Research ArticleCELL BIOLOGY

The structure and global distribution of the endoplasmic reticulum network are actively regulated by lysosomes

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Science Advances  16 Dec 2020:
Vol. 6, no. 51, eabc7209
DOI: 10.1126/sciadv.abc7209
  • Fig. 1 Dynamic ER tubules are coupled to motile lysosomes.

    (A) (i) SIM images of a COS-7 cell with EGFP-VAPA–labeled ER (magenta) and SiR dye-labeled lysosomes (green). (ii) SPT of lysosome motion over 12.5 min at 1.5 s per frame, color-coded by track index. (iii) Track density map extracted from SPT data (see movie S1). (B) Coupled motion (white arrows) of the growing tip of a newly formed ER tubule and associated lysosome. The tip forms three-way junctions (yellow stars). See movie S3. (C) An ER tubule, without an associated lysosome, is unstable and retracts (yellow arrows) after a period of elongation (white arrows). See movie S4. (D) Violin plot of ER tubule growth, in a 90-s interval, for growing tips with or without lysosomes attached. (E) Violin plot of maximum duration of the elongation phases of newly formed lysosome-coupled or lysosome-free ER tubules. (F) Sum of the total tubule growth lengths for lysosome-coupled or lysosome-free growing ER tips. Light green, tubules making network connections; red, tubules failing to connect. ****P < 0.0001, Student’s t test in (D) and (E). Scale bars, 2 μm (A to C). For (D) to (F), N = 481 events in 39 cells; see table S1.

  • Fig. 2 Remodeling of peripheral ER networks requires contacts with motile lysosomes.

    (A) Time-lapse images showing breakage of the connection between the growing tip of a newly formed ER tubule and a lysosome in EGFP-VAPA (KD/MD)–expressing cells. The ER is visualized via EGFP-VAPA (KD/MD), shown in magenta, and lysosomes (green color) are labeled by SiR-Lysosome. See table S2 and movie S5. (B) Average velocities of initially ER-tethered lysosomes that become detached (red arrow) from associated growing ER tips. An example of detachment event is shown in (A). N = 22 events, see table S3. (C) Left: Diagram depicting the regions defined as perinuclear and peripheral regions for the following quantification of lysosome distribution. Right: Violin plots of SiR-Lysosome fluorescent intensity in peripheral versus perinuclear region and percentage of the ER comprising tubules upon knockdown of lysosome motion adaptors. N = 20 cells, see table S4. (D) Representative images showing the distribution of lysosomes and ER tubules in untreated cells and in siRNA-treated cells. The ER is visualized via EGFP-VAPA, shown in magenta, and lysosomes (green color) are labeled by SiR-Lysosome. **P < 0.01 and ****P < 0.0001, Tukey’s one-way analysis of variance (ANOVA) in (B) and (C). Scale bars, 1 μm (A) and 5 μm (D).

  • Fig. 3 Manipulation of lysosome positionings drives the redistribution of ER via reshaping.

    (A) Molecular components of the chemogenetic system. (B) Violin plots of SiR-Lysosome fluorescent intensity and percentage of the ER comprising tubules after 1 hour of vehicle or inducer treatment. N = 10 cells (lysosome intensity) and 20 cells (ER tubule). See table S5. ****P < 0.0001, Student’s t test. (C) Representative images showing the distribution of lysosomes and ER tubules in control and induced cells. The ER is visualized via EGFP-VAPA, shown in magenta, and lysosomes (green color) are labeled by SiR-Lysosome. (D) Molecular components of the optogenetic system for repositioning LAMP1-associated lysosomes. (E to H) Live-cell imaging (E), representative zoom-ins (F), representative kymograph (G), and quantification (H) of LAMP1-mCherry-iLID and YFP-Sec61β in COS-7 cells expressing opto-kinesin before or during activation. White arrows indicate lysosomes pulling ER tubules (yellow asterisks). Quantification shows mean (±SEM) normalized peripheral Sec61β and LAMP1 intensity of eight cells. Blue box indicates illumination with blue light. Scale bars, 5 μm (C) and 1 μm (E to G).

  • Fig. 4 Lysosome-mediated ER reshaping is responsive to nutritional status.

    (A) Schematic representation of the nutritional regulation of lysosome positioning. (B) Representative SIM images showing the distribution of lysosomes (green) and ER (magenta) upon disruption of metabolic pathways, same for (D). From top to bottom: no treatment control; 4-hour serum starvation (SS; same in the following text); 24-hour serum starvation (prolonged starvation), see movie S7; siRNA against Arl8b or SKIP in prolonged starvation. (C) Violin plots of SiR-Lysosome fluorescent intensity and percentage of the ER comprising tubules with indicated treatments. (D) From top to bottom: 3-hour U18666A (10 μM) treatment to block lysosome-to-ER cholesterol transfer; 3-hour MβCD (100 μM) treatment to perturb cholesterol sensing; siRNA against Arl8b or SKIP in 3-hour MβCD (100 μM) treatment. (E) Violin plots of SiR-Lysosome fluorescent intensity and percentage of the ER comprising tubules with indicated treatments. ****P < 0.0001, Tukey’s one-way ANOVA. n ≥ 20 cells per condition from three independent experiments, same for (C). See table S6. Scale bars, 5 μm. ns, not significant.

