EMBO/soda-nel huggingface dataset:
Tudor Staphylococcal Nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis (2021)
Figure 6

Figure 6. Identification of Arabidopsis salt-induced TSN2 interactome

A, Venn diagram showing a comparison between TSN2_NS, TSN2_HS and TSN2_NaCl protein pools.

B, Co-immunoprecipitation (Co-Ip) of TSN and RBP47 in protein extracts prepared from 10-d-old Arabidopsis seedlings expressing Pro35S:GFP-RBP47 and grown under no stress (NS), HS (39ºC for 60 min) or salt (NaCl) stress (150 mM NaCl for 60 min) conditions. The GFP-expressing line was used as a negative control. Endogenous TSN (107 kD) was detected in total fractions (Input) and fractions co-immunoprecipitated (Co-Ip) with RBP47 but not with free GFP in all three conditions. Input and Co-Ip fractions were analyzed by immunoblotting using α-TSN and α-GFP.

C, Localization of GFP-tagged proteins in root cells of 5-d-old Arabidopsis seedlings expressing Pro35S:GFP-RBP47, Pro35S:GFP-UBP1, Pro35S:GFP-TCTP and ProUBQ:GFP-SnRK1α2. The seedlings were grown under 23°C (NS), incubated at 39°C for 60 min (HS) or treated with 200 mM NaCl at 23°C for 60 min (NaCl). Scale bars = 10 μm
Tudor Staphylococcal Nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis (2021)
Figure 8

Figure 8. TSN and SGs confer heat-induced activation of SnRK1.

A, B, E, F Immunoblot analysis with indicated antibodies of protein extracts prepared from root tips of 10-d-old Arabidopsis WT heat-stressed seedlings (39ºC) 0, 20, 40 and 60 min after the onset of HS. For CHX treatment in B, the seedlings were pre-treated with 200 ng μL-1 CHX for 30 min before HS. The charts show SnRK1 activity, expressed as the ratio of phosphorylated to total SnRK1 protein. The data represent mean ratios of integrated band intensities (for both isoforms) normalized to 0 min ± SD from at least four different experiments. P values denote statistically significant differences for comparisons to 0 min (two-tailed t-test).

C, Localization of GFP-SnRK1α1 and GFP-SnRK1α2 in root cells of 5-d-old Arabidopsis WT seedlings incubated at 39°C and imaged at the indicated time points. Scale bars = 10 μm.

D, Quantification of GFP-SnRK1α1 and GFP-SnRK1α2 foci in the experiment shown in C. Data represent means ± SD of at least 16 replicate measurements. The experiment was repeated three times with similar results. ***P < 0.05 (two-tailed t-test).

E, F, Immunoblot analysis with indicated antibodies of protein extracts prepared from root tips of 10-d-old Arabidopsis tsn1 tsn2 (E) or tsn1 tsn2 expressing ProTSN2:GFP-TSN2 (F) heat-stressed seedlings (39ºC) 0, 20, 40 and 60 min after the onset of HS. The charts show SnRK1 activity, expressed as the ratio of phosphorylated to total SnRK1 protein. The data represent mean ratios of integrated band intensities (for both isoforms) normalized to 0 min ± SD from at least four different experiments. P values denote statistically significant differences for comparisons to 0 min (two-tailed t-test).

G, Expression levels of DIN2 and DIN6 in Arabidopsis WT, tsn1 tsn2, tsn1 tsn2;TSN2 and snrk1α1-/- snrk1α2-/+ 10-d-old heat-stressed seedlings relative to unstressed controls. For CHX treatment, the WT seedlings were pre-treated with 200 ng μL-1 CHX for 30 min before HS. Upper and lower box boundaries represent the first and third quantiles, respectively. Horizontal lines mark the median of five replicate measurements, and whiskers mark the highest and lowest values. Means with different letters are significantly different at P < 0.05 (one-way ANOVA).
Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration (2022)
Figure 1

Figure 1. Neuronal stress increases DLK recruitment to membranes.

A. Schematic diagram of MAPK signaling cascades in mammalian cells. Only representative signaling molecules are shown. MAPK cascade components detected in 3 days in vitro (DIV) cultured embryonic DRG neurons by TMT-based mass spectrometry analysis are colored. In grey, MAPKs not detected (n = 2 independent experiments).

