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Addgene inc pdendra2-er-5
Pdendra2 Er 5, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pdendra2-er-5/pdendra2+er+5/pm35051356-261-2-28
Average 90 stars, based on 1 article reviews

pdendra2-er-5 - by Bioz Stars, 2026-09

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Reverse Transcription Polymerase Chain Reaction:

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport
Article Snippet: The following plasmids were from Addgene: pDendra2-ER-5 (57716), pmEmerald-ER-3 (54082), pAcGFP1-Sec61β (15108), pEGFP-ERGIC-53 (38270), pEGFP-Rab1A (49467), pEGFP-Rab5B (61802), pEGFP-Rab11A (12674), pEGFP-Rab7A (12605), pClover-LAMP1 (56528), pEGFP-p62 (38277), pEGFP-Sec23A (66609), pStr-KDEL_SBP-EGFP-Ecadherin (65286), pcDNA3-FLAG-Rab1A-N124I (46778).

Article Title: SURF4-induced tubular ERGIC selectively expedites ER-to-Golgi transport.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER SURF4 RT-PCR R This work TGCATGGGCTTGTAGACTG GAPDH RT-PCR F This work CATCACCATCTTCCA GGAGC GAPDH RT-PCR R This work GGATGATGTTCTGGAGA GCC Recombinant DNA pDendra2-ER-5 Addgene 57716 pmEmerald-ER-3 Addgene 54082 pAcGFP1-Sec61b Addgene 15108 pRTN4A-AcGFP1 Addgene 61807 pEGFP-ERGIC-53 (low expression) Addgene 38270 pEGFP-Rab1A Addgene 49467 pEGFP-Rab5B Addgene 61802 pEGFP-Rab11A Addgene 12674 pEGFP-Rab7A Addgene 12605 pClover-LAMP1 Addgene 56528 pEGFP-p62 Addgene 38277 pEGFP-Sec23A Addgene 66609 pStr-KDEL_SBP-EGFP-Ecadherin Addgene 65286 pcDNA3-FLAG-Rab1A-N124I Addgene 46778 pTol2-elavl3-H2B-GCaMP6s Addgene 59530 pTwist-CMV BetaGlobin-KDELR3-HA (HA tag inserted between E143 and A144 of human KDELR3) Twist Bioscience This work pTwist-CMV BetaGlobin-SURF4-HA (HA tag inserted between D263 and K265 of human SURF4) Twist Bioscience This work pDsRed2-ER-5 (pAPV-DsRed2-ER-5) Generated here This work pEPV-DsRed2-ER-5 Generated here This work pDsRed2-ER-3 Generated here This work pAPV-SBP-DsRed2-ER-5 Generated here This work pEPV-SBP-DsRed2-ER-5 Generated here This work pmEmerald-ERGIC-53 (high expression) Generated here This work pAcGFP1-SURF4 Generated here This work pFLAG-SURF4 Generated here This work pStr-KDEL_APV-SBP-DsRed2-ER-5 Generated here This work pStr-KDEL_EPV-SBP-DsRed2-ER-5 Generated here This work pHaloTag-Sec23A Generated here This work pStr-KDEL_TPV-CXCL9-mCherry-SBP Generated here This work pStr-KDEL_EPV-CXCL9-mCherry-SBP Generated here This work pAPV-DsRed2-mEmerald-ER-5 Generated here This work pAPV-mEmerald-DsRed2-ER-5 Generated here This work pAPV-EGFP(1-228)-ER-5 Generated here This work pAPV-DsRed2-ER-5-KDELKO Generated here This work pEPV-DsRed2-ER-5-KDELKO Generated here This work Software and algorithms ImageJ (Fiji) NIH https://imagej.net/software/fiji/ MATLAB MathWorks https://www.mathworks.com/products/ matlab.html Micro-Manager Edelstein et al., 2014 https://micro-manager.org/ (Continued on next page) e3 Developmental Cell 57, 512–525.e1–e8, February 28, 2022

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(A) Schematics of the sequences of DsRed2-ER-3 and DsRed2-ER-5. SP: signal peptide; L: linker. (B) Representative live-cell images of DsRed2-ER-3 and DsRed2-ER-5 expressed in COS-7 cells. (C) Time-lapse series of the boxed region in (B), showing the morphology and fast motion of the tubular organelles (TOs) (arrows). See also Video S1. (D) Classification of the dominating distribution modes of DsRed2-ER-3 and DsRed2-ER-5 in each cell: ER, TO, vesicles (Vesi), and cytoplasm (Cyto). Error bars: SEM (n = 4 with ~50 cells in each replicate). (E) 3D-STORM image of immunolabeled DsRed2-ER-5 in a COS-7 cell. Colors encode axial positions. (F) Close-ups of the TOs in the three colored boxes in (E). (G) STORM intensity profiles across the widths of two TOs at the magenta and red arrows in (F). Black curves: Gaussian fits with FWHM (full width at half maximum) of 34 and 30 nm, respectively. (H-K) Dual-color live-cell images of DsRed2-ER-5 (green) with the ER marker AcGFP1-Sec61β (H), the Golgi marker Golgi-GFP (I), the canonical ERGIC marker EGFP-ERGIC-53 (J), and the small GTPase EGFP-Rab1A (K). Arrows point to TOs. Scale bars: 5 μm (B,H-K); 2 μm (C,E); 500 nm (F). See also .

