Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Sar1 membrane association is efficiently detected using the Split mNeonGreen system. (A) Schematic representation of the SAIYAN system. The membrane-spanning regions of TANGO1S and HA-tag fused to 10 of the 11 strands of mNG (mNG 1–10 ) were expressed in cells. In addition, Sar1A constructs with a FLAG-tag and a glycine linker fused to the 11th strand of mNG (mNG 11 ) were also expressed. Upon Sar1A activation, mNG 1–10 and mNG 11 combined to form the complete mNG proteins, inducing mNG signals. (B) HA-mNG 1–10 cells transfected with the indicated Sar1A constructs were fixed and stained with anti-Sec16-C and anti-FLAG antibodies. Scale bar = 10 µm. (C) Quantification of mNG intensity from B (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Membrane, Construct, FLAG-tag, Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Validation of SAIYAN technology. (A) Doxycycline-inducible HeLa cells expressing the membrane-spanning region of TANGO1S and HA-tag fused to 10 of the 11 strands of mNG (HA-mNG 1–10 cells) were either non-transfected or transfected with Sar1A constructs with a FLAG tag and a glycine linker fused to the 11th strand of mNG (Sar1A-FLAG-mNG 11 ). The cells were fixed and stained with anti-HA and anti-PDI antibodies. Scale bar = 10 µm. (B) HA-mNG 1–10 cells, treated with or without doxycycline, were transfected with Sar1A-FLAG-mNG 11 . The cells were fixed and stained with anti-HA and anti-FLAG antibodies. Scale bar = 10 µm.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Expressing, Membrane, Transfection, Construct, FLAG-tag, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Production of Sar1A/SAIYAN cells. (A) Doxycycline-inducible stable cell lines expressing the membrane-spanning regions of TANGO1S and HA-tag fused to 10 of the 11 strands of mNG were established using a lentiviral system and G418 selection (HA-mNG 1–10 cells). Stable cells were subsequently electroporated with Cas9 protein, sgRNA, and ssDNA to facilitate the knock-in of FLAG-mNG 11 into the Sar1A locus of the genome. Cells were treated with doxycycline for 24 h and further sorted via FACS to isolate single cells exhibiting mNG signals into 96-well plates. The expanded cell population was then collected and subjected to genomic sequencing. Positive clones were identified and selected for further analysis (Sar1A/SAIYAN cells). (B) Sar1A/SAIYAN (HeLa) cells, either treated or non-treated with doxycycline, were fixed and stained with anti-HA and anti-PDI antibodies. Boxed areas in the middle panels are shown at high magnification in the bottom panels. Scale bars: 10 µm (main), 5 µm (magnification). (C) Sar1A/SAIYAN (HeLa) cells, either treated or non-treated with doxycycline, were fixed and stained with anti-HA and anti-FLAG antibodies. Boxed areas in the middle panels are shown at high magnification in the bottom panels. Scale bar:10 µm (main), 5 μm (magnification). (D) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were fixed and stained with an anti-Sec16-C antibody. Scale bar = 10 µm. (E) Quantification of mNG intensity from D (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells. (F) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sar1A, and anti-GAPDH antibodies. (G) Sar1A/SAIYAN (HeLa) cells, either treated or non-treated with doxycycline, were fractionated via centrifugation. The lysates, the supernatants, and the pellets were subjected to SDS-PAGE, followed by western blotting with anti-FLAG, Sar1A, HA, ERK1, and calnexin antibodies. Source data are available for this figure: .