B. Volcano plot of differently-regulated phosphosites of MAPK cascade components from 3DIV cultured embryonic DRG neurons upon 4h of NGF deprivation, with protein-level cutoffs set at log2 fold change >1.0 and -log10 adjusted P value >1.3 (P< 0.05), marked by dashed lines. Downregulated sites represented as blue points, and up-regulated sites as orange points (n = 2 independent experiments).

C. Representative Western blots of the different MAPKs expressed in DRG neurons (DIV3) cultured in the presence or absence of NGF for 3h and subjected to a membrane/cytosolic fractionation. α-tubulin and STMN-1 are used as controls of cytosolic proteins and Na+/K+-ATPase and STMN-2 as controls of membrane proteins.

D. Quantification of the ratio of protein localized in the membrane fraction after 3h of NGF deprivation (n = 6-14 biological replicates; series of unpaired t tests followed by a Holm-Sidak correction).

E. Flow chart showing the approach used to detect palmitoylation levels in DLK from cultured embryonic DRG neurons. Palmitoylated proteins were labeled with click-it palmitic acid, conjugated with biotin and subjected to immunoprecipitation using streptavidin beads.

F. Representative Western blots of total and palmitoylated DLK (upper panel) and quantification of DLK palmitoylation levels (lower panel) from cultured DRG neurons subjected to 3h of NGF deprivation (n = 5 independent experiments; Mann-Whitney U test).

Data information: Graphs represent mean ± SEM in (D) and (F). *p < 0.05 and **p < 0.01. Diagrams in A and E were created with BioRender.com. See also Appendix Figure S1.
Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration (2022)
Figure 3

Figure 3. DLK- vesicle localization is independent of its activation state.

A. Schematic diagram of DLK indicating point mutations generated for the different GFP-tagged DLK constructs.

B-C. Representative Western blots of GFP, p-cJun and GAPDH (B) and quantification of relative c-Jun phosphorylation levels (C) from HeLa cells non transfected (NT) or transfected with GFP alone (GFP), GFP-tagged DLK wild-type (DLK-WT-GFP), kinase dead mutant (DLK-KD-GFP), leucine zipper domain mutant (DLK-LZ-GFP) and a palmitoyl-site mutant (DLK-CS-GFP) (n = 4-7 biological replicates; series of unpaired t tests /Mann-Whitney U tests with DLK-WT-GFP followed by a Holm-Sidak correction).

D. Representative images and zooms of HeLa cells expressing GFP alone or the different GFP-tagged DLK mutants.

E-F. Representative Western blots of GFP, α-tubulin and Na+/K+-ATPase from cytosolic and membrane fractions (E) and quantification of the percentage of DLK localized in the membrane and cytosolic fraction (F) from HeLa cells expressing GFP alone or the different GFP-tagged DLK mutants (n = 4-5 biological replicates; series of unpaired t tests /Mann-Whitney U tests with DLK-WT-GFP followed by a Holm-Sidak correction).

G-I. Representative stills (G), representative kymographs (H) and quantification of the number of GFP-positive particles (I) from time-lapse recordings of the different GFP-tagged DLK mutants in DIV6 cultured embryonic DRG neurons (n = 31-65 neurons/condition; series of Mann-Whitney U tests with DLK-WT-GFP followed by a Holm-Sidak correction).

J-L. Representative stills (J), kymographs (K) and quantification of the number of DLK-GFP positive particles (L) from time-lapse recordings of DIV6 DRG neurons overexpressing GFP-tagged DLK and treated with control (DMSO) or DLK inhibitor GNE-3511 (DLKi) for 3h (n = 22-46 neurons/condition; Unpaired t test).

Data information: Arrowheads point to individual DLK-GFP positive structures in G and J. All graphs represent mean ± SEM. *p < 0.05 and ***p < 0.001. Scale bar represents 100 μm in (D), 10 μm in (G) and (J), 5 μm in (H), (K) and zooms in (D). See also Figure S2.
Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration (2022)
Figure 4

Figure 4. Sphingomyelin controls DLK - vesicle localization and kinase activity.