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) Schematics of the sequences of DsRed2-ER-3 and DsRed2-ER-5. SP: signal peptide; L: linker. (B) Representative live-cell images of DsRed2-ER-3 and DsRed2-ER-5 expressed in COS-7 cells. (C) Time-lapse series of the boxed region in (B), showing the morphology and fast motion of the tubular organelles (TOs) (arrows). See also Video S1. (D) Classification of the dominating distribution modes of DsRed2-ER-3 and DsRed2-ER-5 in each cell: ER, TO, vesicles (Vesi), and cytoplasm (Cyto). Error bars: SEM (n = 4 with ~50 cells in each replicate). (E) 3D-STORM image of immunolabeled DsRed2-ER-5 in a COS-7 cell. Colors encode axial positions. (F) Close-ups of the TOs in the three colored boxes in (E). (G) STORM intensity profiles across the widths of two TOs at the magenta and red arrows in (F). Black curves: Gaussian fits with FWHM (full width at half maximum) of 34 and 30 nm, respectively. (H-K) Dual-color live-cell images of DsRed2-ER-5 (green) with the ER marker AcGFP1-Sec61β (H), the Golgi marker Golgi-GFP (I), the canonical ERGIC marker EGFP-ERGIC-53 (J), and the small GTPase EGFP-Rab1A (K). Arrows point to TOs. Scale bars: 5 μm (B,H-K); 2 μm (C,E); 500 nm (F). See also .

Article Snippet: The following plasmids were from Addgene: pDendra2-ER-5 (57716), pmEmerald-ER-3 (54082), pAcGFP1-Sec61β (15108), pEGFP-ERGIC-53 (38270), pEGFP-Rab1A (49467), pEGFP-Rab5B (61802), pEGFP-Rab11A (12674), pEGFP-Rab7A (12605), pClover-LAMP1 (56528), pEGFP-p62 (38277), pEGFP-Sec23A (66609), pStr-KDEL_SBP-EGFP-Ecadherin (65286), pcDNA3-FLAG-Rab1A-N124I (46778).

Techniques: Immunolabeling, Marker

(A) Representative images of the four types of subcellular distribution of DsRed2-ER-5 in COS-7 cells: cytoplasm (Cyto), ER, tubular organelles (TOs), and vesicles (Vesi). (B,C) Representative images of DsRed2-ER-5 TOs in U2OS (B) and HeLa (C) cells. (D-K) Dual-color fluorescence micrographs of DsRed2-ER-5 (green) in live COS-7 cells with the ER markers mEmerald-ER-3 (D), the early endosome marker EGFP-Rab5B (E), the recycling endosome marker EGFP-Rab11A (F), the late endosome marker EGFP-Rab7A (G), the lysosome marker Clover-LAMP1 (H), the autophagosome marker EGFP-p62 (I), the highly expressed ERGIC marker AcGFP1-ERGIC-53 (J), and the ERES marker EGFP-Sec23A (K). Arrowheads in (J) indicate TOs negative for ERGIC-53. (L) Immunofluorescence of the COPI vesicle marker β-COP vs. DsRed2-ER-5. (M) Two-color STORM of immunolabeled DsRed2-ER-5 and EGFP-Rab1A showing that Rab1A decorates the surface of the TO. Inset: cross-sectional intensity profiles of the boxed region in the overlay. (N,O) Immunofluorescence of endogenous Rab1A (N) and Rab1B (O) in COS-7 cells, showing good colocalization with the DsRed2-ER-5 TOs. (P) Time-lapse imaging of DsRed2-ER-5 in an unsynchronized COS-7 cell, showing its redistribution from the ER to the TOs, Golgi, and vesicles accompanied by a reduction of fluorescence intensity. Scale bars: 5 μm except for the labeled scale bars in (M). Arrows point to TOs.

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) Representative images of the four types of subcellular distribution of DsRed2-ER-5 in COS-7 cells: cytoplasm (Cyto), ER, tubular organelles (TOs), and vesicles (Vesi). (B,C) Representative images of DsRed2-ER-5 TOs in U2OS (B) and HeLa (C) cells. (D-K) Dual-color fluorescence micrographs of DsRed2-ER-5 (green) in live COS-7 cells with the ER markers mEmerald-ER-3 (D), the early endosome marker EGFP-Rab5B (E), the recycling endosome marker EGFP-Rab11A (F), the late endosome marker EGFP-Rab7A (G), the lysosome marker Clover-LAMP1 (H), the autophagosome marker EGFP-p62 (I), the highly expressed ERGIC marker AcGFP1-ERGIC-53 (J), and the ERES marker EGFP-Sec23A (K). Arrowheads in (J) indicate TOs negative for ERGIC-53. (L) Immunofluorescence of the COPI vesicle marker β-COP vs. DsRed2-ER-5. (M) Two-color STORM of immunolabeled DsRed2-ER-5 and EGFP-Rab1A showing that Rab1A decorates the surface of the TO. Inset: cross-sectional intensity profiles of the boxed region in the overlay. (N,O) Immunofluorescence of endogenous Rab1A (N) and Rab1B (O) in COS-7 cells, showing good colocalization with the DsRed2-ER-5 TOs. (P) Time-lapse imaging of DsRed2-ER-5 in an unsynchronized COS-7 cell, showing its redistribution from the ER to the TOs, Golgi, and vesicles accompanied by a reduction of fluorescence intensity. Scale bars: 5 μm except for the labeled scale bars in (M). Arrows point to TOs.

