EMBO/soda-nel huggingface dataset:
An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome (2023)
Figure 10

Fig 10 The SCAR-WAVE-DCP1 nexus at the vertex can modulate growth anisotropy.

A Representative images showing the phenotypes of dcp1 mutants and mutants in other PB core components or SCAR-WAVE components (5-day-old seedlings). The arrowheads show the growth defects of homozygous dcp1-1 or dcp2-1 mutants (denoted -/-; heterozygous denoted dcp1-1/DCP1; details are also shown). Lower: graph showing relative root length (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 3-4 roots, bars show means + s.d).

B DCP1 regulates cell expansion anisotropy. Representative confocal micrographs showing FM4-64 staining of the WT, dcp1-1 and dcp2-1 mutants (2 μM, 10 min). Scale bars, 20 μm. Right: percentage of isotropic cells per root meristem (%, epidermal cells) in each genotype (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 3-5 roots, bars show means ± s.d). Examples of isotropic or anisotropic cells are shown, along with the developmental axis offset at the x- and y-axes.

Data information: In A, and B, P values were determined by ordinary one-way ANOVA (Kruskal-Wallis produced similar results, with Dunn's or FDR corrections).

Expanded View Figure legends
The SARS-unique domain (SUD) of SARS-CoV and SARS-CoV-2 interacts with human Paip1 to enhance viral RNA translation (2021)
Figure 1

Figure 1. SUD interacts with Paip1

(A) Genome organization of SARS-CoV and schematic illustration of Nsp3 domains. Ubl1: ubiquitin-like domain 1; Mac: macrodomain; DPUP: Domain Preceding Ubl2 and PLpro; PLpro: papain-like protease; NAB: nucleic-acid binding domain.

(B) SUD binding to Paip1 in HEK-293 cells as demonstrated by the split-YFP assay.

(C) Schematic presentation of SUD, Paip1, and PABP. Interactions between SUD and Paip1, as well as PAPB and Paip1 are indicated by red and black lines, respectively. PAM: PABP-binding motif; Paip1M: the middle domain of Paip1; RRM: RNA-recognition motif; PABC: the C-terminal domain of PABP.

(D) Paip1M alone does interact with SUD.

(E) SUD binds to Paip1 and Paip1M with the same binding intensity. n. s.: not significant.

(F) The Mac2 subdomain is crucial for interacting with Paip1M.

(G) Paip1M and SUD interaction was confirmed in vitro by a gel-filtration assay.

Scale bars represent 50 µM.
The SARS-unique domain (SUD) of SARS-CoV and SARS-CoV-2 interacts with human Paip1 to enhance viral RNA translation (2021)
Figure 3

Figure 3. The N-terminal 16 residues of Mac2 are important for Paip1M binding and Mac1 does not affect Mac2:Paip1 interaction

(A) The Mac2 and Paip1M interaction was impaired by removing the N-terminal 16 residues as revealed by the gel-filtration assay.

(B) Isothermal Titration Calorimetry (ITC) assay of Mac2 binding Paip1M. The raw calorimetric curve is displayed on top while the fitted binding isotherm curve is at the bottom. The measured values are: N = 0.94 ± 0.01 (binding stoichiometry); Kd = 15.5 ± 1.5 μM (dissociation constant); ΔH = -6778 ± 149 cal/mol (standard molar enthalpy change for binding); ΔS = -0.716 cal/mol/K (standard molar entropy change).

(C) Δ16-Mac2 lost all binding affinity to Paip1M in the ITC experiment.

(D) ITC result of Mac1−2 interacting with Paip1M. The experimental results are: N = 1.14 ± 0.02; Kd = 18.6 ± 2.5 μM; ΔH = -3730 ± 100 cal/mol; ΔS = 9.14 cal/mol/K.

(E, F) Δ16-Mac2 cannot bind Paip1M, Mac1−2 binds to Paip1M in the split-YFP assay. Scale bars represent 50 µM.
The SARS-unique domain (SUD) of SARS-CoV and SARS-CoV-2 interacts with human Paip1 to enhance viral RNA translation (2021)
Figure 4

Figure 4. SUD, Paip1, and PABP form a ternary complex

(A, B) SUD and PABP do not compete with each other for interacting with Paip1. (A) Excess expression of SUD-RFP does not impair the split-YFP signal between PABP and Paip1 (lower images), compared to the control (upper images). (B) Co-expression of excess RFP-PABP does not result in an impaired interaction between Paip1 and SUD either. RFP: red fluorescent protein. Scale bars represent 50 µM.