  • Fig. 5 Disruption of ER-lysosome contacts impairs ER continuity in growing RGC axons.

    (A) Experimental protocol for Xenopus eye-targeted electroporation, RGC culture, and imaging. (B) Cotransport of a lysosome (green) and an ER tubule tip (magenta) in a representative EGFP-VAPA–expressing axon segment, see movie S8. (C) A representative EGFP-VAPA (KD/MD)–expressing axon fails to join ends of separate ER tubules, see movie S9. (D) Percentage of separate axonal ER tubules successfully connected within 90 s. n = 113 pairs of separate ER tubules. See table S7. (E) Average ER fragment lengths and (F) average axon lengths in EGFP-VAPA (KD/MD)–expressing (n = 152) and EGFP-VAPA–expressing (n = 118) axons. See table S7. (G) Correlation between axon and ER fragment lengths in indicated axons with discontinued ER only. Linear regression analyses are shown by solid lines, and 95% confidence intervals fall between dashed lines. (H) Representative images of cultured EGFP-VAPA (KD/MD)–expressing and EGFP-VAPA–expressing axons from single explants. Yellow dashed lines indicate where axons exit the eye (starting points of length quantification). Mean ± SEM. ****P < 0.0001, Student’s t test. Scale bars, 5 μm (B, left), 1 μm (B, middle), 200 nm (B, right), 1 μm (C), and 20 μm (H).

  • Table 1 Plasmids.

    pEGFPC1-hVAP-AGift from
    C. Tomasetto
    Addgene plasmid
    #104447
    pEGFPC1-hVAP-A KD/MDGift from M. DavidsonAddgene plasmid
    #104449
    mEmerald-Rab5a-7Gift from M. DavidsonAddgene plasmid
    #54243
    EGFP-Rab7AGift from Q. ZhongAddgene plasmid
    #28047
    mEmerald-Sec61β-C1Gift from
    J. Lippincott-Schwartz
    Addgene plasmid
    #90992
    mApple-Sec61β-C1Gift from
    J. Lippincott-Schwartz
    Addgene plasmid
    #90993
    ER-GFPCellLight ER-GFP,
    BacMam 2.0
    Cat#C10590
    Lysosomes-GFPCellLight Lysosomes-
    GFP, BacMam 2.0
    Cat#C10507
    Early endosome-GFPCellLight Early
    Endosome-GFP,
    BacMam 2.0
    Cat#C10586
    pB80-KIF1A(1-365)-
    VVD fast-FLAG-
    SSPB(micro)
    Gift from L.K.(15)
    pB80-LAMP1-mCherry-
    iLID
    Gift from L.K.(15)
    β-actin YFP-Sec61Gift from L.K.(15)
    pBa-flag-BicD2
    594-FKBP
    Gift from G. BankerAddgene plasmid
    #64206
  • Table 2 Antibodies and chemicals.

    SiR-LysosomeCytoskeleton Inc.Cat#CY-SC012
    Bafilomycin A1Sigma-AldrichCat#19-148
    NocodazoleSigma-AldrichCat#M1404-10MG
    Dulbecco’s modified Eagle’s mediumSigma-AldrichCat#D6546
    A/C heterodimerizerTakaraCat#635056
    Fetal bovine serumSigma-AldrichCat#10270-106
    Radioimmunoprecipitation assay bufferSigma-AldrichCat#R0278
    Opti-MEM I Reduced Serum MediumInvitrogenCat#31985062
    Anti-VAPAAbcamCat#ab181067
    Anti-GAPDHSigma-AldrichCat#G8795
    Anti-tubulinAbcamCat#ab131205
    Anti-PLEKHME (SKIP)AbcamCat#ab91581
    Anti-Arl8bProteintechCat#13049-1-AP
    Methyl-β-cyclodextrinSigma-AldrichCat#332615
    U18666ASigma-AldrichCat#U3633
  • Table 3 Critical commercial assays.

    QIAprep Spin Miniprep
    Kit
    QIAGENCat#27104
    Lipofectamine 2000
    Transfection
    Reagent
    InvitrogenCat#11668030
    Lipofectamine
    RNAiMAX
    Transfection
    Reagent
    InvitrogenCat#13778030
    SuperSignal West Pico
    PLUS
    Chemiluminescent
    Substrate
    Thermo Fisher
    Scientific
    Cat#34580
  • Table 4 Experimental models.

    Cercopithecus aethiops:
    COS-7
    ATCCCat#CRL-1651
    XenopusGurdon Institute,
    Cambridge
    Not available
  • Table 5 Oligonucleotides.

    siVAPASMARTpool: ON-TARGETplusCat#L-021382-00-0005
    MISSION siRNA Universal negative controlSigma-AldrichCat#SIC001
    siPLEKHM2 (SKIP)SMARTpool: ON-TARGETplusCat#L-022168-01-0005
    siArl8bSMARTpool: ON-TARGETplusCat#L-020294-01-0005
  • Table 6 Software.

    FijiFijihttps://imagej.net/Fiji
    MATLAB 2016aMathWorkshttps://uk.mathworks.com/products/matlab.html
    GraphPad PrismGraphPad Softwarehttps://graphpad.com/scientific-software/prism/

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