Techniques: Fluorescence, Marker, Immunofluorescence, Immunolabeling, Imaging, Labeling

(A) Flow cytometry histograms of DsRed2-ER-5-transfected COS-7 cells treated with 0.1% DMSO (Ctrl), 1 μM thapsigargin (TG), 7 mM dithiothreitol (DTT), 5 μM MG132, 1 μM CB-5083, or 1 μM brefeldin A (BFA) for 4 hr. (B) Representative fluorescence micrographs of DsRed2-ER-5 in COS-7 cells with brefeldin A treatments of 0, 4, and 8 hr. (C-E) Schematic of SBP-DsRed2-ER-5 (C), a representative image of its presence in the t-ERGIC (D), and its subcellular distribution (E) in transfected COS-7 cells. Error bars: SEM (n = 3 with ~50 cells in each replicate). (F) Single-particle tracking of post-ER carriers in . The color of each trajectory encodes the maximum speed reached. (G) Displacement map of the trajectories in (F). The Golgi apparatus is marked blue. Arrows point to the direction of displacement (from the initial position to the final position), and their magnitudes are scaled according to the legend. (H) Another example of de novo formation of SBP-DsRed2-ER-5 t-ERGIC (arrow) from the ERES (arrowhead) in a RUSH experiment, similar to . 80 μM biotin was added at time −11 min for cargo release. (I) RUSH image sequence showing that the same COPII-coated ERES (arrowhead) sequentially generates two t-ERGICs (white and yellow arrows) in opposite directions. 80 μM biotin was added at time 0. (J) RUSH image sequence showing that a t-ERGIC (arrow) buds from the Golgi apparatus, fuses with an ERES (arrowhead), and carries the cargo back to the Golgi apparatus. 80 μM biotin was added 30 min before time 0. (K) Co-transfection of SBP-DsRed2-ER-5 with a dominant negative Rab1A (Rab1A-N124I) inhibited the generation of t-ERGIC in RUSH, and the cargo was stuck at the ERES. 80 μM biotin was added at time 0. Scale bars: 10 μm (B,F,G); 5 μm (D,K); 2 μm (H-J). Arrows in (B,D) indicate t-ERGICs.

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) Flow cytometry histograms of DsRed2-ER-5-transfected COS-7 cells treated with 0.1% DMSO (Ctrl), 1 μM thapsigargin (TG), 7 mM dithiothreitol (DTT), 5 μM MG132, 1 μM CB-5083, or 1 μM brefeldin A (BFA) for 4 hr. (B) Representative fluorescence micrographs of DsRed2-ER-5 in COS-7 cells with brefeldin A treatments of 0, 4, and 8 hr. (C-E) Schematic of SBP-DsRed2-ER-5 (C), a representative image of its presence in the t-ERGIC (D), and its subcellular distribution (E) in transfected COS-7 cells. Error bars: SEM (n = 3 with ~50 cells in each replicate). (F) Single-particle tracking of post-ER carriers in . The color of each trajectory encodes the maximum speed reached. (G) Displacement map of the trajectories in (F). The Golgi apparatus is marked blue. Arrows point to the direction of displacement (from the initial position to the final position), and their magnitudes are scaled according to the legend. (H) Another example of de novo formation of SBP-DsRed2-ER-5 t-ERGIC (arrow) from the ERES (arrowhead) in a RUSH experiment, similar to . 80 μM biotin was added at time −11 min for cargo release. (I) RUSH image sequence showing that the same COPII-coated ERES (arrowhead) sequentially generates two t-ERGICs (white and yellow arrows) in opposite directions. 80 μM biotin was added at time 0. (J) RUSH image sequence showing that a t-ERGIC (arrow) buds from the Golgi apparatus, fuses with an ERES (arrowhead), and carries the cargo back to the Golgi apparatus. 80 μM biotin was added 30 min before time 0. (K) Co-transfection of SBP-DsRed2-ER-5 with a dominant negative Rab1A (Rab1A-N124I) inhibited the generation of t-ERGIC in RUSH, and the cargo was stuck at the ERES. 80 μM biotin was added at time 0. Scale bars: 10 μm (B,F,G); 5 μm (D,K); 2 μm (H-J). Arrows in (B,D) indicate t-ERGICs.

Techniques: Flow Cytometry, Transfection, Fluorescence, Single-particle Tracking, Sequencing, Cotransfection, Dominant Negative Mutation

(A-C) Flow cytometry histograms (A), lysate immunoblots (B), and subcellular distribution (C) of DsRed2-ER-5 for transfected COS-7 cells treated with 1 μM brefeldin A (BFA) for 0, 4, and 8 hr. Error bars: SEM (n = 3 with ~50 cells in each replicate). (D) Representative RUSH image sequence of SBP-DsRed2-ER-5 after the addition of 80 μM biotin at time 0. Arrows indicate t-ERGICs. See also Video S3. (E) De novo generation of SBP-DsRed2-ER-5-positive t-ERGIC (arrow) in RUSH. Time 0 corresponds to when budding occurred. Biotin was added at −5 min for cargo release. (F) Image sequences of EGFP-Rab1A, SBP-DsRed2-ER-5, and JF635-labeled HaloTag-Sec23A in a RUSH experiment showing the de novo generation of a t-ERGIC (arrow) from the ERES (arrowhead). Time 0 corresponds to when budding occurred. Biotin addition corresponded to −20 min. (G) Fluorescence intensity time traces of the three color channels for the ERES indicated by the arrowhead in (F). (H) Another image sequence of an ERES (arrowhead) in the same RUSH experiment as in (F), showing its fusion with a pre-existing t-ERGIC (arrow). Time 0 corresponds to when fusion occurred. Biotin addition corresponded to −28 min. (I) Fluorescence intensity time traces of the three color channels for the ERES indicated by the arrowhead in (H). Scale bars: 5 μm (D,F,H); 2 μm (E). See also .

