EMBO/soda-nel huggingface dataset:
Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport (2018)
Figure 1

Figure 1. Excessive tubulin polyglutamylation induces Purkinje cell degeneration in a cell-autonomous manner.

(A) Fate of Purkinje cells in mice with Purkinje-cell-specific knockout of Ccp1. Ccp1flox/flox L7-cre mice show a massive but incomplete degeneration of Purkinje cells (calbindin-stained; red) at 4 months, while all Purkinje cells are degenerated at 18 months. Scale bar: 500 µm. (B) Schematic representation of enzymatic tubulin polyglutamylation (red points are glutamate residues that are posttranslationally added to C-terminal tails of α- and β-tubulin). In neurons, TTLL1 is generating a large share of the overall polyglutamylation (Janke et al, 2005), and CCP1 is reversing this PTM. Loss of CCP1 induces TTLL1-mediated hyperglutamylation, which can be avoided by TTLL1 inactivation. (C) Fate of Purkinje cells in mice with combinatorial knockout alleles. Knockout of Ccp1 (pcd-mouse; Ccp1-/-) results in a complete degeneration of Purkinje cells (calbindin-stained; red), while the additional knockout of Ttll1 selectively in the Purkinje cells (L7-cre) protects the entire Purkinje cell layer from degeneration up to 18 months. Scale bar: 500 µm. (D) Nissl staining of the Purkinje cell layer of the brains from various knockout mice shown in (C) confirms the absence of Purkinje cells in Ccp1-/-, and the presence of a wild-type-like Purkinje-cell density in Ccp1-/- Ttll1flox/flox L7-cre mice. Scale bar: 20 µm. Purkinje cell layer (PC) and granule cell layer (GC) are indicated. (E) Immunoblot of tissue extracts from cerebella of Ttll1-/- and wild type mice, probed with the polyE, anti-∆2-tubulin and 12G10 antibodies. Increasing amounts of extracts were loaded for detection of polyglutamylated proteins with polyE. (F) Schematic representation of the experimental paradigm applied in this figure. The thick line in the cerebella symbolises the Purkinje cell layer.
Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport (2018)
Figure 2

Figure 2. Excessive polyglutamylation causes neurodegeneration of the cerebral cortex.

(A) Analyses of tubulin polyglutamylation (antibody: polyE) in extracts of different brain regions of wild type, Ccp1-/-, Ccp6-/- and Ccp1-/-Ccp6-/- mice with immunoblot. In 3-weeks old mice, hyperglutamylation is observed in cerebral cortex, cerebellum and hippocampus of Ccp1-/- and Ccp1-/-Ccp6-/- mice. In 2- and 5-month-old mice, hyperglutamylation is restricted to the cerebellum in Ccp1-/- mice, however it persists in all three brain regions of the Ccp1-/-Ccp6-/- mice (complete analysis in Fig. EV1; α- and β-tubulin bands indicated). (B) Nissl-stained frontal sections of wild type, Ccp1-/-, Ccp6-/- and Ccp1-/-Ccp6-/- mice at 1 and 5 months, showing age-dependent thinning (arrow heads) of the cortex in Ccp1-/-Ccp6-/- mice (complete analysis in Appendix Fig. S2A). (C) Quantification of the thickness of motor and somatosensory cortex of 1- and 5-month-old mice shown in Appendix Fig. S2. The single values shown in Appendix Fig. S2B were averaged, and values from each genotype-specific group were tested against the average values of the remaining three genotypes (Appendix Fig. S2C). Only the cortical thickness of 5-month-old Ccp1-/-Ccp6-/- mice is significantly decreased (mean ±SEM; Mann-Whitney t-test). (D) Immunohistochemistry of brain sections with anti-MAP2 antibody for visualization of neurons (layer V) and apical dendritic (layer IV) and with anti-GFAP for reactive astrocytes (layer V). The complete analysis is shown in Fig. EV2A, EV3A. Scale bars: 50 µm. (E) Quantifications of number of MAP2-positive neurons in layer V, and of number of apical dendrites in layer IV (Fig. EV2B) reveal reduced neuron number in layer V, and reduced dendritic density in layer IV of 5-month-old Ccp1-/-Ccp6-/- mice. Quantification of the number of reactive astrocytes in layer V (Fig. EV3B) shows a significant increase over wild type in Ccp1-/- and in Ccp6-/- mice, however a much stronger increase is seen in Ccp1-/-Ccp6-/- mice. Complete analyses are shown in Fig. EV2C, EV3C (mean ±SEM; student's t-test). (F) Immunohistochemistry of brain sections stained with SMI-32 antibody (a representative section of one brain per genotype group is shown, complete analysis in Appendix Fig. S3). Neuronal SMI-32 labelling indicates increased axonal damage in the cortex of Ccp1-/-Ccp6-/- mice at 5 months. Scale bar: 100 µm. (G) Schematic representation of the experimental paradigm applied in this figure.
Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport (2018)
Figure 3

