EMBO/soda-nel huggingface dataset:
Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP (2022)
Figure 2

Figure 2. Inhibition of CDK5 activity normalises SV pools in BsnGT neurons.

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons treated with a CDK5 inhibitor roscovitine (rosc, 100µM, gray and pink trace) or vehicle (black and red trace) for 30 minutes before stimulation with 40 and 900 APs in the presence of bafilomycin A.

C, D. Plots show mean values of RRP (C) and TRP (D) fraction for both genotypes before and after treatment.

E. Roscovitine treatment has a significantly higher effect on RRP and TRP in BsnGT neurons compared to WT.

Data information: n corresponding to the number of imaging experiments done on 4 independent cell preparations is given in brackets for each analysis. In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. Significance was assessed by two-way ANOVA with Tukey´s multiple comparison test (C, D) and by Student's t-test (E) *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 2µm.
Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP (2022)
Figure 3

Figure 3. CDK5-dependent phosphorylation of Ser551 of Syn1 is increased in BsnGT in vivo and in vitro and not regulated by neuronal activity in vitro.

A. Representative immunoblot with antibody against CDK5-dependent pSer551 of Syn1 (pSer551Syn1) and total Syn1 and total protein stain used as loading control for quantification of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT and BsnGT mice.

B, C. Quantification of pSer551Syn1 / Syn1 ratio (in B) and total Syn1 (in C) abundance on blots from A.

D. Scheme depicts the regulation of phosphorylation of Ser551 of Syn1 by CDK5/calcineurin balance that mediates recruitment of SVs from TRP to the ResP. Changes in this signalling detected in BsnGT neurons are depicted in red.

E. Representative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunostained for pSer551Syn1 and total Syn1.

F. Quantification of IF intensity of pSer551Syn1 in synapses from WT, BsnGT, and WT cells treated with roscovitine.

G. Representative images of 21 DIV hippocampal WT and BsnGT neurons at baseline activity, upon network activity silencing (TTX, 1 µM, 72 h) or in conditions of increased activity (BIC, 30 µM, 48 h)

H. Quantification of experiment in G.

Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independently processed animals in B and C or to the number of independent images (E, G) quantified from two (F) and four (H) independent cultures per condition. For statistics, student's t-test in B and C one-way ANOVA with Sidak´s multiple comparisons test in F and two-way ANOVA with Tukey´s multiple comparisons test in H were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in E and G.
Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP (2022)
Figure 4

Figure 4. Decreased PKA and increased calcineurin activity in BsnGT neurons.

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted from WT and BsnGT hippocampal neurons treated with calcineurin inhibitor FK506.

C, D. Quantification of the RRP and TRP fractions in WT and BsnGT neurons from experiment in A, B.

E. Representative Western blot on P2 fractions prepared from hippocampal tissue of WT and BsnGT mice probed with antibodies against SNAP25 phosphorylated onThr138 and total SNAP25.

F. Quantification of blot in E shows lower pThr138SNAP25 to total SNAP25 IF ratio in BsnGT compared to WT.

G. Scheme illustrates the PKA/calcineurin-dependent regulation of SNAP25 phosphorylation on its Thr138 that promotes enlargement of RRP via an increase in SV release competence. The changes detected in BsnGT are depicted in red for elevated and in blue for lowered abundance/activity.

H, I. Direct activity measurements reveal significant increase in calcineurin and drop in PKA activity in samples prepared from hippocampal tissue of BsnGT mice as compared to their WT littermates.

Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of analysed imaging experiments done on 3 independently prepared cultures per genotype in B-D and number of animals per genotype used for WB or enzyme activity measurements in F, H and I. Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test for C and D and Student's t-test in F, H and I; significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 2 µm.
Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP (2022)
Figure 5

Figure 5. PKA-dependent phosphorylation of Syn1 is decreased and insensitive to forskolin in BsnGT.

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons without treatment (black and red) and upon treatment with adenylyl cyclase activator forskolin (gray and pink) or PKA inhibitor H89 (light grey and orange).

C, D. Quantification of the RRP and TRP fraction from experiment in A and B.

E. Representative immunoblot with antibody against pSer9Syn1 and total Syn1 and total protein stain of hippocampal P2 fraction from WT and BsnGT mice prepared in the presence of KN 93 to isolate PKA-dependent regulation of pSer9Syn1.