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A-C) Flow cytometry histograms (A), lysate immunoblots (B), and subcellular distribution (C) of DsRed2-ER-5 for transfected COS-7 cells treated with 1 μM brefeldin A (BFA) for 0, 4, and 8 hr. Error bars: SEM (n = 3 with ~50 cells in each replicate). (D) Representative RUSH image sequence of SBP-DsRed2-ER-5 after the addition of 80 μM biotin at time 0. Arrows indicate t-ERGICs. See also Video S3. (E) De novo generation of SBP-DsRed2-ER-5-positive t-ERGIC (arrow) in RUSH. Time 0 corresponds to when budding occurred. Biotin was added at −5 min for cargo release. (F) Image sequences of EGFP-Rab1A, SBP-DsRed2-ER-5, and JF635-labeled HaloTag-Sec23A in a RUSH experiment showing the de novo generation of a t-ERGIC (arrow) from the ERES (arrowhead). Time 0 corresponds to when budding occurred. Biotin addition corresponded to −20 min. (G) Fluorescence intensity time traces of the three color channels for the ERES indicated by the arrowhead in (F). (H) Another image sequence of an ERES (arrowhead) in the same RUSH experiment as in (F), showing its fusion with a pre-existing t-ERGIC (arrow). Time 0 corresponds to when fusion occurred. Biotin addition corresponded to −28 min. (I) Fluorescence intensity time traces of the three color channels for the ERES indicated by the arrowhead in (H). Scale bars: 5 μm (D,F,H); 2 μm (E). See also .

Techniques: Flow Cytometry, Western Blot, Transfection, Sequencing, Labeling, Fluorescence

(A) Sequences of the XPV-DsRed2-ER-5 mutations we examined, with varied N-termini after the signal peptide (SP). The original DsRed2-ER-5 has X=A (APV-DsRed2-ER-5). (B) Representative fluorescence micrographs of APV/EPV-DsRed2-ER-5 in COS-7 cells. Arrows point to t-ERGICs. (C) Subcellular distribution of EPV-DsRed2-ER-5. Error bars: SEM (n = 3 with ~50 cells in each replicate). (D) Flow cytometry histograms of APV/EPV-DsRed2-ER-5. (E) Azidohomoalanine-biotin-alkyne pulse-chase of APV/EPV-DsRed2-ER-5. Newly synthesized proteins were labeled by azidohomoalanine click chemistry and detected by NeutrAvidin (see Methods). (F) Immunoblots of intracellular (cell lysate) and secreted (anti-FLAG immunoprecipitation from the culture medium) APV/EPV-FLAG-DsRed2-ER-5. (G,H) Golgi (G) and peripheral ER (H) fluorescence intensity time traces of APV/EPV-SBP-DsRed2-ER-5 in RUSH, pooled from 70 cells from 5 independent runs (APV) or 17 cells from 3 independent runs (EPV). Error bars: SEM. 80 μM biotin was added at time 0. (I,J) Comparison of the time to the peak fluorescence in the Golgi (I) and the time of fluorescence decay to 75% of the start in the ER (J) of APV/EPV-SBP-DsRed2-ER-5 in RUSH. Whiskers and boxes show 10%, 25%, 50%, 75%, and 90% quantiles. (K) Median intracellular fluorescence of different XPV-DsRed2-ER-5 variants expressed in COS-7 cells, as determined by flow cytometry. (L) Representative fluorescence micrographs of TPV/EPV-CXCL9-mCherry-SBP in RUSH. 80 μM biotin was added at time 0. Arrows point to t-ERGICs. See also Video S4. (M,N) Comparison of the time to the peak fluorescence in the Golgi (M) and the time of fluorescence decay to 75% of the start in the ER (N) of TPV/EPV-CXCL9-mCherry-SBP in RUSH. Whiskers and boxes show 10%, 25%, 50%, 75%, and 90% quantiles. Scale bars: 5 μm. P values are calculated by two-tailed t test. See also .

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) Sequences of the XPV-DsRed2-ER-5 mutations we examined, with varied N-termini after the signal peptide (SP). The original DsRed2-ER-5 has X=A (APV-DsRed2-ER-5). (B) Representative fluorescence micrographs of APV/EPV-DsRed2-ER-5 in COS-7 cells. Arrows point to t-ERGICs. (C) Subcellular distribution of EPV-DsRed2-ER-5. Error bars: SEM (n = 3 with ~50 cells in each replicate). (D) Flow cytometry histograms of APV/EPV-DsRed2-ER-5. (E) Azidohomoalanine-biotin-alkyne pulse-chase of APV/EPV-DsRed2-ER-5. Newly synthesized proteins were labeled by azidohomoalanine click chemistry and detected by NeutrAvidin (see Methods). (F) Immunoblots of intracellular (cell lysate) and secreted (anti-FLAG immunoprecipitation from the culture medium) APV/EPV-FLAG-DsRed2-ER-5. (G,H) Golgi (G) and peripheral ER (H) fluorescence intensity time traces of APV/EPV-SBP-DsRed2-ER-5 in RUSH, pooled from 70 cells from 5 independent runs (APV) or 17 cells from 3 independent runs (EPV). Error bars: SEM. 80 μM biotin was added at time 0. (I,J) Comparison of the time to the peak fluorescence in the Golgi (I) and the time of fluorescence decay to 75% of the start in the ER (J) of APV/EPV-SBP-DsRed2-ER-5 in RUSH. Whiskers and boxes show 10%, 25%, 50%, 75%, and 90% quantiles. (K) Median intracellular fluorescence of different XPV-DsRed2-ER-5 variants expressed in COS-7 cells, as determined by flow cytometry. (L) Representative fluorescence micrographs of TPV/EPV-CXCL9-mCherry-SBP in RUSH. 80 μM biotin was added at time 0. Arrows point to t-ERGICs. See also Video S4. (M,N) Comparison of the time to the peak fluorescence in the Golgi (M) and the time of fluorescence decay to 75% of the start in the ER (N) of TPV/EPV-CXCL9-mCherry-SBP in RUSH. Whiskers and boxes show 10%, 25%, 50%, 75%, and 90% quantiles. Scale bars: 5 μm. P values are calculated by two-tailed t test. See also .