A. Representative confocal deconvolved and thresholded images of DIV3 cultured embryonic DRG neurons co-stained for endogenous DLK (green) and the marker for synaptic vesicles Rab3 (magenta).

B. Colocalization analysis between DLK (black values) or same image after 90° rotation (grey values), and markers for different intracellular compartments (Rab3, Rab6, Rab7, Rab11 and mitochondria) in DIV3 cultured embryonic DRG neurons (n = 13-17 images/condition).

C. Representative images of HeLa cells expressing HA alone and the different HA-tagged DLK constructs: a wild-type form of DLK (DLK-WT-HA) and the palmitoyl-mutant DLK (DLK-CS-HA).

D. Western blot analysis from total lysate (Input) and immunoprecipitates (IP) of HA-tag protein immunoprecipitations in HeLa cells overexpressing HA alone and the different HA-tagged DLK constructs, DLK-WT-HA and DLK-CS-HA in the absence of any detergent.

E-F. Lipidomic analysis of the immunoprecipitates obtained from the HA-tagged protein immunoprecipitations from extracts of HeLa cells overexpressing HA alone, DLK-WT-HA and DLK-CS-HA. Quantification of lipid classes (E) and sphingomyelin fatty acid species (F) measured in the immunoprecipitates (n = 3-4 independent experiments; series of unpaired t tests /Mann-Whitney U tests followed by a Holm-Sidak correction).

G-I. Representative Western blots of DLK, p-cJun and GAPDH (G) and quantification of relative c-Jun phosphorylation (H) and DLK levels (I) from 3DIV cultured embryonic DRG neurons treated with D609 for 3h in the presence or absence of NGF (n = 8 biological replicates; series of unpaired t tests with control NGF withdrawal condition followed by a Holm-Sidak correction).

J-K. Representative stills (J), kymographs and quantification of the number of DLK-GFP positive particles (K) from time-lapse recordings from 6DIV cultured embryonic DRG neurons overexpressing GFP-tagged DLK and treated with D609 for 3h (n = 29-46 neurons; Mann-Whitney U test). Arrowheads in J point to individual DLK-GFP positive structures.

L. Representative Western blots of DLK, α-tubulin and Na+/K+-ATPase from cytosolic, membrane and insoluble fractions of 3DIV cultured embryonic DRG neurons treated with D609 for 3h.

Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. CE, cholesteryl ester; CER, ceramide; DAG, diacylglycerol; DCER, dihydroceramides; HCER, hexosylceramides, LCER, lactosylceramides; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; TAG, triacylglycerol. Scale bar represents 20 μm in (C), 10 μm in (J), 5 μm in (A) and (K), and 2 μm in zooms in (A). See also Figure Appendix Figure S3.
Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration (2022)
Figure 5

Figure 5. DLK uses the endocytic pathway as a platform for signaling.

A. Western blot analysis of endogenous DLK, p-cJun, Rab6 and GAPDH, and quantification of relative c-Jun phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with dynasore (DYN) or brefeldin A (BFA) for 3h in the presence or absence of NGF (n = 6-12 biological replicates; series of unpaired t tests /Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction).

B. Representative Western blots of p-MKK4, MKK4 and GAPDH, and quantifications of the relative MKK4 phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with dynasore (DYN) for 3h in the presence or absence of NGF (n = 6 biological replicates, series of unpaired t tests with control NGF withdrawal followed by a Holm-Sidak correction).

C-F. Representative stills (C), kymographs (D) and quantification of the orientation (E) and number of DLK-GFP positive particles (F) from time-lapse recordings of DIV6 DRG neurons overexpressing GFP-tagged DLK and treated with dynasore (DYN) or brefeldin A (BFA) for 3h (n = 34-101 neurons, Chi-square test in E and series of Mann-Whitney tests with control followed by a Holm-Sidak correction in F). Arrowheads in C point to individual DLK-GFP positive structures.

G-H. Representative Western blots of DLK, α-tubulin and Na+/K+-ATPase (G) and quantifications (H) from cytosolic and membrane fractions of 3DIV cultured embryonic DRG neurons with dynasore (DYN) for 3h in the absence of NGF (n = 5-11 biological replicates, series of unpaired t tests /Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction).