Figure 3. Ultrastructural analyses of axon degeneration in the cerebral cortices of mice with hyperglutamylation.

Representative electron microscopy images of sections of wild type and Ccp1-/-Ccp6-/- cerebral cortices with myelinated axons. In Ccp1-/-Ccp6-/- sections, several myelinated axons show signs of degeneration, such as accumulation of cellular organelles and cytoplasmic densification (arrows), or axonal lysis (stars). Note that only some axons in the observed sections displayed these phenotypes Ccp1-/-Ccp6-/-, while such figures were never observed in wild type cortices. Scale bar: 1 µm.
Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport (2018)
Figure 4

Figure 4. Spastin knockout does not protect Purkinje cells from degeneration in the pcd mouse.

(A) Fate of Purkinje cells in 1-month-old wild type, spastin-/-, Ccp1-/- and Ccp1-/-spastin-/- mice visualized with anti-calbindin antibody. Ccp1-/- and Ccp1-/-spastin-/- mice show almost complete loss of the Purkinje-cell layer. Scale bar: 500 µm. (B) Nissl staining shows the complete loss of Purkinje cells in Ccp1-/- and Ccp1-/-spastin-/- mice. Scale bar: 20 µm. Purkinje cell layer (PC) and granule cell layer (GC) are indicated. (C) Calbindin staining of sections showing axons of Purkinje cells as in (A). While wild type and spastin-/- show normal axons, Ccp1-/- and Ccp1-/-spastin-/- mice show axonal swellings (arrow heads) in the remaining Purkinje cells. Scale bars: 20 µm.
Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport (2018)
Figure 5

Figure 5. Axonal cargo transport is perturbed in neurons with excessive polyglutamylation. (A) Representation of the analysis scheme for axonal transport of mitochondria in Ccp1flox/floxCcp6flox/flox neurons. Hippocampal neurons from E17.5 embryos were cultured and transduced (on DIV0) with lentivirus expressing either GFP (control) or GFP-2A-cre (inducing conversion to Ccp1-/-Ccp6-/-). At DIV4, live neurons were stained with MitoTrackerTM and mitochondria were imaged in GFP-positive cells for 1 min with a spinning-disk microscope. Mitochondria movements were plotted in kymographs for all the cells analyzed in each set and broken down into single runs without change in direction or speed. Anterograde and retrograde runs were color-coded in green and red, respectively, while immotile events are shown in blue. (B) Immunoblot of cell extracts from Ccp1flox/floxCcp6flox/flox hippocampal neurons without, or after transduction with lentivirus expressing either GFP or GFP-2A-cre after 4 days in culture. Note the specific increase of polyglutamylation (polyE) in neurons transduced with GFP-2A-cre lentivirus. (C) All kymographs of one representative set of experiments. Mitochondria movements (Movie S1) were plotted in kymographs and analyzed as described in (A). Scale bars: 60 sec, 20 µm. Analyses of (D) run length and (E) speed distribution, represented as scatter dot plots with each point representing a single run extracted from the kymographs shown in (C). Each group represents the pool of measurement of multiple neurons made in the experiment. The black line indicates the median of the distribution with whiskers at interquartile range. Numerical value of the medians is indicated below each plot. (F) Overall motility of mitochondria represented as fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s. Values (below plots) from one set of experiment (C) show a decrease of about 50% in Ccp1-/-Ccp6-/- conditions, while (G) mitochondria density is unaltered. (H-K) Statistical analyses of mitochondria transport parameters of experiments shown in (D-G; Fig. EV4A-D). Each bar represents the mean (±SEM) of medians of single experiments for run length (H) and speed (I) distributions, and average (±SEM) mitochondria motility (J) and density (K) of all seven experimental sets. Values of fold change between control and Ccp1-/-Ccp6-/- conditions are indicated below the graphs. Green bars are for anterograde, and red for retrograde transport events. Significance was tested using unpaired t-test. Average run length (H) as well as speed (I) showed no significant differences in these two parameters between control and Ccp1-/-Ccp6-/- conditions. (J) The average of overall motility reveals an about 50% decrease in overall mitochondria motility, while mitochondria density (K) is unaltered between control and Ccp1-/-Ccp6-/- conditions. Expanded View Figure Legends
The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L (2016)
Figure 1

Figure 1. PARL is part of a large assembly in the IM.