Techniques: Fluorescence, Flow Cytometry, Pulse Chase, Synthesized, Labeling, Western Blot, Immunoprecipitation, Two Tailed Test

(A) Sequences of APV/EPV-FLAG-DsRed2-ER-5 and APV/EPV-SBP-DsRed2-ER-5. (B,C) Subcellular distributions (B) and flow cytometry histograms (C) of APV/EPV-FLAG-DsRed2-ER-5. (D) Immunoblots of APV/EPV-DsRed2-ER-5-transfected cells and non-transfected cells with or without 1 μM thapsigargin (positive control for ER stress) treatment for 5 hr. (E) Flow cytometry histograms of APV/EPV-DsRed2-ER-5 in HeLa and U2OS cells. (F,G) Representative fluorescence micrographs (F) and flow cytometry histograms (G) of APV/EPV-GCaMP6s-ER-5 in COS-7 cells. (H) Representative RUSH image sequence of EPV-SBP-DsRed2-ER-5. 80 μM biotin was added at time 0. (I) Representative fluorescence micrographs of DPV/QPV/NPV-DsRed2-ER-5 in COS-7 cells. (J) Subcellular distributions of different XPV-DsRed2-ER-5 variants. The “A” and “E” data duplicates that of “ER-5” in and that of , respectively. Scale bars: 5 μm. Error bars: SEM (n = 3 with ~50 cells in each replicate). Arrows point to t-ERGICs.

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) Sequences of APV/EPV-FLAG-DsRed2-ER-5 and APV/EPV-SBP-DsRed2-ER-5. (B,C) Subcellular distributions (B) and flow cytometry histograms (C) of APV/EPV-FLAG-DsRed2-ER-5. (D) Immunoblots of APV/EPV-DsRed2-ER-5-transfected cells and non-transfected cells with or without 1 μM thapsigargin (positive control for ER stress) treatment for 5 hr. (E) Flow cytometry histograms of APV/EPV-DsRed2-ER-5 in HeLa and U2OS cells. (F,G) Representative fluorescence micrographs (F) and flow cytometry histograms (G) of APV/EPV-GCaMP6s-ER-5 in COS-7 cells. (H) Representative RUSH image sequence of EPV-SBP-DsRed2-ER-5. 80 μM biotin was added at time 0. (I) Representative fluorescence micrographs of DPV/QPV/NPV-DsRed2-ER-5 in COS-7 cells. (J) Subcellular distributions of different XPV-DsRed2-ER-5 variants. The “A” and “E” data duplicates that of “ER-5” in and that of , respectively. Scale bars: 5 μm. Error bars: SEM (n = 3 with ~50 cells in each replicate). Arrows point to t-ERGICs.

Techniques: Flow Cytometry, Western Blot, Transfection, Positive Control, Fluorescence, Sequencing

(A,B) Representative fluorescence micrographs (A) and flow cytometry histograms (B) of APV/APE-DsRed2-ER-5 in COS-7 cells. (C) Image sequence showing de novo generation of a t-ERGIC through the co-budding of AcGFP1-SURF4 (magenta) and APV-DsRed2-ER-5 (yellow) but not JF635-labeled HaloTag-Sec23A (cyan). Arrowhead marks the ERES. Arrow indicates the t-ERGIC. (D) Representative immunofluorescence images of FLAG-SURF4 (magenta) in COS-7 cells co-transfected with APV/EPV-DsRed2-ER-5 (green). (E) RT-PCR of SURF4 mRNA in control and SURF4 siRNA-treated cells. (F) Representative live-cell images of APV-DsRed2-ER-5 in control and SURF4 siRNA-treated cells. Arrows indicate t-ERGICs. Scale bars: 5 μm (A,D,F); 2 μm (C).

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A,B) Representative fluorescence micrographs (A) and flow cytometry histograms (B) of APV/APE-DsRed2-ER-5 in COS-7 cells. (C) Image sequence showing de novo generation of a t-ERGIC through the co-budding of AcGFP1-SURF4 (magenta) and APV-DsRed2-ER-5 (yellow) but not JF635-labeled HaloTag-Sec23A (cyan). Arrowhead marks the ERES. Arrow indicates the t-ERGIC. (D) Representative immunofluorescence images of FLAG-SURF4 (magenta) in COS-7 cells co-transfected with APV/EPV-DsRed2-ER-5 (green). (E) RT-PCR of SURF4 mRNA in control and SURF4 siRNA-treated cells. (F) Representative live-cell images of APV-DsRed2-ER-5 in control and SURF4 siRNA-treated cells. Arrows indicate t-ERGICs. Scale bars: 5 μm (A,D,F); 2 μm (C).

Techniques: Fluorescence, Flow Cytometry, Sequencing, Labeling, Immunofluorescence, Transfection, Reverse Transcription Polymerase Chain Reaction

(A) Live-cell images of co-transfected AcGFP1-SURF4 and APV-DsRed2-ER-5 in a COS-7 cell. (B) Co-immunoprecipitation of APV/EPV-FLAG-DsRed2-ER-5 with co-expressed SURF4-HA. (C-E) Subcellular distributions (C), flow cytometry histograms (D), and lysate immunoblots (E) of APV/EPV-DsRed2-ER-5 for COS-7 cells co-transfected with control siRNA or SURF4 siRNA. Error bars: SEM (n = 3 with ~50 cells in each replicate). (F,G) Representative fluorescence micrographs (F) and counts per cell (G) of Rab1A-positive tubules in control or SURF4 siRNA-treated COS-7 cells. Whiskers and boxes show 10%, 25%, 50%, 75%, and 90% quantiles. Open squares indicate means. 287 and 321 cells were quantified for siCtrl and siSURF4, respectively. (H,I) Comparison of the time to the peak fluorescence in the Golgi (H) and the time of fluorescence decay to 75% of the start in the ER (I) of APV/EPV-SBP-DsRed2-ER-5 with control siRNA or SURF4 siRNA in RUSH. (J,K) Comparison of the time to the peak fluorescence in the Golgi (J) and the time of fluorescence decay to 75% of the start in the ER (K) of APV/EPV-SBP-DsRed2-ER-5 with or without co-expression of FLAG-SURF4 in RUSH. Scale bars: 5 μm. Arrows point to t-ERGICs. P values are calculated by Mann-Whitney test (G), two-way ANOVA (APV vs. EPV in [H-K]), or two-tailed t test (siRNA or SURF4 overexpression in [H-K]). See also .

