EMBO/soda-nel huggingface dataset:
Peroxisomal protein PEX13 functions in selective autophagy (2016)
Figure 2

Figure 2. PEX13 is Required for Mitophagy.

A. Representative images of Parkin-mediated clearance of mitochondrial outer membrane protein TOMM20 in HeLa/Parkin cells treated with indicated siRNA 16 h after treatment with 10 μM CCCP or DMSO vehicle control. siPEX13 oligo #2 is shown; similar results were observed with three other siPEX13 oligos. Scale bars, 20 μm.

B. Quantification of TOMM20 clearance in the experiment shown in (A). Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with adjustment for multiple comparisons.

C. Representative images of Parkin-mediated clearance of mitochondrial double-stranded DNA (mtDNA) in HeLa/HA-Parkin cells treated with indicated siRNA 8 h after treatment with 2.5 μM oligomycin and 250 nM antimycin A (OA) or DMSO vehicle control. siPEX13 oligo #1 is shown; similar results were observed with three other siPEX13 oligos. Nuclear DNA staining was masked using DAPI. Scale bars, 20 μm.

D. Quantification of mtDNA clearance in the experiment shown in (C). Results represent box plots of ~300 cells analyzed per sample. Whiskers represent 5%-95% range, and each outlier is represented by a dot. Similar results were observed in three independent experiments. ****p<0.0001; Kruskal-Wallis H-test.

E. Representative images of TOMM20 fragmentation or compaction around perinuclear region in MEFs of the indicated genotype 24 h after treatment with 30 μM CCCP or DMSO vehicle control. White lines indicate cell borders. Scale bars, 20 μm.

F. Quantification of percentage of cells in experiment shown in (E) with accumulation of fragmented mitochondria after CCCP treatment. Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. **p<0.01; two-tailed unpaired t-test.

G. Electron microscopic analysis of E18.5 mouse embryonic liver, heart, and skeletal muscle. Shown are representative images from one mouse. Similar results were observed in three mice per genotype. Arrowheads indicate damaged mitochondria. Scale bars, 1 μm.
Peroxisomal protein PEX13 functions in selective autophagy (2016)
Figure 3

Figure 3. siRNA-Resistant WT PEX13, but not Disease-Associated Mutants PEX13 I326T and W313G Rescue the Mitophagy Defect in PEX13 Knockdown Cells.

A. Western blot detection of PEX13 in HeLa/Parkin cells transfected with indicated siRNA and siRNA-resistant PEX13 plasmid. To achieve similar PEX13 protein expression levels, 0.75 μg PEX13 I326T and PEX13 W313G plasmids were transfected compared to 0.25 μg WT PEX13. Total plasmid level was adjusted using empty vector. Asterisk denotes non-specific band.

B. Representative images of Parkin-mediated clearance of TOMM20 in HeLa/Parkin cells treated with PEX13 siRNA and transfected with indicated PEX13 siRNA-resistant plasmid and then treated with CCCP (10 μM, 16 h). Scale bars, 20 μm. See Fig EV3B for representative images of mitochondrial morphology (TOMM20 staining) in control cells treated with DMSO.

C. Quantification of experiment shown in (B). Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. *p<0.05, **p<0.01,***p<0.001; two-tailed unpaired t-test.

D. Western blot detection of PEX13 expression in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts.

E. Representative images of Parkin-mediated clearance of TOMM20 in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts treated with Oligomycin (2.5 μM) + Antimycin A (250 nM) for 24 h. Scale bars, 20 μm. W313G cells varied in morphology; outlined cell in upper right panel shows a representative cell with larger size and abnormal mitochondrial morphology (see Fig EV3C for higher resolution imaging) and outlined cell in lower right panel shows a representative cell with accumulation of fragmented mitochondria that would be scored as positive in (F).

F. Quantification of experiment shown in (E). Results represent mean ± SEM of 5 groups of 10 images of random fields of cells (>350 cells analyzed per sample). Similar results were observed in two independent experiments. ***p<0.0001; two-tailed unpaired t-test.
Peroxisomal protein PEX13 functions in selective autophagy (2016)
Figure 4

Figure 4. PEX13 Colocalizes with Peroxisomes, but not with Mitochondria or Early Autophagosomes during CCCP-Induced Mitophagy.

A-C Representative images of PEX13 and PMP70 (A), PEX13 and TOMM20 (B), or PEX13-Flag and WIPI2 (C) colocalization in HeLa/Parkin cells transfected with PEX13 after 4 h DMSO or CCCP treatment. Scale bars, 20 μm (A-B) and 5 μm (C).