(A) Volcano plot representation of PARL interaction partners. Mitochondria isolated from PARL-/- FITR293T cells expressing PARL-FLAG were subjected to co-immunoprecipitation (IP). Co-purifying proteins were separated by SDS-PAGE and identified by quantitative MS (n=3)(Dataset EV1).

(B) Heat map of Log2 transformed LFQ intensities for three independent experiments. All significant (FDR < 0.05) proteins showing a positive ratio between PARL IP and control in (A) are shown. Grey color shows missing quantitative information. Clustering was performed using the complete method with Euclidean distance.

(C) Complexome analysis of MEF mitochondria (n=3). Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARL immunoprecipitates at a FDR level of 0.05 (A) and identified by mass spectrometry after BN-PAGE analysis are shown after hierarchical clustering.

(D) BN-PAGE analysis of mitochondria lacking SLP2, YME1L or PARL. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-13% blue native gradient gel and analyzed using SLP2-, YME1L- and PARL-specific antibodies.

(E) High resolution complexome analysis of SPY complex members SLP2, YME1L and PARL using MEF mitochondria (n=3). Heat maps and migration profiles are shown after separation using a 3-9% BN-PAGE. SLP2, YME1L and PARL migrate in a high molecular weight complex (2 MDa), whereas SLP2 additionally is present in complexes of 1.6 MDa).
The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L (2016)
Figure 2

Figure 2. SLP2 forms a proteolytic hub in the IM.

(A) Co-immunoprecipitation of endogenous SLP2, PARL and YME1L in mitochondria isolated from human FITR293T cells using either SLP2- or PARL-specific antibodies. IgG was used as a negative control. In, input (10%).

(B, C) Co-immunoprecipitation of endogenous SLP2 and YME1L with PARL-specific antibodies in mitochondria isolated from wild type MEFs (WT) and MEFs lacking YME1L, SLP2 or PARL. In, input (10%).

(D) Assembly of SLP2, PARL and YME1L into a high molecular weight complex. Mitochondria isolated from FITR293T cells (Con) and cells inducibly expressing SLP2-FLAG (FLAG) were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, PARL-, YME1L- and PHB2-specific antibodies. In, input (8%); E, eluate (100%).

(E) High resolution BN-PAGE analysis of mitochondria lacking PARL, YME1L or SLP2. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-9% gradient gel containing 0-10% glycerol and analyzed using SLP2-specific antibodies.

(F, G) Submitochondrial localization of SLP2. (F) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated. (G) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet (P) and supernatant (S) fraction by centrifugation. Fractions were analyzed by SDS-PAGE and immunoblotting. MFN2 served as OM marker, TIMM23, SMAC/DIABLO, and YME1L as IMS markers, and AFG3L1 and AFG2L2 as matrix marker proteins.

(H) Topology of the SLP2-PARL-YME1L (SPY) complex in the IM. IM, inner membrane; IMS, intermembrane space.
The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L (2016)
Figure 3

Figure 3. SLP2 modulates PGAM5 processing by PARL in the SPY complex

(A) Steady state levels of proteolytic substrates of YME1L. Whole cell extracts of Yme1l-/-, Parl-/-, and Slp2-/- MEFs were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies.

(B) PGAM5 processing depends on proteolytically active PARL. PARL and catalytic inactive PARLS277A harboring C-terminal FLAG-epitopes were expressed under the control of a tetracylcine (tet)-inducible promoter in wild type (WT) and PARL-/- FITR293T cells as indicated. Processing of L-PGAM5 to S-PGAM5 was monitored by immunoblotting.

(C) Accelerated processing of L-PGAM5 in Slp2-/- cells. Processing of L-PGAM5 was analyzed by immunoblotting in wild type (WT), Yme1l-/-, Parl-/- and Slp2-/- MEFs after inhibition of cytosolic protein synthesis by cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown in the lower panel. Two-way ANOVA analysis (n=3; ****, p<0.0001).