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) Live-cell images of co-transfected AcGFP1-SURF4 and APV-DsRed2-ER-5 in a COS-7 cell. (B) Co-immunoprecipitation of APV/EPV-FLAG-DsRed2-ER-5 with co-expressed SURF4-HA. (C-E) Subcellular distributions (C), flow cytometry histograms (D), and lysate immunoblots (E) of APV/EPV-DsRed2-ER-5 for COS-7 cells co-transfected with control siRNA or SURF4 siRNA. Error bars: SEM (n = 3 with ~50 cells in each replicate). (F,G) Representative fluorescence micrographs (F) and counts per cell (G) of Rab1A-positive tubules in control or SURF4 siRNA-treated COS-7 cells. Whiskers and boxes show 10%, 25%, 50%, 75%, and 90% quantiles. Open squares indicate means. 287 and 321 cells were quantified for siCtrl and siSURF4, respectively. (H,I) Comparison of the time to the peak fluorescence in the Golgi (H) and the time of fluorescence decay to 75% of the start in the ER (I) of APV/EPV-SBP-DsRed2-ER-5 with control siRNA or SURF4 siRNA in RUSH. (J,K) Comparison of the time to the peak fluorescence in the Golgi (J) and the time of fluorescence decay to 75% of the start in the ER (K) of APV/EPV-SBP-DsRed2-ER-5 with or without co-expression of FLAG-SURF4 in RUSH. Scale bars: 5 μm. Arrows point to t-ERGICs. P values are calculated by Mann-Whitney test (G), two-way ANOVA (APV vs. EPV in [H-K]), or two-tailed t test (siRNA or SURF4 overexpression in [H-K]). See also .

Techniques: Transfection, Immunoprecipitation, Flow Cytometry, Western Blot, Fluorescence, Expressing, MANN-WHITNEY, Two Tailed Test, Over Expression

(A) Epifluorescence of immunolabeled Sec31A (top) and EGFP-Sec23A (bottom) in a COS-7 cell co-expressing EGFP-Sec23A and APV-DsRed2-ER-5. (B) Two-color STORM image of Sec31A and APV-DsRed2-ER-5 for the same view as (A) (left), as well as zoom-ins (right) of the cyan-boxed region of the STORM image and the EGFP-Sec23A epifluorescence image. Yellow arrowheads in (A,B) indicate examples of ERES labeled with both EGFP-Sec23A and Sec31A. Filled and open arrowheads indicate DsRed2-loaded and non-loaded ERESs, respectively. Arrows in (B) indicate t-ERGICs. (C) Statistics of the sizes of DsRed2-loaded and non-loaded, Sec23A-positive ERESs, based on the STORM-determined sizes of the Sec31A clusters. Whiskers and boxes show 10%, 25%, 50%, 75%, and 90% quantiles. P value is calculated by a two-tailed t test. n = 5 STORM images were quantified. (D) Representative RUSH image sequence showing the formation and fusion of LLPS-like domains of co-clustered AcGFP1-SURF4 and APV-SBP-DsRed2-ER-5. Biotin was added at time 0 for cargo release. See also Video S7. (E) Dual-color FRAP image sequence of AcGFP1-SURF4 and APV-SBP-DsRed2-ER-5 in a condensate. The gray circle indicates the illuminated area as defined by a pinhole. Asterisks mark random DsRed2-containing vesicles entering the illuminated area. 80 μM biotin was added at −30 min. (F) Fluorescence recovery time trace for the condensate pointed to by the arrow in (E). (G) Dissolution of the AcGFP1-SURF4 and APV-SBP-DsRed2-ER-5 condensates in RUSH by adding 3% 1,6-hexanediol at time 0. Arrows mark the gradually dissolved condensates. 80 μM biotin was added at −30 min. See also Video S8. Scale bars: 2 μm (A,B,D,G), 200 nm (zoom-ins of B), 1 μm (E). See also .

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) Epifluorescence of immunolabeled Sec31A (top) and EGFP-Sec23A (bottom) in a COS-7 cell co-expressing EGFP-Sec23A and APV-DsRed2-ER-5. (B) Two-color STORM image of Sec31A and APV-DsRed2-ER-5 for the same view as (A) (left), as well as zoom-ins (right) of the cyan-boxed region of the STORM image and the EGFP-Sec23A epifluorescence image. Yellow arrowheads in (A,B) indicate examples of ERES labeled with both EGFP-Sec23A and Sec31A. Filled and open arrowheads indicate DsRed2-loaded and non-loaded ERESs, respectively. Arrows in (B) indicate t-ERGICs. (C) Statistics of the sizes of DsRed2-loaded and non-loaded, Sec23A-positive ERESs, based on the STORM-determined sizes of the Sec31A clusters. Whiskers and boxes show 10%, 25%, 50%, 75%, and 90% quantiles. P value is calculated by a two-tailed t test. n = 5 STORM images were quantified. (D) Representative RUSH image sequence showing the formation and fusion of LLPS-like domains of co-clustered AcGFP1-SURF4 and APV-SBP-DsRed2-ER-5. Biotin was added at time 0 for cargo release. See also Video S7. (E) Dual-color FRAP image sequence of AcGFP1-SURF4 and APV-SBP-DsRed2-ER-5 in a condensate. The gray circle indicates the illuminated area as defined by a pinhole. Asterisks mark random DsRed2-containing vesicles entering the illuminated area. 80 μM biotin was added at −30 min. (F) Fluorescence recovery time trace for the condensate pointed to by the arrow in (E). (G) Dissolution of the AcGFP1-SURF4 and APV-SBP-DsRed2-ER-5 condensates in RUSH by adding 3% 1,6-hexanediol at time 0. Arrows mark the gradually dissolved condensates. 80 μM biotin was added at −30 min. See also Video S8. Scale bars: 2 μm (A,B,D,G), 200 nm (zoom-ins of B), 1 μm (E). See also .