(C) GFP-trap-based co-immunoprecipitation (CoIP) assay demonstrating a complex formed by PABP, Paip1, and SUD. GFP: green fluorescent protein.
The SARS-unique domain (SUD) of SARS-CoV and SARS-CoV-2 interacts with human Paip1 to enhance viral RNA translation (2021)
Figure 6

Figure 6. The corresponding SUD:Paip1 interaction exists in SARS-CoV-2

(A) Sequence alignment of SUD between SARS-CoV (Genbank: AY291315) and SARS-CoV-2 (GenBank: MN908947). Domains Mac2 (SUD-N), Mac3 (SUD-M) and DPUP (SUD-C) range from K389 to L526, G527 to S652 and K653 to S720, respectively. The first 16 residues of Mac2 are boxed in blue. This figure was generated by using the ESPript server (http://espript.ibcp.fr/ESPript/ESPript/).

(B) SARS-CoV-2 SUD (SARS2-SUD) interacts with Paip1 in HEK-293 cells as demonstrated by the split-YFP assay.

(C) SARS2-Mac2 binds to Paip1. SARS2-Mac2 but not Δ16-SARS2-Mac2 interacts with Paip1M.

(D) MERS-CoV SUD-MC (Mac3−DPUP) or XMC (Mac1−Mac3−DPUP) do not bind to Paip1. Scale bars represent 50 µM.
cGAS facilitates sensing of extracellular cyclic dinucleotides to
activate innate immunity (2019)
Figure 1

Figure 1. eCDNs trigger innate immune responses.

A qRT-PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in different cell types. Cells were stimulated with ecGAMP (5 μg/ml) for 4 h.

B qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with different eCDNs (5 μg/ml) for 4 h.

C, D ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular 2'3'-cGAMP (C) or c-di-AMP (D) at indicated concentrations.

E qRT-PCR detection of IFNB1 mRNA in THP-1 cells stimulated with indicated eCDNs (5 μg/ml) for 4 h.

F ELISA detection of IFNβ in supernatants of THP-1 cells stimulated with indicated eCDNs at indicated concentrations for 4 h.

G qRT-PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in human CD14+ monocytes derived from PBMC stimulated with indicated eCDNs (5 μg/ml) for 4 h and 8 h. Each symbol represents one individual donor. Data are means+SD averaged from 10 healthy donors.

H ELISA detection of IFNβ in supernatants of human CD14+ monocytes derived from PBMC stimulated with indicated eCDNs (5 μg/ml) for 4 h. Each symbol represents result from one individual donor. Data are means+SD averaged from 10 healthy donors.

Data in (A-F) are means+SD averaged from at least 2 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. One-way ANOVA (B, E) and Two-way ANOVA (C, D, F) were used for statistical analysis, respectively. **, p<0.01; ***, p<0.001.
cGAS facilitates sensing of extracellular cyclic dinucleotides to
activate innate immunity (2019)
Figure 2

Figure 2. eCDNs are less potent than iCDNs in inducing innate immune responses.

A,B qRT-PCR detection of IFNB1 mRNA abundance in THP-1 cells treated with ecGAMP and icGAMP (A) or ec-di-AMP and ic-di-AMP (B) at indicated concentrations for 4 h.

C,D ELISA detection of IFNβ release from THP-1 cells stimulated with ecGAMP and icGAMP (C) or ec-di-AMP and ic-di-AMP (D) at indicated concentrations for 4 h.

E,F qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ecGAMP and icGAMP (E) or ec-di-AMP and ic-di-AMP (F) at indicated concentrations for 4 h.

G,H ELISA detection of IFNβ release from mBMDMs stimulated with ecGAMP and icGAMP (G) or ec-di-AMP and ic-di-AMP (H) at indicated concentrations for 4 h.

Data are means+SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001; ns, not significant.
cGAS facilitates sensing of extracellular cyclic dinucleotides to
activate innate immunity (2019)
Figure 3

Figure 3. eCDNs require endocytosis to activate type I IFN.

A Frequencies of FITC+ THP-1 cells stimulated with FITC-ecGAMP for 1 h in presence of DMSO or indicated inhibitors including Dynasore (10 μM), chlorpromazine (CPZ, 10 μM), dimethylamiloride (DMA, 100 μM), polyinosinic acid (Poly I, 50 μg/ml) or mannans from Sacharomyces cerevesiae (Mannans, 1 mg/ml).

B, C qRT-PCR detection of IFNB1 (B) and IL6 (C) mRNA in THP-1 cells stimulated with ecGAMP (5 μg/ml) or intracellular 2′3′-cGAMP (icGAMP) (0.1 μg/ml) for 4 h in presence of DMSO or dynasore (10 μM).

