EMBO/soda-nel huggingface dataset:
Phosphorylation by casein kinase 2 enhances the interaction between ER-phagy receptor TEX264 and ATG8 proteins (2022)
Figure 1

Figure 1. Phosphorylation of Ser271 and Ser272 upstream of the TEX264 LIR is required for ATG8 binding and autophagosomal localization

(A) Alignment of the LIR motif of TEX264 homologs in vertebrates. Asterisks show the phosphorylation sites identified by mass spectrometry.

(B) HEK293T cells transiently expressing FLAG and FLAG-tagged TEX264 were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies.

(C) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.

(D and E) MEFs stably expressing TEX264-GFP or its mutant were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 1 µm (insets) (D). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 48 cells for each cell type (E).

(F and G) WT and TEX264-KO HeLa cells (with or without the indicated TEX264 mutants with a C-terminal FLAG tag) stably expressing the ER-phagy reporter (ss-RFP-GFP-KDEL) were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured in starvation medium lacking amino acids and serum for 9 h (F). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± standard error of the mean (SEM) of three independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (G).
Phosphorylation by casein kinase 2 enhances the interaction between ER-phagy receptor TEX264 and ATG8 proteins (2022)
Figure 2

Figure 2. Phosphorylated TEX264 localizes to autophagosomes

(A and B) MEFs stably expressing TEX264-GFP were cultured in starvation media with or without bafilomycin A1 (Baf A1) and immunostained with anti-pTEX264 and anti-LC3 antibodies. Bars: 10 µm and 1 µm (insets) (A). Quantification of the number of pTEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 35 cells for each cell type (B).

(C) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h, and cell homogenates were subjected to an OptiPrep membrane flotation analysis.

(D, E and F) WT, ATG3-KO, and FIP200-KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies (D). Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown (E and F). Data represent the mean ± SEM of three independent experiments.
Phosphorylation by casein kinase 2 enhances the interaction between ER-phagy receptor TEX264 and ATG8 proteins (2022)
Figure 3

Figure 3. CK2 is important for starvation-induced ER-phagy through TEX264 phosphorylation

(A) HeLa cells were cultured with 20 nM CK1 inhibitor (D4476) or CK2 inhibitor (CX4945) for 3 h, followed by starvation (with the inhibitors) for 3 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies.

(B) Recombinant TEX264-FLAG was incubated with or without CK2 and the CK2 inhibitor (CX4945) in the presence of 200 µM ATP for 1 h.

(C) HEK293T cells transiently expressing TEX264-FLAG and one of the three Halo-CK2A isoforms or Halo-CK2B were subjected to immunoprecipitation with an anti-FLAG antibody and detected with anti-Halo and anti-FLAG antibodies.

(D and E) HEK293T cells transiently expressing WT or TEX264-FLAG mutants were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies (D). The band intensities in the IP fractions were quantified, and the phospho-serine:FLAG ratio was calculated. Data represent the mean ± SEM of five independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (E).

(F and G) MEFs stably expressing TEX264-GFP were cultured with the CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) and immunostained with an anti-LC3 antibody. Scale bars represent 10 μm and 1 μm (insets) (F). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by an unpaired two-tailed Student's t-test. Data were collected from 85 cells for each cell type (G).

(H and I) WT HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) for 6 h (H). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of six independent experiments. Differences were statistically analyzed by an unpaired two-tailed Student's t-test (I).

(J) Model of the CK2-mediated phosphorylation of TEX264 in ER-phagy.
Phosphorylation by casein kinase 2 enhances the interaction between ER-phagy receptor TEX264 and ATG8 proteins (2022)
Figure 4

Figure 4. Phosphorylation of TEX264 LIR increases its affinity to ATG8s

(A) Binding affinity of TEX264 LIR peptides (TEX264269-278) to LC3 and GABARAP was measured by ITC (n = 1). The error bars show the fitting error.

(B) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.