F, G. Quantification of pSer9Syn1/Syn1 ratio (in F) and Syn1 (in G) on blots from E.

H. Representative images of hippocampal neurons from WT and BsnGT mice labelled with antibodies against pSer9Syn1 and total Syn1. Neurons were pre-treated (4 µM, 1hr) with KN93 to isolate PKA-dependent phosphorylation and then treated in addition with forskolin or H89 before fixation.

I. Quantification of staining in H.

J. Scheme illustrates the PKA-dependent phosphorylation of Syn1 on Ser9 that promotes recruitment of SVs to the recycling pool. Changes in this signalling confirmed in BsnGT are depicted in red.

Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments performed on three independently prepared culture batches in (C and D), samples prepared from individual animals in F and G, or quantified independent visual fields obtained from 2 independent culture preparations in I. The statistical significance was assessed in C, D and I using two-way ANOVA with Tukey´s multiple comparison test and in F and G using Student's t-test as is depicted in graphs as *p≤0.05, **p < 0.01, ***p< 0.001. The scale bar is 2 µm in A and 5 µm in H.
Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP (2022)
Figure 6

Figure 6. PDE4 activity inhibition rescues BsnGT phenotype.

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for untreated WT and BsnGT neurons (black and red) and cultures treated with of 6-Bnz-cAMPS (50 µM, 1 hour; gray and pink) and rolipram (1 µM, 30 min, light grey and orange).

C, D. Quantifications of RRP and TRP fractions in experiment in A and B.

E. Effect of treatment is significantly higher in BsnGT than in WT.

F, G. Quantification of cAMP levels and PDE4-associated phosphodiesterase activity assessed in hippocampal tissue of WT and BsnGT animals.

H. Quantification of pSer145PDE4B to the total PDE4B ratio from experiment in I.

I. Representative immunoblot with antibody against CDK5-dependent pSer145PDE4B and total PDE4B on hippocampal tissue from WT and BsnGT animals.

J. The scheme illustrates the CDK5-dependent regulation of cAMP hydrolysis by PDE4 and the downstream effect of PKA affecting the TRP size. The changes in this signalling detected in BsnGT are shown; blue indicates decreased and red an increased activity or abundance.

Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the number of imaging experiments performed on three independently prepared cultures in (C-E) or individual animals used for sample preparation in F-H. Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test in C, D and E and Student's t-test in F,G and H and is given as: *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 2µm.
FIBCD1 is an endocytic GAG receptor associated with a novel neurodevelopmental disorder (2022)
Figure 1

Figure 1: Expression of FIBCD1 in human tissues and properties of FIBCD1 variants identified in 2 cases of undiagnosed neurodevelopmental disorders.

(A) Top, schematic of FIBCD1 protein, with labelled intracellular domain (IC, red), transmembrane domain (TM, pink), coiled coil (CC, dark blue) and FReD (light blue). Location of patient variants denoted in red, blue variant denotes the control used in later experiments. Left, family pedigrees of P1 (top) and P2 (bottom) showing affected proband (filled, arrow) and carriers (half-filled). Right, representative traces of Sanger sequencing to confirm segregation within the family. P1 variants are inherited in autosomal recessive manner; P2 exhibits inheritance by uniparental disomy.

(B) P2 MRI images (axial, sagittal and coronal plane) showing ventriculomegaly (red asterisk), slightly thickened cortex and bilateral enlarged gyri (white asterisk).

(C) Amino acid sequence conservation sites of patient variants Gly29Ser, Arg406Cys and Pro456Leu in different species, as labelled.

(D) FIBCD1 expression in various human visceral tissues and brain regions (inset). Expression is plotted relative to the tissue with lowest detectable expression (trachea; inset, choroid plexus). n represents technical replicates (n = 2).

(E) In-situ hybridisation with probe pairs specific to Fibcd1 mRNA (purple) in mouse whole-brain coronal section, left hemisphere shown. Insets of high Fibcd1 expressing regions are (i) cortex, (ii) pyramidal cell layer of hippocampus, (iii) medial habenula, (iv) granule cell layer of the dentate gyrus and (v) hypothalamus. Scale bar sizes are as indicated, (n=3)

(F) Relative mRNA expression levels of mouse Fibcd1 (primers binding to exon 4 and 5) normalised to Gapdh, in the indicated adult brain regions, analysed by RT-qPCR (n = 3). Olf.Bulb = olfactory bulb; Ctx. = cortex; Hipp. = hippocampus; Midbr. = midbrain; Pons & Med = pons and medulla; Crb. = cerebellum.