D, E qRT-PCR detection of Ifnb1 (D) and Il6 (E) mRNA in mBMDMs stimulated with ecGAMP (5 μg/ml) or icGAMP (0.1 μg/ml) for 4 h in presence of DMSO or dynasore (10 μM).

F, G Immunostaining for EEA1 (red) (F) and LAMP2 (red) (G) in THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 30 min, nucleus in blue (DAPI). Data are representative of 3 independent experiments. Scale bar, 10 μm.

H-K qRT-PCR detection of IFNB1 and IL6 mRNA in THP-1 cells (H, I) and mBMDMs (J, K) stimulated with ecGAMP (5 μg/ml) and icGAMP (0.1 μg/ml), respectively, for 4 h in presence of DMSO or bafilomycin A1 (BafA) (1 μM).

Data are means+SD (A-E, H-K) averaged from 3 independent experiments performed in technical triplicates and each symbol represents mean of technical triplicates. One-way ANOVA followed by Dunnett's post hoc test (A) or Two-way ANOVA followed by Tukey's post hoc test (B-E, H-K) were used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001; ns, not significant.
cGAS facilitates sensing of extracellular cyclic dinucleotides to
activate innate immunity (2019)
Figure 4

Figure 4. STING is important but not sufficient for eCDNs-induced type I IFN response.

A qRT-PCR detection of IFNB1 mRNA in scrambled (Scramble) or STING shRNA stably transfected (STING KD) THP-1 cells stimulated with ec-di-AMP (5 μg/ml) and ecGAMP (5 μg/ml) or transfected with ISD or poly(I:C).

B ELISA detection of IFNβ protein in supernatants of Scramble or STING KD THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C).

C qRT-PCR detection of Ifnb1 mRNA in WT and Sting-/- (Sting KO) mBMDMs stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD.

D ELISA detection of IFNβ protein secretion in supernatants of mBMDMs stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly (I:C).

E qRT-PCR detection of Ifnb1 mRNA in WT, Sting KO or Sting KO complemented with mouse STING (Sting KO+mSTING) RAW264.7 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD.

F Western blot detection of indicated proteins in lysates of WT and Sting-/- (KO) mBMDMs stimulated with ecGAMP (5 μg/ml) or transfected with ISD. Data are representative of 3 independent experiments.

G qRT-PCR detection of IFNB1 mRNA levels in STING stable HEK293T cells (HA-STING-HEK293T) stimulated with ecGAMP or icGAMP at 5 μg/ml for indicated times.

H Western blot detection of indicated proteins in HA-STING-HEK293T cells stimulated with increasing amounts of ecGAMP and icGAMP at 5 μg/ml for 24 h. Data are representative of 3 independent experiments.

I qRT-PCR detection of the induction of IFNB1 mRNA in HEK293T cells stably transfected with pcDNA3.1-HA (HA), HA-STING and HA-STING+HA-cGAS stimulated with ecGAMP (5 μg/ml), icGAMP (0.1 μg/ml) or transfected with ISD.

Data in (A-E, G, I) are means+SD averaged from at least 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001; ns, not significant.
cGAS facilitates sensing of extracellular cyclic dinucleotides to
activate innate immunity (2019)
Figure 5

Figure 5. cGAS facilitates eCDN detection in macrophages.

A qRT-PCR detection of IFNB1 mRNA in WT and CGAS KO THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly (I:C) for 4 h.

B ELISA detection of IFNβ in supernatants of WT and CGAS KO THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.

C qRT-PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas-/- (Cgas KO) mice stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.

D ELISA detection of Ifnb1 in supernatants of mBMDMs from WT and Cgas KO mice stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.

E qRT-PCR detection of IFNB1 mRNA in WT and CGAS KO THP-1 cells stimulated with ecGAMP (5 μg/ml) or icGMAP (0.1 μg/ml) or transfected with ISD.

F qRT-PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas KO stimulated with ecGAMP (5 μg/ml) or icGMAP (0.1 μg/ml) or transfected with ISD.

G Western blot detection of indicated proteins in lysates of mBMDMs from WT and Cgas KO mice stimulated with ecGAMP (5 μg/ml), icGAMP (0.1 μg/ml) or transfected with ISD. Data are representative of 3 independent experiments.

H, I qRT-PCR detection of IFNB1 mRNA (H) and ELISA detection of IFNβ in supernatants (I) of WT, CGAS KO, CGAS KO complemented with cGAS (CGAS KO+cGAS) or CGAS KO stably transfected with the empty vector (CGAS KO+Vector) THP-1 cells stimulated with ecGAMP (5 μg/ml).