I-K. Representative stills (I), kymographs (J) and quantification of the number of DLK-GFP positive particles (K) from cultured embryonic DRG neurons treated with chlorpromazine (CHP), dansylcadaverine (DCV) or methyl-β-cyclodextrin (MCD) for 3h (n = 12 neurons, series of Mann-Whitney U tests with control situation followed by a Holm-Sidak correction). Arrowheads in I point to individual DLK-GFP positive structures.

L. Representative Western blots of p-cJun and GAPDH, and quantification of relative c-Jun phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with chlorpromazine (CHP), dansylcadaverine (DCV) or methyl-β-cyclodextrin (MCD) for 3h in the absence of NGF (n = 6-24 biological replicates, series of Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction).

M. Representative Western blots of p-cJun and GAPDH, and quantification of relative c-Jun phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with vincristine (VNC) and dynasore (DYN) for 3h (n = 7 biological replicates, series of Mann-Whitney U tests with vincristine condition followed by a Holm-Sidak correction).

Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (C) and (I), 5 μm in (D) and (J). See also Figure Appendix Figure S3.
Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration (2022)
Figure 6

Figure 6. Inhibition of endocytosis protects against neuronal apoptosis and axon degeneration.

A. Western blot analysis of cleaved caspase-3 (cCasp3) and GAPDH, and quantifications of cCasp3 levels in 3DIV cultured embryonic DRG neurons treated with dynasore (DYN) for 8h in the absence of NGF (n = 6-13 biological replicates, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction).

B. Quantifications of cCasp3 levels from 3DIV DRG neurons treated with chlorpromazine (CHP), dansylcadaverine (DCV) or methyl-β-cyclodextrin (MCD) for 8h in absence of NGF (n = 6-13 biological replicates, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction).

C. Representative images of DRG neurons expressing GFP, Dnm-1-WT-HA or Dnm1-DN-HA form, cultured in the absence of NGF for 8h, and stained with antibodies against cCasp3 (magenta) and HA tag (green). On the right, quantification of the ratio of cCasp3 positive cells over GFP or HA positive cells (n = 10-15 images from 3 independent cultures, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction).

D. Representative images of axons from E12.5 DRG explants cultured for 2 days, subsequently deprived of NGF and treated with different concentrations of dynasore (DYN) for 24 h, and visualized by immunostaining for βIII-tubulin. Quantification of the axonal degeneration index as the ratio of fragmented axon area over total axon area (n = 13-15 explants, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction).

E. Representative images of axons from cultured embryonic DRG neurons cultured for 2 days, subsequently deprived of NGF and treated with different concentrations of dynasore (DYN) for 24 h, and visualized by imaging SiR-tubulin probe. On the right, quantification of fragmented axons using the degeneration index (n = 15 images from 3 independent cultures, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction).

F. Representative images of axons from cultured embryonic DRG neurons expressing GFP, Dnm-1-WT-HA or Dnm1-DN-HA form and deprived of NGF for 24h, and visualized with an antibody against neurofilament heavy chain (NFH). On the right, quantification of fragmented axons using the degeneration index in (n = 8-13 images from 3 independent cultures, series of unpaired t-test tests with control NGF withdrawal followed by a Holm-Sidak correction).

Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 100 μm in (E), 40 μm in (F), 20 μm in (C) and (D) and 10 μm in zooms in (D). See also Figure Appendix Figure S4.
MECP2 mutations affect ciliogenesis: a novel perspective for Rett syndrome and related disorders (2020)
Figure 1

Figure 1. MeCP2 deficiency affects primary cilium formation.

(A) Representative immunofluorescence of primary cilium in WT or Mecp2 null MEF cells. Cells were starved for 48 h before staining with anti-γ-tubulin (green) and anti-acetylated α-tubulin (red) antibodies. Merge of all channels and DAPI staining (blue) is depicted for both genotypes. Scale bar 20 µm, and 10 µm in the enlarged image. Arrows indicate not ciliated cells.

(B, C) Cilia were counted and analysed with ImageJ to quantify the percentage of ciliated cells (n=134 WT and n=140 Mecp2 null cells) (B) and the cilium length (n=104 WT and n=43 Mecp2 null cells) (C). The graphs show the average of three independent experiments (mean ± S.E., **p<0.01, by Mann-Whitney test; ***p<0.001, by Student's t-test).