(C and D) MEFs stably expressing TEX264-GFP or its mutants were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 5 µm (insets) (C). The intensity of the GFP signal under starvation conditions was quantified at TEX264-GFP puncta and the ER. The puncta:ER signal ratio was calculated. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 70 puncta for each cell type (D).

(E and F) WT and TEX264-KO (expressing WT or TEX264-FLAG mutants) HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, the cells were cultured in starvation medium lacking amino acids and serum for 9 h (E). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of four independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (F).
Mitochondria mediate septin cage assembly to promote autophagy of Shigella (2016)
Figure 1

Figure 1. SEPT7 cages inhibit bacterial replication.

A Model depicting the x-light system used for single bacterial analysis. S. flexneri M90T carry an inducible plasmid (x-light Shigella) engineered with the LacI repressor system from Escherichia coli, and a GFP or mCherry gene downstream of the operator. In the presence of IPTG, metabolically active bacteria stop LacI repressor activity. As a consequence the fluorescent protein is synthesised when bacteria are metabolically active.

B HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 4 independent experiments. Student’s t-test, *** = p<0.001.

C HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for p62. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG without (-) or with (+) p62 from at least 4 independent experiments. Student’s t-test, *** = p<0.001.

D Control (CTRL) or isopropanol-treated x-light Shigella GFP were induced with IPTG for 30 min, then labelled with SYTOX Orange for 10 min. The scale bar represents 5 μm.

E HeLa cells were infected for 1 h 30 min, treated with ethanol (CTRL) or erythromycin (EM) for 2 h prior to fixation, and then labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments. Student’s t-test, *** = p<0.001.
Mitochondria mediate septin cage assembly to promote autophagy of Shigella (2016)
Figure 2

Figure 2. Proteins enriched at the Shigella-septin cage.

A Schematic representation of the proteomic experiments used to isolate novel proteins enriched at the Shigella-septin cage. (a) A SEPT6-STREP-FLAG construct was (b) transfected in HeLa cells for 24h. (c) Cells were infected with S. flexneri AfaI for 4 h, harvested, and (d) SEPT6-STREP-FLAG cage-associated proteins isolated through co-immunoprecipitation (Co-IP).

B HeLa cells stably expressing SEPT6-GFP were infected with S. flexneri AfaI for 4 h, harvested, and then tested with Co-IP. Empty vector and/or uninfected HeLa cells were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for SEPT6, GFP, SEPT7, or p62. The lysates were immunoblotted for GAPDH as control for cellular protein levels.

C The protein pulldown experiments identified 56 proteins putatively associated to the Shigella-septin cage, and then categorised into 6 groups based on analysis from the Gene ontology database. See also Dataset EV1.
Mitochondria mediate septin cage assembly to promote autophagy of Shigella (2016)
Figure 3

Figure 3. Mitochondria promote septin cage assembly.

A HeLa cells were labelled with MitoTracker Red CMXRos, treated with cytochalasin D for 30 min, then fixed and labelled for endogenous SEPT7 for confocal microscopy. The scale bar represents 0.5 μm. See also Fig EV3A.

B HeLa cells were infected with S. flexneri for 4 h 40 min, labelled with MitoTracker Red CMXRos, fixed and labelled with antibody for endogenous SEPT7 for quantitative confocal microscopy. The scale bar represents 1 μm.

C HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, infected with Shigella-mCherry for 4 h 40 min, and processed for CLEM. SEPT6 is shown in green, mitochondria in red, and Shigella-mCherry in blue. Septin cages identified by fluorescent light microscopy (FLM) were processed for TEM. The right-most image is enlarged from the boxed region in the TEM image, and shows the bacterium (Sf) surrounded by a double membrane of autophagy (Au) and the mitochondrial membrane (Mt). The scale bar represents 1 μm. See also Fig EV3B.

D HeLa cells stably expressing SEPT6-GFP were transfected with Mito-BFP for 24 h, then infected with Shigella-mCherry for 2 h for live confocal microscopy. Time frame sequence shows a septin cage assembling around Shigella. Mitochondria (circle) support sites of septin ring assembly around the bacterium. Each frame was acquired every 2 min. The scale bar represents 1 μm. See also Movie EV1.

