeling with JFX646-HaloTag ligand. Violin plots show the relative displacement of each marker over time (n = 10 cells; three biological replicates). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test, compared with Sec61b displacement. (C) Cells lacking both Sar1 isoforms and expressing native EGFP-Sec61b and HaloTag-Sec23a were transfected with a construct encoding ss-DsRed and imaged live using spinning disk confocal microscopy following treatment with SLF (50 mM) and labeling with JFX646-HaloTag ligand. Representative images are shown (n = 7 cells; three biological replicates). Arrowheads indicate accumulation of ss-DsRed with COPII condensates, which ultimately move away from their site of origin. Scale bar, 2 mm. (D) Relative fluorescence intensities of cargo (ss-DsRed) and COPII (HaloTag-Sec23a) were measured at ER subdomains over time. Error bars represent mean ± SEM. An asterisk highlights the time point at which cargo and COPII undergo a >1 mm displacement, and all measurements are aligned with respect to this time point (n = 10 cells; three biological replicates). (E) Expression of 43FM-HaloTag-L1CAM was transiently induced in control cells co-expressing Sar1 (H79G) or in cells lacking Sar1a and depleted of Sar1b, each labeled with JFX646-HaloTag ligand, and subjected to photobleaching after treatment with DDS for 60 min. Normalized fluorescence recovery in each case is shown (n = 10 cells each; at least three biological replicates each), and error bars represent mean ± SEM. An asterisk highlights the time point at which individual sites exhibiting elevated 43FM-HaloTag-L1CAM fluorescence were bleached. (F) Cells lacking Sar1a and natively co-expressing EGFP-Sec61b and HaloTag-Sec23a in the presence and absence of Sar1b were transfected with a construct encoding ss-DsRed and imaged as described for (B). The length of time ss-DsRed remained associated with HaloTag-Sec23A prior to undergoing displacement (more than 1 mm) was determined in each case. **p < 0.01, calculated using a t test, compared with cells lacking only Sar1a. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: Expressing, Microscopy, Transfection, Construct, Confocal Microscopy, Labeling, Marker, Control

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 4. The absence of Sar1 destabilizes ERGIC membranes (A, B, D, E, and F) Cells lacking Sar1a (mock transfected or depleted of Sar1b for 72 h) were immunostained using antibodies directed against Sec16a (A), Tango1 (B), ERGIC-53 (and Sec16a shown in insets, with arrows highlighting their juxtaposed distribution) (D), TFG (E), or COPB1 (and GM130 shown in insets) (F). Representative confocal images (maximum intensity projections) are shown. Scale bars, 5 mm; inset bars, 2 mm. (C) Quantification of the fold change in the fluorescence intensities of Sec31a, Sec16a, and Tango1 in the absence of Sar1 (relative to mock siRNA treatment). Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test. (G) Representative immunoblots of extracts generated from control and CRISPR-modified cell lines lacking Sar1a and either in the presence or in the absence of Sar1b, using antibodies directed against COPB1 and actin. (H) Quantification of the fold change in COPB1 levels in the absence of Sar1 (relative to mock siRNA treatment of cells lacking only Sar1a). Error bar represents mean ± SEM (n = 4 biological replicates). See also Figure S8.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: Transfection, Western Blot, Generated, Control, CRISPR

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 5. The absence of Sar1 leads to the formation of COPII condensates that inter- fere with secretory cargo movement through the Golgi (A) Time-lapse spinning disk confocal microscopy was used to image cargo (ss-DsRed) accumulated in the perinuclear region of cells lacking Sar1 following addition of 1,6-hexanediol (top) or 2,5- hexanediol (bottom). Representative images are shown (n = 10 cells; three biological replicates). Scale bar, 5 mm. (B) Quantification of cargo (ss-DsRed) remaining within the perinuclear region of cells lacking Sar1 following 30 min of incubation with 1,6-hexanediol or 2,5-hexanediol (relative to the 0 min time point). Error bars represent mean ± SEM (n = 10 cells each; three biological replicates). ***p < 0.001, calculated using a t test, compared with treatment with 2,5-hexanediol. (C) Representative electron micrographs taken within the perinuclear region of high-pressure- frozen cells lacking Sar1a following a mock siRNA treatment or Sar1b depletion. The nuclear enve- lope (NE) is indicated in each image (n = 5 cells each; at least three biological replicates). Scale bar, 500 nm. See also Figure S9.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: Confocal Microscopy, Incubation