Data are means+SD (A-F, H-I) averaged from at least 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001; ns, not significant.
cGAS facilitates sensing of extracellular cyclic dinucleotides to
activate innate immunity (2019)
Figure 6

Figure 6. CDNs bind cGAS directly leading to its dimerization.

A Purified h-cGAS protein was precipitated with Ctrl beads or cGAMP beads and then immunoblotted.

B THP-1 cells were stimulated with biotin-cGAMP for indicated times and then whole cell lysates (WCL) were precipitated with streptavidin beads followed by immunoblotting.

C Immunofluorescent staining of cGAS (red) in THP-1 cells treated with FITC-ecGAMP (5 μg/ml) (green) or FITC-icGAMP (0.1 μg/ml) (green) for 4 h, nucleus in blue (DAPI). Scale bar, 10 μm.

D Purified h-cGAS protein was precipitated with beads coupled with cGAMP, c-di-GMP or c-di-AMP followed by immunoblotting.

E Binding curves of surface-immobilized 2'3'-cGAMP with His-cGAS at indicated concentrations. Vertical lines mark the start of association and dissociation phases of the binding events. The dashed lines are global fits to a Langmuir reaction model; global fitting parameters are listed in the table below the plot. (n=4 independent experiment).

F The small-angle X-ray scattering analysis of the full-length cGAS with cGAMP. The EOM fit of the measured SAXS data. The goodness-of-the fit χ2 = 1.1.

G Structural alignment of the representative structures from cGAS-cGAMP EOM analysis. cGAMP subunits are colored red and blue, respectively. Different conformations of the cGAS N-termini are highlighted in additional shades of red and blue.

H The Dmax distributions (the maximum distance within a particle) derived from the EOM analysis of the measured SAXS profile (pool - the white histogram, the selected structures - the black histogram). The distribution of the selected structures shows a bimodal behaviour with an average value of 166.9 Å.

Data (A-E) are representative of at least 3 independent experiments.
cGAS facilitates sensing of extracellular cyclic dinucleotides to
activate innate immunity (2019)
Figure 7

Figure 7. CDNs promote formation of cGAS/STING complex.

A Immunostaining of STING (anti-STING, red) in THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are representative of 3 independent experiments. Scale bar, 10 μm.

B Cellular localization of ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are presentative of 5 independent experiments. Scale bar, 10 μm.

C Frequency of perinuclear accumulation of FITC-ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml) for 2 h. Data are means+SD averaged from 5 independent experiments and approximately 100 cells were imaged and counted in each experiment. Each symbol represents the percentage of THP-1 cells with perinuclear cGAMP aggregates in every independent experiment. Mann-Whitney U test was used for statistical analysis. **p<0.01.

D Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of cGAMP agarose. Purified GST-cGAS and His-STING were incubated separately or together with cGAMP agarose. The input and immunoprecipitates were immunoblotted.

E Western blot detection of the presence of His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in IP lysis buffer overnight at 4°C.

F Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in the absence or presence of increasing 2′3′-cGAMP.

G Western blot detection of the presence of cGAS and STING in the immunoprecipitates of streptavidin beads. THP-1 cells were stimulated with biotin-ecGAMP (5 μg/ml) or biotin-icGAMP (1 μg/ml) for indicated time and cell lysates were harvested for IP with streptavidin beads. Data (A-B, D-G) are representative of 3 independent experiments.
cGAS facilitates sensing of extracellular cyclic dinucleotides to
activate innate immunity (2019)
Figure 8

Figure 8. eCDNs promote cGAS-mediated DNA sensing.

A, B qRT-PCR detection of Ifnb1 mRNA (A) or ELISA detection of IFNβ in supernatants (B) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001.

C Western blot detection of indicated proteins in lysates of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are representative of 3 independent experiments.

Expanded View Figure Legends
ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport (2018)
Figure 1

Figure 1. Reversible accumulation of polymeric ATZ within LAMP1-positive endolysosomes on acute lysosomal inhibition.

A Intracellular localization of total (HA) and polymeric ATZ (2C1) in WT MEF mock-treated, Confocal Laser Scanning Microscopy (CLSM).

B Same as A for MEF exposed to 50 nM BafA1 for 12 h.

C Same as A, 4 h after BafA1 wash-out.

D Quantification of ATZ-positive, LAMP1-positive endolysosomes (EL) (n=13, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.

E ATZ polymers immunoisolated from lysates of WT MEF mock treated (lane 1), incubated for 12 h with BafA1 (lane 2) and 4 h after BafA1 wash-out (lane 3). Immunoprecipitation (IP) of ATZ polymers with polymer-specific 2C1 antibody, transfer on PVDF membrane, revealed with anti-HA antibody on western blot (WB).