D Western blot detection of endogenous PEX13 in HeLa/Parkin cells treated with 10 μM CCCP for the indicated time.
Peroxisomal protein PEX13 functions in selective autophagy (2016)
Figure 5

Figure 5. PEX13 and PEX3 are Required for Selective Mitophagy, whereas PEX14 and PEX19 are Required for General Autophagy.

A. Quantitative real-time PCR detection of mRNA levels for PEX3, PEX13, PEX14, and PEX19 in HeLa cells transfected with the indicated siRNA. ***p<0.001; two-tailed unpaired t-test.

B. Quantification of Parkin-mediated TOMM20 clearance in HeLa/Parkin cells transfected with the indicated siRNA 16 h after treatment with 10 µM CCCP. Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. **p<0.01, ***p<0.001; one-way ANOVA with adjustment for multiple comparisons.

C. Quantification of mtDNA clearance in HeLa/HA-Parkin cells transfected with the indicated siRNA 8 h after treatment with 2.5 μM oligomycin and 250 nM antimycin A (OA). Results represent box plots of ~300 cells analyzed per sample. Whiskers represent 5%-95% range, and each outlier is represented by a dot. Similar results were observed in three independent experiments. ****p<0.0001, NS= not significant; Kruskal-Wallis H-test.

D. Quantification of GFP-LC3 puncta in HeLa/GFP-LC3 cells treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or 3 h HBSS starvation media. Results represent mean ± SEM in triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. *p<0.05, **p<0.01, ***p<0.001, NS= not significant; one-way ANOVA with adjustment for multiple comparisons. Statistical analyses refer to the differences between Pex or ATG7 siRNAs vs. NC siRNA within each treatment group.
HIV-1 capsids mimic a microtubule regulator to coordinate early stages of infection (2020)
Figure 1

Figure 1. EB1-associated +TIP, CLIP170 promotes early HIV-1 infection.

(A) Schematic representation of EB1 constructs and domain functions. CLIP170 (lower) uses CAP-Gly domains to interact with EB1's EEY motif, and secondary SxIP-mediated interactions with EB1's EBH domain.

(B) Representative IF images in CHME3 stably expressing Flag alone control or the Flag-tagged EB1 constructs depicted in A co-stained for Tyrosinated-MTs (Tyr-MTs), and the nucleus (Hoechst). Scale bar, 10 μm.

(C) HIV-1-VSV-luc infection is enhanced in CHME3 depleted of endogenous EB1 and stably expressing full-length (FL) EB1, but not in control Flag or any of the other EB1 constructs depicted in A.

(D) Increasing amount of FL-EB1, but not ∆EEY results in a dose-dependent enhancement of HIV-1-VSV-luc infection in transiently transfected CHME3 cells.

(E-F) HIV-1-VSV-luc (E) or HIV-1-VSV-ZsGreen at various dilutions (corresponding to multiplicities of infection (m.o.i) 0.06, 0.03 and 0.02) (F) infection is reduced in differentiated THP-1 depleted of CLIP170 determined by luciferase assays or FACS analysis, respectively.

(G) HIV-1-WT-luc infection is decreased in CLIP170 depleted CHME3 cells.

Data information: Data in C-G are mean values from three independent experiments +/- SEM. Statistical significance was determined by one-way ANOVA (C-D) or t test (E-G). * = p<0.05, ** = p<0.01, *** = p<0.001. Molecular weight markers (in kDa) are shown to the right of WBs.
HIV-1 capsids mimic a microtubule regulator to coordinate early stages of infection (2020)
Figure 2

Figure 2. CLIP170 promotes multiple stages of early HIV-1 infection.

(A-D) Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells either treated with control or CLIP170 siRNAs (A) or expressing Flag control or Flag-CLIP170 (D) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing ∼ 9% (Control siRNA) and 3% (CLIP170 siRNA) shift (A) or no difference (D) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot. (B-C) HIV-1-VSV-luc infection is enhanced in Flag-CLIP170, but not in Flag control, expressing CHME3 cells. Statistical significance was determined by t test (mean ±SEM). * = p<0.05. Molecular weight markers (in kDa) are shown to the right of WBs in C.

(E) The percentage fused double-labelled (GFP-Vpr+/S15-tomato-) HIV-1-DL-WT are not affected in CHME3 cells depleted of CLIP170. Data represent mean of ≥895 virus particles analyzed in ≥11 fields of view (≥17 cells) for each condition. One-way ANOVA was used to calculate statistical significance (mean ±SEM).