E HeLa cells were treated with control (CTRL), Drp1, or Mfn1 siRNA for 72 h. Whole cell lysates were immunoblotted for Drp1 or Mfn1 to show the efficiency of depletion. GAPDH was used as loading control. siRNA-treated cells were infected with S. flexneri for 4 h 40 min, then fixed for microscopy and stained for endogenous SEPT7 for quantitative confocal microscopy. Graph represents the mean % ± SEM of Shigella inside SEPT7 cages from at least 4 independent experiments per treatment. Student’s t-test, ** = p <0.01; *** = p<0.001.
Mitochondria mediate septin cage assembly to promote autophagy of Shigella (2016)
Figure 4

Figure 4. A role for septins in mitochondrial fission.

A HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos, and fixed for confocal microscopy. Inset image highlights location of septins at sites of mitochondrial fission. The scale bar represents 5 μm.

B HeLa cells were fixed and labelled with antibody against SEPT7 and secondary antibody coupled to HRP, and processed for TEM. The right image is enlarged from the boxed region in the TEM image, and shows a SEPT7 (S7) filament (labelled in black) intersecting with the mitochondrial (Mt) fission site. The scale bar represents 100 nm. See also Fig EV4.

C HeLa cells expressing SEPT6-GFP were stained with MitoTracker Red CMXRos for live SOFI-based super resolution microscopy. Each image was reconstructed from 100 raw TIRF frames using a custom made SOFI-based algorithm. Arrows highlight SEPT6 association with location of mitochondrial fission. The scale bar represents 1 µm. See also Movie EV3.

D HeLa cells expressing SEPT6-GFP were labelled with MitoTracker Red CMXRos, fixed for widefield microscopy, and labelled with antibodies to Drp1. Arrows highlight one example of Drp1 and SEPT6 association with location of mitochondrial fission. The scale bar represents 1 μm.

E HeLa cells were transfected with Mito-BFP and ActA-SEPT7-mCherry, fixed and labelled with antibody to Drp1 for widefield microscopy. The scale bar represents 5 μm. Inset images highlight Drp1 and SEPT7 associated with sites of mitochondrial fission. Pearson’s correlation coefficient from 3 independent experiments (mean ± SEM) shows that mitochondria in ActA-SEPT7 transfected cells recruit significantly more Drp1 than control cells (Mito-BFP transfected alone). Student’s t-test, * = p<0.05.
Mitochondria mediate septin cage assembly to promote autophagy of Shigella (2016)
Figure 5

Figure 5. Drp1 interacts with septins to enhance mitochondrial fission.

A HeLa cells were treated with control (CTRL), SEPT7, or Drp1 siRNA for 72 h. Whole cell lysate of siRNA-treated cells were immunoblotted for GAPDH, SEPT7, or Drp1 to show the efficiency of siRNA depletion. GAPDH was used as loading control. siRNA-treated cells were labelled with MitoTracker Red CMXRos, and fixed for confocal microscopy. The scale bar represents 5 μm.

B HeLa cells were treated with control (CTRL), SEPT2, SEPT7, SEPT9, Drp1, or SEPT7+Drp1 siRNA for 72 h, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Graph represents the distribution in length (μm; whiskers from min to max) of mitochondria in siRNA-treated cells from 3 independent experiments (analysis of at least 100 measurements per biological replicate). Student’s t-test, *** = p<0.001.

C HeLa cells stably expressing SEPT6-GFP were harvested, and then tested with Co-IP. HeLa cells expressing GFP were used in parallel as control. GFP-trap magnetic agarose beads were used to isolate SEPT6-GFP from cells, and extracts were immunoblotted for Drp1, SEPT2, SEPT7, or GFP. The lysates were immunoblotted for GAPDH as control for cellular protein levels.

D HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, and whole cell lysates were immunoblotted for SEPT7, Drp1, phosphorylated Drp1 (P-Drp1), or Mfn1. GAPDH was used as loading control. Graph represents the mean % ± SEM of the relative amount of Drp1, P-Drp1, Mfn1, or SEPT7 proteins quantified by densitometry from 4 independent experiments. Student’s t-test, ns = non-significant; ** = p<0.01, *** = p<0.001.

E HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous P-Drp1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of P-Drp1 to mitochondria was significantly decreased as compared to control cells as measured by Pearson’s correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student’s t-test, * = p<0.05.

F HeLa cells were treated with control (CTRL) or SEPT7 siRNA for 72 h, labelled with MitoTracker Red CMXRos, then fixed and labelled for endogenous Mfn1 for quantitative confocal microscopy. Representative images shown here. The scale bar represents 1 μm. The recruitment of Mfn1 to mitochondria was significantly increased as compared to control cells as measured by Pearson’s correlation coefficient from at least 5 independent experiments (mean ± SEM, analysis of at least 250 cells per biological replicate). Student’s t-test, * = p<0.05.
Mitochondria mediate septin cage assembly to promote autophagy of Shigella (2016)
Figure 6

Figure 6. Shigella fragment mitochondria to counteract septin cage assembly.

A HeLa cells stably expressing SEPT6-GFP were treated with Drp1 siRNA for 72 h, transfected with mito-BFP 24 h, and then infected with S. flexneri-mCherry for 2 h for live confocal microscopy. Representative image shows interplay between two Shigella-septin cages and fused mitochondria. The scale bar represents 1 μm. See also Movie EV3.

B HeLa cells were treated with Drp1 siRNA for 72 h, and then infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 3 independent experiments. Student’s t-test, *** = p<0.001.

C HeLa cells were infected with S. flexneri M90T for 4 h 40 min, labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show the length of mitochondrial (μm; whiskers from min to max) surrounding Shigella outside (-) or inside (+) SEPT7 cages from 3 independent experiments (analysis of at least 130 measurements per biological replicate). Student’s t-test; *** = p<0.001.

D HeLa cells were either kept uninfected as a control or infected with S. flexneri M90T or S. flexneri ΔIcsA for 4 h 40 min. Samples were labelled with MitoTracker Red CMXRos, and fixed for quantitative confocal microscopy. Boxplots show length of mitochondria (μm; whiskers from min to max) in uninfected cells (CTRL), surrounding S. flexneri M90T (+M90T) or S. flexneri ΔIcsA (+ΔIcsA) from 3 independent experiments (analysis of at least 250 measurements per biological replicate). Student’s t-test; *** = p<0.001.

E A potential model for the Shigella septin cage and actin tail assembly pathways. Based on our findings we propose that (i) mitochondria promote septin cage assembly for antibacterial autophagy or (ii) Shigella fragment mitochondria to counteract septin cage entrapment. Depending on the fragmentation of mitochondria by IcsA, Shigella will be compartmentalised in septin cages or spread cell-to-cell via actin-based motility.
Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation (2010)
figf1

(a) Rapamycin (Rap) treatment or nutrient deprivation attenuated the expression of the Wnt reporter TopFlash in HEK-293T cells. (n = 3; two asterisks, P 0.01). DMSO, dimethylsulphoxide. (b) Expression analysis of Wnt target genes by quantitative RT–PCR after 4 h of treatment with rapamycin. (n = 3; asterisk, P 0.05; two asterisks, P 0.01). Wnt3a CM, Wnt3a conditioned medium. (c) Knockdown of LC3 or Beclin 1 potentiated Wnt signalling. (n = 3; asterisk, P 0.05). (d) Atg5 knockout enhanced Wnt signalling. TopFlash reporter activity induced by Wnt3a CM in Atg5−/− or wild-type MEFs. Results are means ± s.d. after normalization to Renilla luciferase activity. (n = 3; asterisk, P 0.05).
Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation (2010)
figf2