(D) The accelerated processing of L-PGAM5 in Slp2-/- cells is mediated by PARL. PARL was depleted from Slp2-/- cells by RNAi prior to CHX treatment. A quantification of L-PGAM5 levels at different time points is shown. Two-way ANOVA analysis (n=3; **, p<0.01. ****, p<0.0001).

(E) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin were analyzed by BN-PAGE and immunoblotting using FLAG- and PGAM5-specific antibodies.
The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L (2016)
Figure 4

Figure 4. The SPY complex facilitates PINK1 processing by PARL

(A) PARL dependent cleavage of PINK1-HA is reduced in Slp2-/- and Yme1l-/- MEFs. Whole cell extracts of Slp2-/-, Parl-/ -and Yme1l-/- MEFs expressing PINK1-HA were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Cells were treated with 20 µM MG132 or 20 µM CCCP for 4 h.

(B) Quantification of the protein ratio (Log2) PINK1-HA 52 kDa/63 kDa (n=3; *, p<0.05; One-way ANOVA). n.s., not significant. Error bars indicate SEM.

(C) PINK1-HA associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using FLAG-, HA- and SLP2-specific antibodies.

(D) PINK1 associates with SLP2. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells, depleted of SLP2 by siRNA as indicated, were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using SLP2- and PINK1-specific antibodies.
The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L (2016)
Figure 5

Figure 5. OMA1 cleaves PGAM5 under stress and is regulated by SLP2

(A) Processing of L-PGAM5 to S-PGAM5 in depolarized mitochondria of wild type (WT), Slp2-/-, Yme11-/-, and Parl-/- MEFs. PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown. L-PGAM5 levels at t=0 was set to 100%. Two-way ANOVA analysis (n=3; *, p<0.05, **, p<0.01. ****, p<0.0001). Arrowheads denote intermediate PGAM5 cleavage products.

(B) OMA1-mediated processing of PGAM5 in depolarized Parl-/- mitochondria. Processing of L-PGAM5 to S-PGAM5 in Parl-/ mitochondria was assessed as in (A) in the presence or absence of CCCP (20 µM), which were depleted of OMA1 by siRNA as indicated. The arrowhead denotes an intermediate PGAM5 cleavage product. SCR, scrambled.

(C) Impaired PGAM5 processing in depolarized Oma1-/- mitochondria lacking PARL. Processing of L-PGAM5 to S-PGAM5 was examined as in (A) after 2 h in wild type (WT) and Oma1-/- mitochondria in the presence or absence of CCCP (20 µM, 2 h), which were depleted of PARL by siRNA as indicated. * unspecific cross-reaction.

(D) OMA1 mediates accelerated processing of L-OPA1 in Slp2-/- MEFs. Processing of OPA1 and PGAM5 was monitored in Slp2-/-, Oma1-/-, and Slp2-/-Oma1-/- cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide (CHX).

(E) OMA1 interacts with SLP2. OMA1-/- FITR293T cells ectopically expressing OMA1-myc were transfected with pcDNA5 (Control) or pcDNA5-SLP2-FLAG (FLAG). Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, YME1L-, PARL-, and myc-specific antibodies. In, input (8%); E, eluate (100%). Samples were analyzed by SDS-PAGE in parallel (lower panel).

(F) Quantification of immunoprecipitation efficiencies in (E).

(G) Model for the regulation of IM proteases by the membrane scaffold SLP2. SLP2 inhibits processing of L-OPA1 by OMA1 and of PGAM5 by PARL, while facilitating PINK1 cleavage by PARL, thus modulating mitochondrial morphology, quality control and cell survival.
Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport (2020)
Figure 1

Figure 1 NPA inhibits PIN1-dependent cellular auxin efflux.

A Normalized firefly luciferase luminescence (compared to renilla luciferase) as a function of external IAA concentration. Arabidopsis protoplast cells were transiently transformed with either AtPIN1 or GFP (in the case of the control) both under the control of a constitutive CaMV 35S promoter. Where indicated, 10 µM NPA was added.

B Normalized firefly luciferase luminescence (compared to renilla luciferase) as a function of external NPA concentration in the presence of 100nM IAA in control GFP- (blue or red) or PIN1- (green or purple) transformed protoplasts.