(F) Percentage HIV-1-WT-GFP-Vpr particles within 2 μm of the nucleus is reduced in CHME3 depleted of CLIP170 at 2 and 4 h.p.i, scatterplot with mean of ≥354 virus particles quantified in ≥13 cells per sample. Statistical significance was determined by one-way ANOVA * = p<0.05, *** = p<0.001.

(G-H) Infected cells in F analyzed by live cell imaging. Scatterplots representing displacement over time (velocity) (G) and cumulative distance travelled over time (speed) (H). Each dot represents one viral particle tracked, red line indicates the mean. 188 trajectories were analyzed from 25 movies. Statistical significance was determined by t test. *** = p<0.001.

(I) The induction of Ac-MTs is blocked in HIV-1-VSV-luc (Virus) infected, but not in uninfected (NI) or Mock infected CHME3 depleted of EB1, but not CLIP170 at 6 h.p.i. Tyrosinated MTs (Tyr-MTs) are not affected. Representative fields are shown. Similar results were obtained in three independent experiments. Scale bar, 50 μm.
HIV-1 capsids mimic a microtubule regulator to coordinate early stages of infection (2020)
Figure 3

Figure 3. EB1 and CLIP170 facilitate uncoating of incoming HIV-1 cores.

(A-B) CHME3 treated with negative control, EB1 or CLIP170 siRNAs were either lysed and analyzed by WB (A) or infected with GFP-Vpr/S15-tomato-labeled VSV-G pseudotyped HIV-1 (B). Molecular weight markers (in kDa) are shown to the right of WBs in A. Scatterplot of maximum p24 intensity of fused viral particles normalized to the control mean. Each dot represents one virion, red lines indicate means. The number of viral particles analyzed per condition from three independent experiments is shown. Statistical significance was determined by one-way ANOVA (* = p<0.05, ** = p<0.01, *** = p<0.001).

(C-D) Amounts of pelleted p24 CA normalized to the input CA at 3 h.p.i. with HIV-1-VSV-luc in 293A (C) or CHME3 (D) treated with control, EB1 or CLIP170 siRNAs. Mean +/- SEM from three independent experiments is shown. Statistical significance was determined by one-way ANOVA. Error bars = standard error of the mean (* = p-value ≤0.05, ** = p-value ≤0.01, *** = p-value ≤0.001).
HIV-1 capsids mimic a microtubule regulator to coordinate early stages of infection (2020)
Figure 4

Figure 4. CLIP170, but not EB1, binds to HIV-1 cores.

(A-C) Representative (n=3) WB analysis demonstrating that while GAPDH-HA control and EB1-Flag do not bind (A, upper panels and B, respectively) FEZ1-Flag control and Flag-CLIP170 (A, lower panels and C, respectively) pelleted with in vitro assembled HIV-1 CA-NC complexes.

(D) Quantification of the levels of bound CLIP170 as a ratio of total protein across experimental replicates (n=3) from C. Statistical significance was determined by t test (mean ±SEM) *** = p<0.001.

(E) Representative WB analysis (n=3) showing the levels of HIV-1 p24 CA in mock and virus fractions separated by sucrose gradient centrifugation. Fractions 1-3 contain free p24 not associated with the intact cores, while fractions #8 and # 9 harbouring intact HIV-1 cores.

(F) Flag-CLIP170, but not Flag control, also binds to fractions containing intact HIV-1 cores (Virus #8 and #9) but not with Mock fractions.

(G) HIV-1-WT-luc infection is increased in CHME3 cells stably expressing Flag-CLIP170, but not control Flag or Flag-CLIP170ΔCAPgly. Statistical significance was determined by one-way ANOVA. * = p<0.05. Mean +/- SEM from three independent experiments is shown. Molecular weight markers (in kDa) are shown to the right of WBs.

(H) Representative images (n=3) of CHME3 expressing Flag-CLIP170 or Flag-CLIP170ΔCAPgly stained for Flag and Tyr-MTs. Scale bar, 10 μm.
HIV-1 capsids mimic a microtubule regulator to coordinate early stages of infection (2020)
Figure 5

Figure 5. CLIP170 binds to HIV-1 CA-NC in a manner distinct from currently known capsid binders.

(A) Super resolution microscopy (SIM) imaging of Flag-tagged proteins, cyclophilin A (CypA-Flag), EB1-Flag, or Flag-CLIP170 bound to in vitro assembled CA-NC complexes, stained with anti-p24 AG3.0 (Red), anti-Flag or anti-CLIP170 (Green). Scale bar, 2 μm. Representative images from different fields of view are shown.

(B) WB analysis confirming protein expression in samples from A and C. Molecular weight markers (in kDa) are shown to the right of WBs.