(a) Dvl2 underwent autophagy-mediated degradation. E-cadherin was used as a loading control for membrane fraction, and tubulin as a loading control for cytosolic proteins. The asterisk indicates the phosphorylated form of Dvl2. (b) The half-life of Dvl2 was prolonged in Atg5−/− cells. MEFs were subjected to starvation (starv.) for the indicated durations and harvested for immunoblotting with the indicated antibodies. Dvl2 levels were quantified (right panel). Autophagy induction is indicated by the levels of LC3 II and p62 degradation. (c) Reintroduction of Atg5 in Atg5−/− MEFs restored starvation-induced Dvl2 degradation. MEFs infected with recombinant adenovirus expressing GFP or Atg5 were starved for the indicated durations, and the cell lysates were subjected to immunoblotting with the indicated antibodies. (d) Dvl2 degradation was slowed down in Atg7−/− MEFs. MEFs were subjected to starvation for the indicated durations and harvested for immunoblotting with the indicated antibodies. Dvl2 levels were quantified (right panel). (e) The continued degradation of Dvl2 in Atg5−/− cells is proteasome-dependent. MEFs were starved for 2, 4 or 8 h with MG132 (1 μM) or BFA1 (0.1 μM) for different durations, followed by anti-Dvl2 immunoblotting to detect endogenous Dvl2 protein levels. Tubulin served as a loading control. Uncropped images of blots are shown in Supplementary Information, Fig. S8.
Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation (2010)
figf3

(a) Immunoelectron microscopy analysis of HeLa cells transfected with Flag–Dvl2 under nutrient-rich or starvation conditions. The arrowheads indicate Flag–Dvl2. Scale bars, 1 μm (left); 250 nm (right). (b) Localization of endogenous Dvl proteins (red) and GFP–LC3 (green) in GFP–LC3 stable HeLa cells in nutrient-rich medium or under starvation (starv.) conditions with BFA1 for 4 h. The right panel shows the percentage co-localization between Dvls and GFP–LC3-positive dots. Scale bar, 5 μm. (c) Immunoelectron microscopy analysis of HeLa cells transfected with Flag–Dvl2 and GFP–LC3 under starvation conditions. Arrows indicate Flag–Dvl2; arrowheads indicate GFP–LC3. Scale bar, 250 nm. (d) Dvl2–LC3 interaction in vivo. IB, immunoblotting; IP, immunoprecipitation; S, starvation; WCL, whole-cell lysate; N, nutrient-rich. (e, f) Dvl2 interacts directly with LC3 (e) and GABARAP (f) but not with GATE-16 in vitro. GST pulldown assay using in vitro-translated Flag–Dvl2 and immobilized GST or the indicated GST fusions. Precipitated proteins were detected with anti-Flag antibody. Coomassie staining of GST and GST–LC3 protein is shown. (g) The LC-3-binding motif is important for the Dvl2–LC3 interaction. GST–LC3-immobilized beads were incubated with the lysates from the cells expressing wild-type (WT) Dvl2 or the W444A, I447A mutant (MUT); the pulldowns were then subjected to anti-haemagglutinin (anti-HA) immunoblotting. Uncropped images of blots are shown in Supplementary Information, Fig. S8.
Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation (2010)
figf4

(a) Interaction between LC3 and wild-type Dvl2 or its point mutants. (b) Ubiquitylation of wild-type Dvl2 and its variants. Transfected HEK-293T cells were treated with BFA1 (0.2 μM) for 6 h. Ubiquitylated proteins were precipitated with Ni-nitrilotriacetate (Ni-NTA) beads, followed by anti-HA immunoblotting. Ub, ubiquitin. (c) Degradation kinetics of wild-type Dvl2 and its variants under starvation conditions. Lower panel: quantification of the immunoblotting results. (d) Starvation promotes co-localization of Dvl, ubiquitin and p62. Scale bar, 10 μm. (e) Starvation induces the formation of high-molecular-mass Dvl2 aggregates. (f) p62 knockdown alters Dvl localization. Scale bar, 10 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S8.