(D) Representative immunofluorescence of primary cilium in hTERT-RPE-1 cells silenced with MECP2- or control-siRNAs. Cells were starved for 48 h before staining with anti-γ-tubulin (green) and anti-acetylated α-tubulin (red) antibodies. Scale bar 10 µm, and 5 µm in the enlarged image. Arrows indicate not ciliated cells.

(E) Representative Western blotting showing the MeCP2 expression in CTRL and silenced hTERT-RPE-1 cells, with respect to GAPDH, used as loading control.

(F) The percentage of MeCP2 positive cells was calculated by analysing n=1000 CTRL and silenced hTERT-RPE-1 cells (mean ± S.E., ***p<0.001, by Student's t test).

(G, H) Cilia were counted and analysed with ImageJ to quantify the percentage of ciliated cells (n=191 CTRL and n=197 siMeCP2 cells) (G) and the cilium length (n=50 CTRL and n=50 siMeCP2 cells) (H). The graphs show the mean ± S.E of three independent experiments (***p<0.001, by Mann-Whitney test (G) or Student's t-test (H)).

(I) Representative immunofluorescence of primary cilium in WT and Mecp2 null primary cortical neurons at DIV7 and DIV14. Scale bar 10 µm, and 5 µm in the enlarged image. Arrows indicate not ciliated cells.

(J, K) Neurons were fixed at DIV3, DIV7 and DIV14 and primary cilium detected by immunostaining with anti-AC3 antibody. Percentage of ciliated cells (J) and cilium length (K) were analysed. Histograms represent the average of three independent experiments, analysing neurons derived from 3 different experiments (n=156 WT DIV3; n=49 KO DIV3; n=177 WT DIV7; n=82 KO DIV7; n=59 WT DIV14; n=37 KO DIV14) (mean ± S.E., **p<0.01, ***p<0.001, by two-way ANOVA followed by Bonferroni post hoc test).

(L) Primary cortical astrocytes were fixed at DIV15 and stained with anti-acetylated α-tubulin (red) and anti-ɣ-tubulin (green) antibodies. Scale bar 20 µm, and 10 µm in the enlarged image. Arrow indicates not ciliated cell.

(M, N) Percentage of ciliated cells (M) and cilium length (N) were analysed (n=26 WT and n=34 Mecp2 null astrocytes). Histograms represent the average of three independent experiments (mean ± S.E., *p<0.05, Student's t test).
MECP2 mutations affect ciliogenesis: a novel perspective for Rett syndrome and related disorders (2020)
Figure 2

Figure 2. MeCP2 expression in null neurons rescues primary cilium defects.

(A) Representative immunofluorescence of Mecp2 null cortical neurons infected at DIV0 with lentiviruses expressing GFP or MeCP2iresGFP. Neurons were fixed at DIV7 and immunostained with anti-AC3 antibody to detect the primary cilium. GFP-positive cells represent infected cells. Scale bar 20 µm, and 10 µm in the enlarged image.

(B) Western blot comparing MeCP2 levels in Mecp2 null neurons infected with lentiviruses expressing GFP or MeCP2iresGFP with respect to WT neurons. Data are normalized to total protein content, visualized by a TGX stain-free technology.

(C-D) MECP2 expression in null neurons induced a significant recovery of ciliogenesis. (C) The graph represents the percentage of ciliated cells, counting n=50 WT and n=100 null cells of two different preparations (mean ± S.E., ***p<0.001, by two-way ANOVA followed by Bonferroni post-hoc test). Statistical analysis reported a significant interaction (F (1,135) = 8.264), a significant genotype effect (F (1,135) = 37.59) and a significant infection effect (F (1,135) = 26.89). (D) The graph represents the mean ± S.E. of cilium length in WT and Mecp2 null neurons infected with GFP or MeCP2iresGFP expressing lentiviruses, counting n=28 cells of two different preparations (**p<0.01, ***p<0.001, by two-way ANOVA followed by Bonferroni post-hoc test). Two-way ANOVA reported a significant interaction (F (1,103) = 6.405), a significant genotype effect (F (1,103) = 35) and a significant infection effect (F (1.103) = 4.039).