(C) Quantification (n=3) of the association of each protein with 97 to 137 CA-NC complexes, using untransfected lysates (Control) and EB1-Flag as controls for non-specific background staining.

(D) Lysates from untransfected, CypA-Flag- or Flag-CLIP170-transfected cells were subjected to CA-NC binding assays and WB analysis.

(E) Levels of CypA or CLIP170 bound to WT or R18L CA-NC assemblies in samples from D were quantified using Fiji (n = 4 experimental replicates; data is presented as a ratio of CypA or CLIP170 in bound versus input for each CA-NC assembly, normalized to WT CA-NC arbitrarily set to 1). Statistical significance was determined by t test (mean ±SEM). *** = p<0.001.
HIV-1 capsids mimic a microtubule regulator to coordinate early stages of infection (2020)
Figure 6

Figure 6. The MHR of HIV-1 capsid encodes an EB1-mimetic motif.

(A) Sequence similarities of the EB1 C-terminus with MHR (highlighted in blue) in HIV-1 (NL4-3). Identical residues are highlighted in red and similar residues are printed in red.

(B) Structure modelling of HIV-1 capsid hexamers with focus on MHR alongside C-terminus of EB1 in red boxes. Identical residues are indicated in red and similar residues are indicated in dark orange. UCSF Chimera 1.12 software was used for structure modelling of NL4-3 hexamer (PDB 3J3Q) and EB1 (PDB 2HKQ).

(C-E) Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv), but not HA-tagged GAPDH (GAPDH) binds to Flag-CLIP170 (C), endogenous forms of various human SxIP-containing +TIPs, but not vimentin (D) or to Flag-tagged SxIP fragment (Flag-Clasp2M) (E) in 293T cells. Results are representative of three experimental replicates.

(F-G) HIV-1-VSV-luc (Virus), but not uninfected (NI) or Mock, infection induces the formation of Ac-MTs in CHME3 expressing GAPDH-MHRhiv, but not GAPDH, as shown by WB analysis (F) or imaging at 6 h.p.i (G). Scale bar, 15 μm. Representative fields are shown. Results are representative of three experimental replicates.

(H) GAPDH-MHRhiv expression in CHME3 reduces infection with HIV-1-WT-luc. Statistical significance was determined by t test. * = p<0.05. Data are mean values from three independent experiments +/- SEM. Molecular weight markers (in kDa) are shown to the right of WBs.
HIV-1 capsids mimic a microtubule regulator to coordinate early stages of infection (2020)
Figure 7

Figure 7. Differential EB1-mimicry in MHR domains across retroviruses.

(A) Sequence similarities of MHR in HIV-1 (NL4-3), SIVmac239, and MuLV with the core SxIP-binding motif (bold text) within the EBH domain of EB1 from different species, adapted from (Honnappa et al., 2009). Residues identical to those in EB1 are highlighted in red, similar residues are printed in red.

(B) Schematic representation of HA-tagged GAPDH constructs, with MHR from HIV-1 (GAPDH-MHRhiv), SIVmac239 (GAPDH-MHRsiv), or MuLV (GAPDH-MHRmulv).

(C) Anti-HA co-IP in CHME3 expressing GAPDH-MHRhiv, GAPDH-MHRsiv or GAPDH-MHRmulv showing binding to endogenous CLIP170. Results are representative of three experimental replicates.

(D-E) Anti-HA co-IP in Rhesus macaque FRhK4 (D) or rat fibroblast Rat2 (E) cells expressing GAPDH alongside GAPDH-MHRsiv or GAPDH-MHRmulv, respectively. Anti-EB1 co-IP was included as positive control to demonstrate CLIP170 binding by EB1 in Rat2 cells. Results are representative of three experimental replicates.

(F-I) FRhK4 cells infected with SIV-VSV-luc (F) or Rat2 cells infected with MuLV-VSV-luc (G) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i, or analyzed by WB (H and I, respectively). Scale bar, 20 μm. Representative fields are shown. Results are representative of three experimental replicates.

(J-K) Measurements of SIV-VSV-luc or MuLV-VSV-luc infectivity in FRhK4 (J) or Rat2 (K) cells depleted of CLIP170. Statistical significance was determined by t test. *** = p<0.001. Data are mean values from two independent experiments +/- SEM.

(L) 293T transfected with GAPDH-MHRhiv or R162Q mutant (GAPDH-MHRhivQ) were subjected to anti-HA co-IP. Representative WB analysis is shown (n = 2). Molecular weight markers (in kDa) are shown to the right of WBs.

