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: Polyclonal antibodies against Sec16-N (374–387 aa; RRID: N/A, rabbit), Sec16-C (2,319–2,332 aa; RRID: N/A, rabbit), and TANGO1-CT (1,884–1,898 aa; RRID: N/A, rabbit) were raised in rabbits by immunization with keyhole limpet hemocyanin-conjugated peptides and affinity-purified by columns conjugated with the peptides (Thermo Fisher Scientific; ; ; ; ).

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: 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: Polyclonal antibodies against Sec16-N (374–387 aa; RRID: N/A, rabbit), Sec16-C (2,319–2,332 aa; RRID: N/A, rabbit), and TANGO1-CT (1,884–1,898 aa; RRID: N/A, rabbit) were raised in rabbits by immunization with keyhole limpet hemocyanin-conjugated peptides and affinity-purified by columns conjugated with the peptides (Thermo Fisher Scientific; ; ; ; ).

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: Polyclonal antibodies against Sec16-N (374–387 aa; RRID: N/A, rabbit), Sec16-C (2,319–2,332 aa; RRID: N/A, rabbit), and TANGO1-CT (1,884–1,898 aa; RRID: N/A, rabbit) were raised in rabbits by immunization with keyhole limpet hemocyanin-conjugated peptides and affinity-purified by columns conjugated with the peptides (Thermo Fisher Scientific; ; ; ; ).

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: Polyclonal antibodies against Sec16-N (374–387 aa; RRID: N/A, rabbit), Sec16-C (2,319–2,332 aa; RRID: N/A, rabbit), and TANGO1-CT (1,884–1,898 aa; RRID: N/A, rabbit) were raised in rabbits by immunization with keyhole limpet hemocyanin-conjugated peptides and affinity-purified by columns conjugated with the peptides (Thermo Fisher Scientific; ; ; ; ).

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: Polyclonal antibodies against Sec16-N (374–387 aa; RRID: N/A, rabbit), Sec16-C (2,319–2,332 aa; RRID: N/A, rabbit), and TANGO1-CT (1,884–1,898 aa; RRID: N/A, rabbit) were raised in rabbits by immunization with keyhole limpet hemocyanin-conjugated peptides and affinity-purified by columns conjugated with the peptides (Thermo Fisher Scientific; ; ; ; ).

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: Polyclonal antibodies against Sec16-N (374–387 aa; RRID: N/A, rabbit), Sec16-C (2,319–2,332 aa; RRID: N/A, rabbit), and TANGO1-CT (1,884–1,898 aa; RRID: N/A, rabbit) were raised in rabbits by immunization with keyhole limpet hemocyanin-conjugated peptides and affinity-purified by columns conjugated with the peptides (Thermo Fisher Scientific; ; ; ; ).

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: 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: Polyclonal antibodies against Sec16-N (374–387 aa; RRID: N/A, rabbit), Sec16-C (2,319–2,332 aa; RRID: N/A, rabbit), and TANGO1-CT (1,884–1,898 aa; RRID: N/A, rabbit) were raised in rabbits by immunization with keyhole limpet hemocyanin-conjugated peptides and affinity-purified by columns conjugated with the peptides (Thermo Fisher Scientific; ; ; ; ).

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: Polyclonal antibodies against Sec16-N (374–387 aa; RRID: N/A, rabbit), Sec16-C (2,319–2,332 aa; RRID: N/A, rabbit), and TANGO1-CT (1,884–1,898 aa; RRID: N/A, rabbit) were raised in rabbits by immunization with keyhole limpet hemocyanin-conjugated peptides and affinity-purified by columns conjugated with the peptides (Thermo Fisher Scientific; ; ; ; ).

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: Polyclonal antibodies against Sec16-N (374–387 aa; RRID: N/A, rabbit), Sec16-C (2,319–2,332 aa; RRID: N/A, rabbit), and TANGO1-CT (1,884–1,898 aa; RRID: N/A, rabbit) were raised in rabbits by immunization with keyhole limpet hemocyanin-conjugated peptides and affinity-purified by columns conjugated with the peptides (Thermo Fisher Scientific; ; ; ; ).