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Stable Transfection, Expressing, Membrane, Selection, Knock-In, Genomic Sequencing, Clone Assay, Staining, Transfection, SDS Page, Western Blot, Centrifugation

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Sar1A/SAIYAN (HeLa) cells proliferate and secrete normally. (A) HeLa and Sar1A/SAIYAN (HeLa) cells were treated with or without doxycycline for 24 h, and cell viability was measured and normalized using untreated HeLa cells as control. Error bars represent the means ± SEM. n = 4. (B) Sar1A/SAIYAN (HeLa) cells, treated with or without doxycycline, were fixed and stained with anti-Sec16-C and anti-GM130 antibodies. Scale bars = 10 µm. (C) Sar1A/SAIYAN (HeLa) cells treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry. RUSH chase was started with biotin addition, and live imaging was performed. Scale bars = 10 μm.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Control, Staining, Transfection, Imaging

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: ER exit site organization is required for the efficient activation of Sar1A. (A, C, E, and G) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were fixed and stained with anti-Sec16-C antibodies. Scale bar = 10 µm. (B, D, F, and H) Quantification of mNG signals from A, C, E, and G, respectively (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Western blotting analysis of Sar1A/SAIYAN (HeLa) cells on , , and . (A) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec12, and anti-GAPDH antibodies. (B) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-cTAGE5 CC1, anti-Sec12, and anti-GAPDH antibodies. (C) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-TANGO1 CC1, and anti-GAPDH antibodies. (D) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec16-N, and anti-GAPDH antibodies. (E) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec23A (11D8), and anti-GAPDH antibodies. (F) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec31A (rabbit), and anti-GAPDH antibodies. (G) Sar1A/SAIYAN (HeLa) cells were stably expressed using mCherry-tagged Sec23A constructs as indicated. Cells were lysed and subjected to SDS-PAGE, followed by western blotting with anti-FLAG, anti-HA, anti-Sec23A (11D8), anti-Sec31A (rabbit), and anti-GAPDH antibodies. Source data are available for this figure: .

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Western Blot, Transfection, SDS Page, Stable Transfection, Construct

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Sec23A and Sec31A depletion exerts opposite effects on the activation of Sar1A in living cells. (A and C) Sar1A/SAIYAN (HeLa) cells transfected with the indicated siRNAs were fixed and stained with anti-Sec16-C antibodies. Scale bar = 10 µm. (B and D) Quantification of mNG signals from A and C, respectively (arbitrary units [A.U.]). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells. (E) Sar1A/SAIYAN (HeLa) cells transfected with the indicated plasmids were fixed and processed for microscopic analysis. Scale bar = 10 µm. (F) Quantification of mNG signals from E (A.U.). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Each CLSD mutant of Sec23A exhibits different properties on Sar1 activation. (A) Sar1A/SAIYAN (HeLa) cells stably expressing mCherry-tagged Sec23A constructs, as indicated, were fixed and processed for microscopic analysis. Scale bar = 10 µm. (B) Quantification of mNG signals from A (A.U.). Error bars represent the means ± SEM. Each data point represents the mNG intensity of the analyzed cells.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Mutagenesis, Activation Assay, Stable Transfection, Expressing, Construct