Data information: Each point is the mean of six measurements normalized to the firefly luciferase signal from cells with no external auxin added. n=6, error bars indicate standard error.
Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport (2020)
Figure 2

Figure 2 Definition and composition of the PIN1 core complex.

A, B Native PAGE separation reveals distinct detergent-sensitive PIN1-complexes. Plasma membrane preparations from dark grown Arabidopsis cell suspension cultures were solubilized with (a) Complexiolyte 27 (a mixture of ionic and non-ionic detergents), and (b) Complexiolyte 47 (lower stringency, non-ionic detergent), (both Logopharm); first dimension BN-PAGE, second dimension SDS-PAGE, blots stained with anti-PIN1 antibody revealing distinct PIN1 complex populations at the indicated positions. Values are given in KDa.

c) Molecular abundance (abundancenormspec values calculated as summarized peptide PVs divided by the number of MS-accessible protein specific amino acids) of PIN subunits and ABCB19 in anti-GFP affinity purification of CL47-solubilized PIN1-GFP-expressing roots with a GFP-specific DARPin. Note the significant heteromerization of several endogenous PINs with PIN1-GFP and the absence of other abundant interaction partners (ABC19B shown as an example).
Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport (2020)
Figure 3

Figure 3 NPA and quercetin both stabilize PIN1 complex formation.

a) BN-PAGE was performed with PIN1-containing microsomes prepared from either PIN1-expressing HEK293T cells or a dark-grown Arabidopsis cell suspension culture. Prior to solubilization with 1% dodecyl maltoside, microsomes were incubated with either 10 µM NPA or 10 µM quercetin.

b) PIN1 dimer stability induced by NPA after expression in HEK cells. Relative distribution between monomer and dimer after NPA treatment is given relative to distribution of untreated samples after solubilization with 50% (v/v) CL27. Each measurement given (three for each concentration) represents the mean of three gel lanes for wild type (circles) or triple S2523, S253E, S261E phosphomimetic sequences (crosses) (example images are given in the figure inset).
Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport (2020)
Figure 4

Figure 4 NPA induces PIN1-PIN4 interaction in vivo

a) Summed 2D projections of PLA interactions (left) and co-localisation (right) (n=3) between PIN1 and PIN4 laterally bisected 2D-projected heat map. Treatments as indicated.

b) Quantification of positive PLA interactions. n.t (no treatment) and 10µM NPA, n=4; 10µM IAA and 10µM IAA+ 10µM NPA; n=3. Error bars indicate standard deviation.
Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport (2020)
Figure 5

Figure 5 Inhibition of auxin transport by NPA requires PIN1 dimerization.

a) Microsomes from HEK293 cells expressing PIN1-RFP were separated under native conditions in the presence of NPA or an anti-PIN1 Fab fragment. 1) PIN1-RFP, 2) PIN1-RFP+Fab, 3) PIN1-RFP+10 µM NPA, 4) PIN1-RFP+Fab+10 µM NPA, 5) PIN1-RFP + 10 µM Quercetin (Q), 6) PIN1-RFP+Fab+10 µM Quercetin (Q). Protein complexes were solubilized and separated under native conditions as described before. Western blots were performed with an anti-RFP monoclonal antibody.

b) Fourteen-day-old Arabidopsis seedlings (wild type, brown; pPIN1::Mab9B2; blue) grown on AM containing 0.2 µM NPA. Scale bar = 1 cm.

c) NPA-affected lateral root density of nine-day-old Arabidopsis seedlings expressing Mab9B2 scFv fragments (wild type sample sizes were between 16 and 21 plants; Mab9B2 sample sizes were between 12 and 31 plants). Bars indicate standard error.

d) NPA-affected apical hook angle in dark-grown three-day-old Arabidopsis seedlings expressing Mab9B2 scFv fragments. (wild type sample sizes were between 38 and 50 plants; Mab9B2 sample sizes were between 33 and 45 plants).

e) Gravitropic curvature in four-day old Arabidopsis seedlings expressing Mab9B2 scFv fragments. (wild type sample sizes were between 15 and 21 plants; Mab9B2 sample sizes were between 14 and 20 plants).

Data information: Asterisks indicate significant difference between genotypes (p≤0.05) after either a Student-t (c) or Kolmogorov Smirnov (d, e) test.

Expanded View Figure legends