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: Polyclonal antibodies against Sec16-N (374–387 aa; RRID: N/A, rabbit), Sec16-C (2,319–2,332 aa; RRID: N/A, rabbit), and TANGO1-CT (1,884–1,898 aa; RRID: N/A, rabbit) were raised in rabbits by immunization with keyhole limpet hemocyanin-conjugated peptides and affinity-purified by columns conjugated with the peptides (Thermo Fisher Scientific; ; ; ; ).

Techniques: Incubation, Staining

Journal: Scientific reports

Article Title: Acetyl-CoA flux from the cytosol to the ER regulates engagement and quality of the secretory pathway.

doi: 10.1038/s41598-021-81447-6

Figure Lengend Snippet: Figure 2. Morphological reorganization of ER, ERGIC and Golgi apparatus in aberrant AT-1 models. (a) Representative ER morphology in primary-cultured MEFs using ER3-mCherry (scale bar, 3 µm). White asterisk (*) indicates an enlarged cisternae within the perinuclear rough ER. High-magnification areas for WT and AT-1 sTg are shown (scale bar, 2 µm). (b–f) Quantification of puncta revealed using SIM microscopy in primary- cultured MEFs in (b) Sec13 puncta (n = 14 WT; n = 13 AT-1 sTg; n = 10 AT-1S113R/+), (c) Sec31 puncta (n = 25 WT; n = 20 AT-1 sTg; n = 21 AT-1S113R/+), (d) Sec23 puncta (n = 4 WT; n = 7 AT-1 sTg; n = 5 AT-1S113R/+), (e) Sec24 puncta (n = 12 WT; n = 15 AT-1 sTg; n = 14 AT-1S113R/+), (f) Sec16 puncta (n = 19 WT; n = 15 AT-1 sTg; n = 14 AT-1S113R/+). (g–j) Pearson’s coefficient of puncta revealed using SIM microscopy in primary-cultured MEFs in (g) Sec31 and Sec13 puncta (n = 8 WT; n = 7 AT-1 sTg; n = 5 AT-1S113R/+), (h) Sec23 and Sec24 puncta (n = 4 WT; n = 7 AT-1 sTg; n = 5 AT-1S113R/+), (i) Sec31 and Sec24 puncta (n = 8 WT; n = 8 AT-1 sTg; n = 9 AT-1S113R/+), (j) Sec13 and Sec16 puncta (n = 6 WT; n = 6 AT-1 sTg; n = 5 AT-1S113R/+). (k) ERGIC morphology in primary-cultured MEFs using ERGIC-53 antibody (scale bar, 3 µm) and quantified using Imaris reconstruction of volume and number of puncta (n = 7 WT; n = 7 AT-1 sTg; n = 7 AT-1S113R/+). (l) Golgi apparatus morphology in primary-cultured MEFs using GM130 antibody (scale bar, 3 µm) and quantified using Imaris reconstruction of volume and area (n = 11 WT; n = 12 AT-1 sTg; n = 12 AT-1S113R/+). (m) Representative electron microscopy of MEFs from WT, AT-1 sTg, and AT-1S113R/+. Yellow outlines Golgi structures and orange outlines disorganized secretory structures and vesicles. (scale bar, 500 nm). MEFs from multiple cells from 3 biologically independent animals for each genotype for (b–l). One-way ANOVA. *P < 0.05; **P < 0.005.

Article Snippet: All antibodies were diluted in antibody dilution buffer (1% BSA, 5% goat serum in PBS) and stained with ERGIC-53 (E1031, Sigma Aldrich, 1:80), Sec13 (A303980A, Bethyl Laboratories or H00006396-B02P, Novus Biologicals, 1:50), Sec16 (A300-648A, Bethyl Laboratories.

Techniques: Cell Culture, Microscopy, Electron Microscopy