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: DPD treatment accumulates collagen I within the ER of Sar1A/SAIYAN (BJ-5ta) cells. Sar1A/SAIYAN (BJ-5ta) cells were treated with DMSO or 0.5 mM DPD and incubated for 16 h. Cells were fixed and stained with an anti-collagen I antibody. Scale bar = 10 µm.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Incubation, Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Activated Sar1 prevails in the ERGIC region of Sar1A/SAIYAN (BJ-5ta) cells. (A–O) Sar1A/SAIYAN (BJ-5ta) cells were fixed and stained with anti-Sec16-C (A), anti-ERGIC53 (B), anti-Sec23 (5H2) (C), anti-Sec24B (D), anti-Sec24D (E), anti-p125A (F), anti-TANGO1-CT (G), anti-Sec12 (H), anti-TFG (I), anti-Sec13 (J), anti-Sec31A (mouse) (K), anti-β-COP (L), anti-GM130 (M), anti-PDI (N), and anti-Rab1A (O) antibodies. Images were captured using Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (A–L) Right; top: Magnification of the indicated regions is on the left. Right; bottom: Magnification of the indicated regions on the upper. (P) Pearson’s correlation coefficient was used to quantify the degree of colocalization. n = 5. Cyan; outer COPII coats, blue; inner COPII coats, purple; ER exit site resident proteins, red; ERGIC proteins, orange; COPI protein, magenta; ER and Golgi proteins. Error bars represent the mean 95% CI.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Triple staining of Sar1A/SAIYAN (BJ-5ta) and parental BJ-5ta reveals the organization of the ER-Golgi interface of collagen-secreting cells. (A–C) Sar1A/SAIYAN (BJ-5ta) cells were fixed and stained with anti-Sec16-C and anti-ERGIC53 (A), anti-Sec23 (5H2), and anti-ERGIC53 (B), and anti-Rab1A and anti-ERGIC53 (C) antibodies. Images were captured using the Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (D) BJ-5ta cells were fixed and stained with anti-Sec16-C, anti-Sec23 (5H2), and anti-ERGIC53 antibodies. Images were captured using the Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (A–D) (right; top) Magnification of the indicated regions is on the left. (right; bottom) Double staining of the magnified region on the top.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Staining, Double Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Quantification of Pearson’s correlation coefficient to quantify the degree of colocalization in Sar1A/SAIYAN (HeLa) cells. Sar1A/SAIYAN (HeLa) cells were fixed and stained with anti-Sec16-C, anti-ERGIC53, anti-Sec23, anti-Sec24B, anti-Sec24D, anti-p125A, anti-TANGO1-CT, anti-Sec12, anti-TFG, anti-Sec13, anti-Sec31A (mouse), anti-β-COP, anti-GM130, anti-PDI, and anti-Rab1A antibodies. Images were captured using the Airyscan2. n = 5. Cyan; outer COPII coats, blue; inner COPII coats, purple; endoplasmic reticulum (ER) exit site resident proteins, red; ERGIC proteins, orange; COPI protein, magenta; ER and Golgi proteins. Error bars represent the mean 95% CI.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells

doi: 10.1083/jcb.202403179

Figure Lengend Snippet: Reticular pattern of activated Sar1 signals diminished with DPD treatment in Sar1A/SAIYAN (BJ-5ta) cells. (A) Sar1A/SAIYAN (BJ-5ta) cells were treated with DMSO or 0.5 mM DPD and incubated for 16 h. Cells were fixed and stained with anti-Sec16-C antibodies. Images were captured using the Airyscan2. Scale bars: 10 µm (main), 1 µm (magnification). (B) Pearson’s correlation coefficient was quantified to assess the degree of colocalization. n = 5. Error bars represent the mean 95% CI.

Article Snippet: Sar1A/SAIYAN (HeLa) cells on a 3.5-cm glass bottom dish (Matsunami) treated with or without doxycycline were transfected with Str-KDEL_ManII-SBP-mCherry (Addgene).

Techniques: Incubation, Staining

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

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 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: eLife

Article Title: Dynamic regulation of inter-organelle communication by ubiquitylation controls skeletal muscle development and disease onset

doi: 10.7554/elife.81966

Figure Lengend Snippet: Figure 5. Klhl40a loss results in perturbation of the ER-Golgi vesicle trafficking through secretion-associated Ras GTPase (Sar1a). (A) ER-Golgi vesicle trafficking proteins exhibit altered levels in klhl40a mutant muscle compared to control (+/+) in proteome analysis; ns indicates no significant difference (B) Western blot showing ER-exit site protein Sar1a is upregulated in klhl40a mutant muscle, and downstream COPII and Golgi proteins are downregulated in mutant muscle (3mo) (C) Quantification of the protein by Western blot in klhl40a and control zebrafish. N=3 in each group. Data are mean ± S.E.M; with one-way ANOVA and Tukey’s HSD test (****p<0.001). (D) Transmission electron microscopy (TEM) of zebrafish larva (4 dpf)

Article Snippet: SAR1A cDNA (addgene, #67451) or KLHL40 (WT or mutant cDNAs) were cloned into the pDEST15 vector by gateway cloning.