Techniques: Immunolabeling, Expressing, Labeling, Two Tailed Test, Sequencing, Fluorescence

(A) DsRed2-loaded (arrow) and non-loaded (arrowhead) ERESs in a SURF4 siRNA-treated cell. Sec31A is shown in 3D-STORM, and epifluorescence of EGFP-Sec23A and APV-DsRed2-ER-5 are shown in gray and green, respectively. Note that with SURF4 knockdown, the APV-DsRed2-ER-5 cargo no longer clustered strongly at the ERES, so that DsRed2-loaded and non-loaded ERESs were subjectively assigned based on enhanced contrast of the epifluorescence image. (B) Statistics of the sizes of DsRed2-loaded and non-loaded, Sec23A-positive ERESs, based on the STORM-determined sizes of the Sec31A clusters, in control and SURF4 siRNA-treated cells. Whiskers and boxes show 10%, 25%, 50%, 75%, and 90% quantiles. P values are calculated by the two-tailed t test. n = 4 STORM images were quantified. (C,D) 3D-STORM of immunolabeled APV-SBP-DsRed2-ER-5 (C) or AcGFP1-SURF4 (D), in comparison with epifluorescence images of AcGFP1-SURF4 (C) or APV-SBP-DsRed2-ER-5 (D) of the same views in a RUSH experiment. For cargo release, 80 μM biotin was added 40 min before sample fixation. Arrows indicate SURF4 and DsRed2 condensates. Vertical cross-sections of the STORM images are given for the condensates indicated by the magenta arrows, showing membrane localizations for both proteins. Colors in the 3D-STORM images encode axial positions (depth).

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) DsRed2-loaded (arrow) and non-loaded (arrowhead) ERESs in a SURF4 siRNA-treated cell. Sec31A is shown in 3D-STORM, and epifluorescence of EGFP-Sec23A and APV-DsRed2-ER-5 are shown in gray and green, respectively. Note that with SURF4 knockdown, the APV-DsRed2-ER-5 cargo no longer clustered strongly at the ERES, so that DsRed2-loaded and non-loaded ERESs were subjectively assigned based on enhanced contrast of the epifluorescence image. (B) Statistics of the sizes of DsRed2-loaded and non-loaded, Sec23A-positive ERESs, based on the STORM-determined sizes of the Sec31A clusters, in control and SURF4 siRNA-treated cells. Whiskers and boxes show 10%, 25%, 50%, 75%, and 90% quantiles. P values are calculated by the two-tailed t test. n = 4 STORM images were quantified. (C,D) 3D-STORM of immunolabeled APV-SBP-DsRed2-ER-5 (C) or AcGFP1-SURF4 (D), in comparison with epifluorescence images of AcGFP1-SURF4 (C) or APV-SBP-DsRed2-ER-5 (D) of the same views in a RUSH experiment. For cargo release, 80 μM biotin was added 40 min before sample fixation. Arrows indicate SURF4 and DsRed2 condensates. Vertical cross-sections of the STORM images are given for the condensates indicated by the magenta arrows, showing membrane localizations for both proteins. Colors in the 3D-STORM images encode axial positions (depth).

Techniques: Two Tailed Test, Immunolabeling

$(A) Representative fluorescence micrograph of APV-Dendra2-ER-5 in COS-7 cells. (B) Representative fluorescence micrograph of APV-SBP-Dendra2-ER-5 in RUSH. 80 μM biotin was added at 0 min. (C) Representative fluorescence micrographs of APV-DsRed2-mEmerald-ER-5 and APV-mEmerald-DsRed2-ER-5 in COS-7 cells. (D) Table summarizing the C-terminal linker lengths and the fractions of t-ERGIC-predominant cells for different APV-FP-ER-5 constructs. See Methods for detail. (E,F) Subcellular distributions (E) and flow cytometry histograms (F) of APV-(FLAG-)DsRed2-ER-5 and its C-terminal inserted derivatives. The “FLAG (N-ter)” data in (E) duplicates “APV” in . (G) Co-immunoprecipitation of APV-FLAG-DsRed2-ER-5, APV-DsRed2-ER-5-FLAG, and EPV-FLAG-DsRed2-ER-5 with KDELR3-HA. (H) Dual-color live-cell fluorescence micrographs of APV-DsRed2-ER-5-HA and EGFP-Rab1A in a co-transfected cell. (I-K) Subcellular distribution (I), flow cytometry histograms (J), and lysate immunoblots (K) of APV-mOrange2-ER-5 in COS-7 cells with or without the co-expression of FLAG-SURF4. Scale bars: 5 μm. Error bars: SEM (n = 3 with ~50 cells in each replicate). Arrows indicate t-ERGICs. See also and .$