F Quantification of E, n=3, mean ± SEM. Unpaired two-tailed t-test, ns P>0.05, * P<0.05.

G-K Same as B in WT MEF, in cells exposed to 20 mM CST and in Cnx-, Crt- and ERp57-KO MEF.

L Quantification of ATZ-positive EL (n=10, 9, 11, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.

Data information: Scale bars: 10 μm.
ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport (2018)
Figure 2

Figure 2. Disposal of polymeric ATZ requires a functional LIR domain of FAM134B

A HEK293 cells transfected with empty vector (lanes 1, 6), ATZ-HA (2, 7), FAM134B-V5 (3 and 8), FAM134B-V5 and ATZ-HA (4, 9), or FAM134BLIR-V5 and ATZ-HA (5, 10), incubated for 12 h with 50 nM BafA1 and then treated with the cross-linker DSP before lysis as described in Methods. Lanes 1-5, WB of the total cell extract (TCE); lanes 6-10, WB of antiV5 immunoprecipites to isolate complexes containing ectopically expressed FAM134B or FAM134BLIR. The membranes were probed with antiV5 (upper panels), antiCNX, antiHA and antiLC3 antibodies.

B Quantification of LC3 co-precipitating with FAM134B-V5 (A, lanes 8 and 9). Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05.

C Same as Fig 1B, WT MEF. Scale bar: 10 μm.

D Ectopic expression of FAM134BLIR in WT MEF inhibits ATZ delivery to endolysosomes. Scale bar: 10 μm.

E, F Distribution of gold-labeled ATZ-HA by IEM in BafA1 treated WT MEF and WT MEF overexpressing FAM134BLIR, respectively. EV, ER-derived vesicles, red arrowheads; EL, endolysosome.

G Quantification of ATZ-gold density of E, F (n=75 and 79 EL, respectively). Unpaired two-tailed t-test, **** P <0.0001.

H Max projection of the same cell as in D after deconvolution. Insets show orthogonal section of select regions. Scale bar: 10 μm.

I Decay of ATZ polymers (CHX chase, upper panel) immunoisolated with the polymer-specific 2C1 antibody (visualized with antiHA in WB) in HEK293 cells mock-transfected (lanes 1-4) or expressing FAM134BLIR-V5 (lanes 5-8). Middle panel, expression of FAM134BLIR-V5 assessed by WB; lower panel, loading control.

(J) Quantification of (I) (Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05).

K Flow cytometry analysis of ATZ-HA polymer levels in MEFs mock treated, exposed to BafA1, and co-expressing FAM134LIR. MFI: mean fluorescence intensity (Mean ± SEM, n=5, unpaired two-tailed t-test, ns P>0.05, ** P<0.01, *** P <0.001).
ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport (2018)
Figure 4

Figure 4. Delivery of polymeric ATZ to endolysosomes in cells lacking LC3 lipidation or autophagosome biogenesis.

A-H Intracellular localization of LAMP1-positive EL, total (HA) and polymeric (2C1) ATZ in WT MEF exposed to BafA1 for 12 h (A); in Atg4BKO MEF (B); in Atg7KO MEF (C); in Fip200KO MEF (D); in Ulk1/2 double-KO MEF (E); in Atg13KO MEF (F); in Atg9KO MEF (G); in RubiconKO MEF (H). Scale bars: 10 μm.

I Quantifications of A-H (n=10, 9, 9, 10, 7, 15, 9, 12 cells). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.
ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport (2018)
Figure 5

Figure 5. Dissecting ATZ delivery to LAMP1-positive endolysosomes.

A Distribution of gold-labeled ATZ-HA by IEM in WT MEF exposed to BafA1 (the cell on the right is not transfected, as specificity control for gold labeling). ER, endoplasmic reticulum; EV, ER-derived vesicle; EL, endolysosomes. Scale bar: 1 μm.

B Inset of A. Red asterisks show site of EV:EL membranes fusion

C-E Distribution of gold-labeled ATZ-HA by IEM in WT MEF. ES, ER subdomains. Red asterisk shows site of EV:EL membranes fusion. Scale bar: 1 μm.

F 3D visualization by means of electron tomography of organelles containing gold labeled ATZ-HA (visualized as white dots, see MovieEV1). ES, ER subdomains. Scale bar: 500 nm.

G CLEM showing ATZ (red), LAMP1-positive EL (cyan) and GFP-derived fluorescence (green) in correspondence to heavily labeled vesicles, which were identified as autophagosomes for their double membrane (insets 1-3). GFP-LC3 was also stained by immunogold. Scale bar: 1 μm.