Expanded View Figure Legends
Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway (1999)
Figure 2

Phenotype of the apg16Δ strain. (A) Disruption constructs of YMR159c (APG16). Disruption of YMR159c was achieved by insertion of the LEU2 marker gene at the AccI site or by replacing an AccI–PvuII fragment with the LEU2 gene. (B) Autophagy‐negative phenotype of the apg16Δ strain. The autophagic ability of wild‐type (TN125), apg12Δ (YNM107) and apg16Δ cells (YNM114) was measured by the alkaline phosphatase assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate the standard deviation of three independent experiments. (C) Loss of viability during starvation. Wild‐type (KA311B, ○), apg12Δ (YNM101, ●) and apg16Δ cells (YNM124, ▴) were cultured in nitrogen starvation medium, and their viability was determined by phloxine B staining. (D) Defect in API maturation in apg16Δ cells. Transport of pro‐API to the vacuole was examined by immunoblotting with anti‐API antiserum. The positions of precursor and mature API are indicated. (E) The Apg12p–Apg5p conjugation in apg16Δ cells. Total lysates were prepared by the NaOH/2‐mercaptoethanol extraction method from apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115), both carrying pHA‐APG12 (CEN). Western blot analysis was performed using anti‐HA antibody.
Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway (1999)
Figure 3

Apg16p interacts with the Apg12p–Apg5p conjugate but not with the Apg12p monomer. The apg12Δapg16Δ cells (YNM115) were transformed with high copy (2μ) plasmids encoding MycApg12p and/or HAApg16p as indicated. Total lysates were immunoprecipitated with anti‐Myc (lanes 1–4) or anti‐HA antibody (lanes 5–8) and detected by immunoblotting using anti‐Myc or anti‐HA antibody. The positions of the IgG heavy chain and κ light chain are indicated.
Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway (1999)
Figure 4

Apg16p interacts preferentially with Apg12p‐modified Apg5p. Total lysates were prepared from apg16Δ cells (YNM124) or apg12Δapg16Δ cells (YNM115) harboring both pHA‐APG5 and pMyc‐APG16 CEN plasmids. The lysates were immunoprecipitated and analyzed as described in Figure 3.
Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway (1999)
Figure 5

The N‐terminal region of Apg16p is required for interaction with Apg5p, and the C‐terminal region is required for self‐multimerization. (A) PJ69‐4A cells were co‐transformed with each pGBD and pGAD plasmid as indicated. Transformants were selected on Trp− Leu− plates, and then two‐hybrid interaction (+ or −) was assessed for growth on Ade− Trp− Leu− plates. (B) Self‐multimerization of Apg16p in vivo. Lysates of apg16Δ cells (YNM124) transformed with pMyc‐APG16 (2μ) and/or pHA‐APG16 (2μ) were immunoprecipitated with anti‐Myc antibody and detected with anti‐HA antibody.
Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway (1999)
Figure 6

Apg16p mediates Apg5p assembly. Immunoprecipitation was carried out as described in Figure 3, using apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115) carrying pMyc‐APG12 (CEN) and pHA‐APG12 (CEN) (A), or pMyc‐APG12 (CEN) and pHA‐APG5 (CEN) (B). Following SDS–PAGE, immunoprecipitates were subjected to immunoblot analysis using anti‐HA or anti‐Myc antibody as indicated. A model of the Apg12p–Apg5p–Apg16p complex is illustrated in each panel. Apg16p forms a homo‐oligomer through its C‐terminal coiled‐coil region. It is not clear whether Apg16p forms a dimer or a larger multimer. The N‐terminal region of each Apg16p interacts with Apg5p; most of these complexes are conjugated to Apg12p (A) but a small population are unmodified (B). In the absence of Apg16p, separate Apg5ps do not assemble.
Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway (1999)
Figure 7

Expression levels of Apg16p are dependent on the Apg12p–Apg5p conjugate. Total lysates were prepared by the NaOH/2‐mercaptoethanol extraction method from apg16Δ (YNM124), apg5Δapg16Δ (YNM126) and apg12Δapg16Δ cells (YNM115) carrying pHA‐APG16 (CEN). Western blot analysis was performed using anti‐HA antibody.
Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway (1999)
Figure 8

Apg16p exists in a pellet fraction in an Apg5p‐dependent manner. Spheroplasts were generated from cells harboring pHA‐APG5 (A), pHA‐APG16 (B) or pHA‐APG16 (2μ) with or without pHA‐APG5 (2μ) (C). They were lysed by Dounce homogenization. After unbroken spheroplasts were removed by low‐speed centrifugation, the supernatant (T) was fractionated by 100 000 g centrifugation for 60 min to generate a supernatant (S) and pellet (P) fraction. Each fraction was subjected to immunoblot analysis using anti‐HA antibody.