(G) Relative mRNA expression levels of mouse Fibcd1 (primers binding to exon 4 and 5) normalised to Gapdh in the hippocampus of the indicated developmental time points, analysed by RT-qPCR (n =3).

Data information: Panel E is representative of 3 independent experiments from 3 individual mice, for panels F and G each data point represents an individual mouse. Data is represented as mean and error bars represent SD. p-values were calculated by 1-way ANOVA comparing each sample to the hippocampus region (F) or the time point E14.5 (G). ** =p≤0.01; **** p≤0.0001.
FIBCD1 is an endocytic GAG receptor associated with a novel neurodevelopmental disorder (2022)
Figure 2

Figure 2: Neurological deficits in FIBCD1-deficient mice and flies.

(A) Immunofluorescent images of control and neuronal (Nsyb) CG10359 (dFibcd1) RNAi-mediated knockdown D. melanogaster, 3rd instar larvae NMJ (NMJ6/7) stained with anti-horseradish peroxidase antibodies. Empty control and RNAi-mediated knockdown of CG10359 (dFibcd1-i) lines #1, 2 and 3 shown. Scale bar = 20µm. Representative images of 3 independent experiments.

(B-C) Quantification of (A), control and CG10359 knockdown lines NMJ neuron bouton number (B) and NMJ neuron axon branch points (C). n(empty) = 12; n(line #1) = 20; n(line #2) = 11; n(line #3) = 14.

(D) Negative geotaxis assay of adult Drosophila control and RNAi lines #1, #2 and #3 compared to control lines expressing RNAi targeting luciferase. Climbing index represents the percentage of flies that crossed the 5 cm vial mark within 5 seconds after gentle tapping to the bottom of the vial. N is the number of tested vials: n(luciferase) = 53; n(line #1) = 63; n(line #2) = 36; n(line #3) = 31.For flies per vial, see Figure EV2D.

(E) Representative coronal section images of Golgi-Cox staining of Fibcd1 WT and KO hippocampi (left), Neurolucida tracing of hippocampal CA1 pyramidal neurons (middle) and apical dendrites with spines (right). Scale bars as indicated.

(F-I) Quantifications of (F) dendritic spine density, (G) total length of apical as well as basal dendrites, (H) dendritic nodes in apical as well as basal dendrites and (I) number of dendrites (n = 5).

(J-K) Sholl analysis of apical and basal dendrites (n = 5).

(L) Percentage of mouse spontaneous alterations in the Y-maze (n(Fibcd1 WT) = 9; n(Fibcd1 KO) = 15).

(M) Latency to enter the dark (foot shock) chamber during the inhibitory avoidance task at training and testing (24 hours post-training) days (n(Fibcd1 WT) = 8; n(Fibcd1 KO) = 15).

Data information: For panels B-D each data point represents an individual NMJ, for panels F-I, L and M each data point represents an individual mouse. Data is represented as mean and error bars represent SEM. p values were calculated using 2-way ANOVA (panels B and C), 1-way ANOVA (panel D) or unpaired Student's t-test (panels F-I, L and M).For panels J and K p-values were calculated using 2-way ANOVA and differences at individual distances in the Sholl analysis were corrected for multiple comparisons by Bonferroni's multiple comparisons test.* = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.
FIBCD1 is an endocytic GAG receptor associated with a novel neurodevelopmental disorder (2022)
Figure 5

Figure 5: FIBCD1 mediates responses of primary hippocampal cultures to CSPGs.

(A) Left, representative image of immunofluorescent staining (MAP2, red; DAPI, blue) of primary hippocampal cultures at 2 days in vitro (DIV), plated on +/- CSPG coating with and without prior digestion with ChABC, as indicated. Right, quantification of DIV2 images, showing the number of protruding cells per field normalised to untreated condition. n(Fibcd1 WT) = 4; n(Fibcd1 KO) = 3. Scale bar = 250µm.