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) Representative fluorescence micrograph of APV-Dendra2-ER-5 in COS-7 cells. (B) Representative fluorescence micrograph of APV-SBP-Dendra2-ER-5 in RUSH. 80 μM biotin was added at 0 min. (C) Representative fluorescence micrographs of APV-DsRed2-mEmerald-ER-5 and APV-mEmerald-DsRed2-ER-5 in COS-7 cells. (D) Table summarizing the C-terminal linker lengths and the fractions of t-ERGIC-predominant cells for different APV-FP-ER-5 constructs. See Methods for detail. (E,F) Subcellular distributions (E) and flow cytometry histograms (F) of APV-(FLAG-)DsRed2-ER-5 and its C-terminal inserted derivatives. The “FLAG (N-ter)” data in (E) duplicates “APV” in . (G) Co-immunoprecipitation of APV-FLAG-DsRed2-ER-5, APV-DsRed2-ER-5-FLAG, and EPV-FLAG-DsRed2-ER-5 with KDELR3-HA. (H) Dual-color live-cell fluorescence micrographs of APV-DsRed2-ER-5-HA and EGFP-Rab1A in a co-transfected cell. (I-K) Subcellular distribution (I), flow cytometry histograms (J), and lysate immunoblots (K) of APV-mOrange2-ER-5 in COS-7 cells with or without the co-expression of FLAG-SURF4. Scale bars: 5 μm. Error bars: SEM (n = 3 with ~50 cells in each replicate). Arrows indicate t-ERGICs. See also and .

Techniques: Fluorescence, Construct, Flow Cytometry, Immunoprecipitation, Transfection, Western Blot, Expressing

(A) Representative fluorescence micrographs of APV/EPV-Dendra2-ER-5, APV/EPV-mOrange2-ER-5, APV/EPV-mCherry-ER-5, APV/EPV-EGFP-ER-5, and APV/EPV-mEmerald-ER-5 in COS-7 cells. (B) Subcellular distributions of different APV/EPV-FP-ER-5 constructs. The APV-DsRed2 and EPV-DsRed2 data duplicate that of “ER-5” in and that of , respectively. The APV-mOrange2 data duplicates that in . Error bars: SEM (n = 3 with ~50 cells in each replicate). (C) Sequences and flow cytometry of APV-DsRed2-mEmerald-ER-5 and APV-mEmerald-DsRed2-ER-5. (D,E) RUSH image sequences of APV-SBP-mCherry-ER-5 (D) and APV-SBP-EGFP-ER-5 (E) showing efficient ER exit and the formation of t-ERGIC. Arrows indicate t-ERGICs. Scale bars: 5 μm.

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) Representative fluorescence micrographs of APV/EPV-Dendra2-ER-5, APV/EPV-mOrange2-ER-5, APV/EPV-mCherry-ER-5, APV/EPV-EGFP-ER-5, and APV/EPV-mEmerald-ER-5 in COS-7 cells. (B) Subcellular distributions of different APV/EPV-FP-ER-5 constructs. The APV-DsRed2 and EPV-DsRed2 data duplicate that of “ER-5” in and that of , respectively. The APV-mOrange2 data duplicates that in . Error bars: SEM (n = 3 with ~50 cells in each replicate). (C) Sequences and flow cytometry of APV-DsRed2-mEmerald-ER-5 and APV-mEmerald-DsRed2-ER-5. (D,E) RUSH image sequences of APV-SBP-mCherry-ER-5 (D) and APV-SBP-EGFP-ER-5 (E) showing efficient ER exit and the formation of t-ERGIC. Arrows indicate t-ERGICs. Scale bars: 5 μm.

Techniques: Fluorescence, Construct, Flow Cytometry

(A) Schematics of different C-terminal insertions of APV-DsRed2-ER-5. (B) Representative fluorescence micrographs of APV-FLAG-DsRed2-ER-5, APV-DsRed2-ER-5-FLAG, APV-DsRed2-ER-5-HA, and APV-DsRed2-ER-5-C18 in COS-7 cells. (C) Representative fluorescence micrograph of APV-EGFP(1-228)-ER-5 in COS-7 cells, showing t-ERGICs. (D,E) Subcellular distributions (D) and immunoblots (E) of APV-EGFP(1-238)-ER-5 and the C-terminally truncated APV-EGFP(1-228)-ER-5. Error bar: SEM (n = 3 with ~50 cells in each replicate). The APV-EGFP(1-238)-ER-5 subcellular distribution in (D) duplicates “APV-EGFP-ER-5” in . (F) Dual-color live-cell fluorescence micrographs of EGFP-Rab1A and EPV-DsRed2-ER-5-HA in a co-transfected COS-7 cell. (G) Immunofluorescence of endogenous calumenin vs. fluorescence micrograph of EGFP-Rab1A in a COS-7 cell. Scale bars: 5 μm. Arrows point to t-ERGICs.

Journal: bioRxiv

Article Title: Tubular ERGIC (t-ERGIC): a SURF4-mediated expressway for ER-to-Golgi transport

doi: 10.1101/2021.04.06.438517

Figure Lengend Snippet: (A) Schematics of different C-terminal insertions of APV-DsRed2-ER-5. (B) Representative fluorescence micrographs of APV-FLAG-DsRed2-ER-5, APV-DsRed2-ER-5-FLAG, APV-DsRed2-ER-5-HA, and APV-DsRed2-ER-5-C18 in COS-7 cells. (C) Representative fluorescence micrograph of APV-EGFP(1-228)-ER-5 in COS-7 cells, showing t-ERGICs. (D,E) Subcellular distributions (D) and immunoblots (E) of APV-EGFP(1-238)-ER-5 and the C-terminally truncated APV-EGFP(1-228)-ER-5. Error bar: SEM (n = 3 with ~50 cells in each replicate). The APV-EGFP(1-238)-ER-5 subcellular distribution in (D) duplicates “APV-EGFP-ER-5” in . (F) Dual-color live-cell fluorescence micrographs of EGFP-Rab1A and EPV-DsRed2-ER-5-HA in a co-transfected COS-7 cell. (G) Immunofluorescence of endogenous calumenin vs. fluorescence micrograph of EGFP-Rab1A in a COS-7 cell. Scale bars: 5 μm. Arrows point to t-ERGICs.

Techniques: Fluorescence, Western Blot, Transfection, Immunofluorescence

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