Techniques: Mutagenesis, Control, Western Blot, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: Dynamic regulation of inter-organelle communication by ubiquitylation controls skeletal muscle development and disease onset

doi: 10.7554/elife.81966

Figure Lengend Snippet: Figure 6. Morphological changes in vesicular trafficking compartments in Klhl40a deficient myofibers. Sar1a is increased and co-localized with PDI in Klhl40a deficiency. The number of PDI-positive foci is also increased in the absence of Klhl40a. The fraction of Sec23 and Tango1 positive foci is decreased in klhl40a mutant myofibers. Disruption of the Golgi architecture was observed in a fraction of Klhl40a deficient myofibers. Data are mean ± S.E.M (unpaired t-test, parametric) for each quantification.

Article Snippet: SAR1A cDNA (addgene, #67451) or KLHL40 (WT or mutant cDNAs) were cloned into the pDEST15 vector by gateway cloning.

Techniques: Mutagenesis, Disruption

Journal: eLife

Article Title: Dynamic regulation of inter-organelle communication by ubiquitylation controls skeletal muscle development and disease onset

doi: 10.7554/elife.81966

Figure Lengend Snippet: Figure 7. SAR1A is a direct ubiquitylation target of the KLHL40-CUL3 complex and is differently ubiquitylated by a disease-causing mutation in KLHL40. (A) Coimmunoprecipitation in C2C12 cells showing KLHL40 directly interacts with SAR1A. (B) Co-overexpression of decreasing KLHL40-FLAG and constant SAR1A-V5 in C2C12 myoblasts demonstrates that KLHL40 is a regulator of Sar1A protein. (C) Co-overexpression of decreasing amounts of KLHL40-FLAG and constant amount of SAR1A-V5 in C2C12 myoblasts in the presence of UPS inhibitor MG132 increases the SAR1A protein levels in comparison to MG132- condition. (D) Alignment of the amino acid sequence of the SAR1A ubiquitylation site demonstrates high conservation in vertebrates (K182 in all species, marked by the asterisk). (E) Localization of different disease-causing variants in KLHL40 in the protein domains. (F) In vitro ubiquitylation of human SAR1A by CUL3 protein complex in the presence of wild-type and disease-causing KLHL40 proteins. (G) Quantifying the relative human SAR1A ubiquitylation by wild-type and disease-causing KLHL40-CUL3 complex. (H) Ubiquitylation of overexpressed SAR1A in the presence of KLHL40 in C2C12 myoblasts. Data are mean ± S.E.M; with one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test and Brown-Forsythe test (****p<0.001; n.s. non significant) n=3.

Article Snippet: SAR1A cDNA (addgene, #67451) or KLHL40 (WT or mutant cDNAs) were cloned into the pDEST15 vector by gateway cloning.

Techniques: Mutagenesis, Over Expression, Comparison, Sequencing, In Vitro

Journal: eLife

Article Title: Dynamic regulation of inter-organelle communication by ubiquitylation controls skeletal muscle development and disease onset

doi: 10.7554/elife.81966

Figure Lengend Snippet: Figure 9. KLHL40-NM patients exhibit increased SAR1A protein and vesicle accumulation with ECM defects in skeletal muscle. (A) Immunofluorescence in control and a KLHL40 patient muscle biopsy showing increased SAR1A protein in the patient muscle (white arrows). Moreover, collagen accumulation is seen in the patient muscle (white arrowhead). (B) Transmission electron microscopy of KLHL40 patient muscle showed vesicle accumulation (arrows) and disorganized and damaged extracellular matrix between myofibers (arrows).

Article Snippet: SAR1A cDNA (addgene, #67451) or KLHL40 (WT or mutant cDNAs) were cloned into the pDEST15 vector by gateway cloning.

Techniques: Immunofluorescence, Control, Transmission Assay, Electron Microscopy