(B) Left, representative images of DIV14 neurons, same conditions as in (A). Right, quantification of DIV14 images, representing the percentage of cells per field that are clumped. n(Fibcd1 WT) = 3; n(Fibcd1 KO) = 2. Scale bar = 250µm.

(C) Volcano plots of differential gene expression of transcriptomes at DIV3 hippocampal cultures comparing (left) WTCSPG vs WT and KOCSPG vs KO (FDR < 0.05) (right). Significantly upregulated and downregulated genes are shown in red and blue, respectively. The top 20 DEGs are labelled.

(D) Above, Venn diagram of significant DEGs unique to WTCSPG vs WT (green, 270 genes), KOCSPG vs KO (orange, 193 genes) and common between the two (grey, 153 genes). Below, lists of the 20 most significant DEGs and their fold change for each comparison, showing downregulated DEGs in blue and upregulated in red. n(Fibcd1 WT) = 5; n(Fibcd1 KO) = 4. n represents a prep of cells.

(E) GO term enrichment analysis for significantly downregulated genes (FDR < 0.05) in (left) WTCSPG vs WT and (right) KOCSPG vs KO.

Data information: For panels A and B each data point represents an individual preparations of primary cell culture. Data are represented as mean and error bars represent SEM. p values were calculated by 1-way ANOVA. * = p≤0.05; ** =p≤0.01; *** =p≤0.001.

EXPANDED VIEW FIGURE LEGENDS
The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation (2016)
Figure 1

Figure 1. MAPKKK5 regulates chitin-induced MAPK activation.

A Schematic diagram of MAPKKK5 constructs. N; N-terminal domain, KD; kinase domain, C; C-terminal domain.

B MAPKKK5-C interacts with PBL27 in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 2 mM 3-aminotriazole (3-AT) indicates a positive interaction.

C Chitin-induced MAPK activation was analyzed by immunoblots with α-pMAPK.

D Complementation of chitin-induced MAPK activation in mapkkk5 mutants by expression of MAPKKK5-FLAG. MAPK activation was analyzed by immunoblots with α-pMAPK.

E Flg22-induced MAPK activation was analyzed by immunoblots with α-pMAPK. Experiments (B) - (E) were performed three times with similar results.
The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation (2016)
Figure 2

Figure 2. MAPKKK5-mediated immune response and disease resistance.

A Chitin-induced callose deposition in the mapkkk5 mutants. Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Representative pictures from the biological replicates are presented. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student’s t-test (P < 0.05).

B Leaf discs were treated with 10 μM (GlcNAc)7 in a solution containing 500 μM L-012 and 10 μg/ml horseradish peroxidase. ROS production was quantified using a luminescence microplate reader. Data are means ±SD calculated using three biological replicates, where each biological replicate consisted of two technical replicates.

C mapkkk5 mutations reduce resistance to Alternaria brassiciicola. Lesions sizes were measured 6 days after inoculation. Values are mean ±S.E., n ≥92. Asterisks indicate significant difference from wild-type controls by Student’s t-test (P < 0.01).

D Analysis of chitin-induced transcriptional reprogramming in mapkkk5-1. Total RNA was extracted from seedlings treated with mock or 40 μM chitin for 3 hours and analyzed by RNA-seq. Genes that are significantly induced or suppressed by chitin in the wild type were selected (q-value < 0.05; 12,992 genes). The log2 fold changes of the selected genes compared to mock in the wild type and mapkkk5-1 were plotted. Yellow and blue dots indicate genes that show reduced induction or suppression (506 genes) or enhanced induction or suppression (151 genes) in mapkkk5-1 compared to the wildtype, respectively. The slope of the linear regression line (red) indicates that the overall transcriptional response is weakened in mapkkk5-1 compared to the wild type. As a comparison, the line y=x (black) is shown. For more details, see the Methods.

E A heatmap showing MAPKKK5-dependent genes. Genes showing reduced induction or suppression in mapkkk5-1 compared to the wild type were selected as described in the Methods (339 genes). The log2 fold changes of the selected genes compared to mock were subjected to hierarchical clustering analysis. Yellow indicates positive values, blue indicates negative values and black indicates zero: see the color scale.

F Expression patterns of representative MAPKKK5-dependent genes. qRT–PCR analysis of defense-related genes in 8-day-old seedlings exposed to 40μM (GlcNAc)7 for 3h. Data are shown as the average of three independent biological replicates ± SD. The asterisks indicate statistically significant differences from the WT controls by Student’s t-test (P < 0.05).
The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation (2016)
Figure 3

Figure 3. MAPKKK5 interacts with PBL27 at plasma membrane.

A The MAPKKK5-GFP protein was detected in the membrane fraction. The total (T), soluble (S), and microsomal membrane (M) fractions purified from Nb leaves expressing MAPKKK5-GFP were used for immunoblots with antibodies against GFP, PM-localized aquaporin PIP1 and cytosolic ascorbate peroxidase EAP1.

B Subcellular localization of MAPKKK5-GFP and GFP in Nb leaves. The results were photographed at 38 hpi. Scale bar=50µm.

C Detection of MAPKKK5-GFP and PBL27-HA transiently expressed in Nb leaves. Total proteins were purified from Nb leaves at 38 hpi.

D Localization of MAPKKK5K375M-GFP was analyzed by using total (T), soluble (S), and microsomal membrane (M) fractions purified from Nb leaves expressing MAPKKK5 K375M –GFP

E Co-immunoprecipitation assays show that MAPKKK5K375M-GFP forms the complex with PBL27-HA in Nb leaves. The microsomal fractions purified from Nb leaves co-expressing MAPKKK5K375M-GFP and PBL27-HA at 48 hpi were used for co-immunoprecipitation with α-GFP.

F Visualization of the interaction between PBL27 and MAPKKK5K375M by bimolecular fluorescence complementation (BiFC) analysis in Nb leaves. The results were photographed at 38 hpi. Venus fluorescence indicates the interaction between PBL27 and MAPKKK5K375M. Scale bar=50µm.
The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation (2016)
Figure 4

Figure 4. MAPKKK5 disassociates from PBL27 after chitin perception.

A Detection of MAPKKK5-GFP and MAPKKK5K375M-GFP transiently expressed in Arabidopsis protoplasts.

B Expression of MAPKKK5-GFP reduces the level of PBL27. CBB, Coomassie brilliant blue.

C Arabidopsis protoplasts expressing PBL27-HA and MAPKKK5-GFP were treated with 30 μM MG132 for 3 h and subjected to immunoblots withα-HA and α-GFP.

D The co-immunoprecipitation assay shows that PBL27 forms the complex with MAPKKK5 in Arabidopsis protoplasts. Total proteins purified from protoplasts co-expressing PBL27 and MAPKKK5 were subjected to immunoprecipitation with α-HA.

E Visualization of the interaction between PBL27 and MAPKKK5K375M by BiFC analysis in Arabidopsis protoplasts. FLS-associated RLCK BIK1 was also used for this assay. Venus fluorescence indicates interaction between PBL27 and MAPKKK5K375M. Scale bar=10µm.

F The disassociation of PBL27-MAPKKK5 interaction after chitin treatment in Arabidopsis protoplasts. Protoplasts were co-transfected with MAPKKK5K375M-GFP and PBL27-HA, stimulated with or without 10μM chitin or 1 μM flg22 for 10 min and subjected to the co-immunoprecipitation assay. Experiments (A) - (F) were performed three times with similar results.
The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation (2016)
Figure 5

Figure 5. PBL27 Phosphorylates the C-terminal domain of MAPKKK5.

A PBL27 phosphorylates MAPKKK5-C in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. CBB, Coomassie brilliant blue.

B BIK1 does not phosphorylate MAPKKK5-C in vitro. RBOHD was used as a positive control. Asterisk indicates artificial bands.

C Identification of amino acid residues of MAPKKK5 phosphorylated by PBL27. The C-terminal domain of MAPKKK5 was phosphorylated by PBL27 in vitro and subjected to LS-MS/MS analysis. All MS/MS spectra of phosphorylated peptides were validated manually. The amino acid residues phosphorylated by PBL27 are shown in red.

D The S622A mutation strongly reduces phosphorylation by PBL27. Each of phosphorylation sites was substituted by alanine, and subjected to the in vitro phosphorylation assays.

E PBL27 does not phosphorylate MAPKKK5-C6xA in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography.

F PBL27 interacts with MAPKKK5-C6xA in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 10 mM 3-AT indicated a positive interaction.