EMBO/soda-nel huggingface dataset:
Phosphorylation of Cav1.2 on S1928 Uncouples the L-type Ca2+ Channel from the β2 Adrenergic Receptor (2016)
Figure 5

Figure 5. ISO-induced Displacement of the β2AR from Cav1.2 Blunts Subsequent Cav1.2 Phosphorylation.

Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.

(A) β2AR was IPed before IB for α11.2 (top part of IB), GluA1 (middle part of IB), and β2AR (bottom part of IB), as indicated.

(B,C) For quantification, α11.2 and GluA1 IB signals were normalized to β2AR (**p<0.01, ANOVA).

ISO-induced displacement of the β2AR from α11.2 (A, lanes 2-6) lasted at least 3 min but not 20 min (compare lanes 6 and 7).

(D) α11.2 and GluA1 were concurrently IPed from same samples as in (A) by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 (top three panels) and middle part for pS845, pS831, and total GluA1 (bottom three panels).

(E-H) For quantification, pS1928 and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ANOVA).

ISO-induced displacement of the β2AR from α11.2 (see A) rendered α11.2 (but not GluA1) refractory to re-phosphorylation of S1928 and S1700 upon a second ISO application of α11.2 (D, compare lanes 5 and 6).
Phosphorylation of Cav1.2 on S1928 Uncouples the L-type Ca2+ Channel from the β2 Adrenergic Receptor (2016)
Figure 6

Figure 6. ISO Pre-treatment Does Not Blunt Cav1.2 Phosphorylation by Subsequent ISO Treatment in S1928A KI Mice.

Forebrain slices from S1928A KI mice were treated with vehicle (lanes 1,5) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.

(A) α11.2 and GluA1 were concurrently IPed from same samples by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 and middle part for pS845, pS831, and total GluA1. In S1928A KI mice, S1700 re-phosphorylation after a 3 min wash out of ISO was not blunted (lane 6) in contrast to WT mice but rather augmented (compare to lane 5).

(B-E) For quantification, pS1928 (virtually absent) and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ***p<0.001, ANOVA).
Phosphorylation of Cav1.2 on S1928 Uncouples the L-type Ca2+ Channel from the β2 Adrenergic Receptor (2016)
Figure 8

Figure 8. β2AR Binding to α11.2 is Required for β-adrenergic Stimulation of α11.2 Phosphorylation and L-type Currents.

(A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).

(A) Myr-Pep 2 displaced α11.2 (lane 5 vs. 3, top of blot) but not GluA1 (middle, same blot) from β2AR (bottom, same blot); the inverse was true for Myr-DSPL (lane 1 vs. 3).

(B,C) For quantification, α11.2 and GluA1 immunosignals were normalized to β2AR signals (**p<0.01, ***p<0.001, One Way ANOVA).

(D) Myr-Pep2 blunted ISO-induced phosphorylation of α11.2 S1700 (lane 6 vs. 4, top of blot) but not GluA1 S845 (middle, same blot); the inverse was true for Myr-DSPL (lane 2 vs. 4).

(E,F) For quantification, pS1700 and pS845 immunosignals were normalized to α11.2 and GluA1 signals, respectively (***p<0.001, One Way ANOVA).

(G) Representative cell-attached recordings from hippocampal neurons as in Figure 7. In interleafed experiments cultures were pre-incubated for 30 min with 10 M Myr-Pep2 or scrambled Myr-Pep2 (Myr-Pep2scr). The patch pipette contained either vehicle (H2O; control) or 1 µM ISO. The ISO induced increase in NPo was prevented by Myr-Pep2 but not Myr-Pep2scr. Arrows indicate the 0-current level (i.e. closed channel).

(H) Summary plot for (G). For statistical analysis, the NPo value was determined for each recording and pooled under each condition for comparison (n=10-12 patches; *p<0.05, One Way ANOVA with Tukey post-hoc test).
Phosphorylation of Cav1.2 on S1928 Uncouples the L-type Ca2+ Channel from the β2 Adrenergic Receptor (2016)
Figure 9

Figure 9. β2AR Binding to α11.2 is Required for PTT-LTP.

Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C, G-I) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO and 10 µM of Myr-Pep2 or Myr-Pep2scr. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.

(A-C) Litter-matched WT but not conditional Cav1.2 KO mice showed PTT-LTP (**p < 0.001, one-way ANOVA).

(D-F) WT as well as Cav1.2 KO mice showed NMDAR-dependent 100 Hz LTP.

(G-I) Myr-Pep2 but not Myr-Pep2scr blocked PTT-LTP (**p < 0.001, one-way ANOVA).
CrRLK1L receptor-like kinases HERK1 and ANJEA are female determinants of pollen tube reception (2019)
Figure 1

Figure 1. The herk1 anj fertility defect is caused by maternally-mediated pollen tube overgrowth.

A Representative siliques from wild-type (WT; Col-0), herk1, anj and herk1 anj plants prior to dehiscence. Siliques were placed on double-sided sticky tape and carpel walls separated from the replum to expose the developing seeds. Scale bar = 5 mm.

B Developing seeds per silique in wild-type, herk1, anj and herk1 anj plants. Fully expanded siliques were dissected and photographed under a stereomicroscope. n = 15 (four independent experiments with at least three plants per line and five siliques per plant). Data presented are means ± SEM. *** p<0.001 (Student's t-test).

C Percentage of pollen tubes with normal reception at the female gametophyte (black bars; representative image middle centre of figure) and with overgrowth (grey bars; representative image lower centre) as assessed by aniline blue staining. 15 self-pollinated stage 16 flowers from wild-type, herk1, anj and herk1 anj were analysed. Legend scale bars = 50 µm. *** p<0.001 (χ-square tests).

D Aniline blue staining of pollen tube reception in reciprocal crosses between wild-type and herk1 anj plants with at least two siliques per cross. Legend as per (C). *** p<0.001 (χ-square tests).
CrRLK1L receptor-like kinases HERK1 and ANJEA are female determinants of pollen tube reception (2019)
Figure 2

Figure 2. HERK1 and ANJ are expressed in the female gametophyte and localise to the filiform apparatus of the synergid cells.

A,B Expression of pHERK1::H2B-TdTomato in mature ovules. White dotted lines delineate the egg cell and synergid cells.

C,D Expression of pANJ::H2B-TdTomato in mature ovules. White dotted lines delineate the egg cell and synergid cells.

E,F Localisation of HERK1-GFP in the synergid cell from the pFER::HERK1-GFP construct in (F) and corresponding differential interference contrast (DIC) image in (E). White and red dotted lines delineate the egg cell and synergid cells, respectively.

G,H Localisation of ANJ-GFP in the synergid cell from the pANJ::ANJ-GFP construct in (H) and corresponding DIC image in (G). White and red dotted lines delineate the egg cell and synergid cells, respectively.

Data information : Scale bars = 50 µm. M, micropyle. Arrows, filiform apparatus. CC, central cell. EC, egg cell. SC, synergid cell.
CrRLK1L receptor-like kinases HERK1 and ANJEA are female determinants of pollen tube reception (2019)
Figure 3

Figure 3. Normal synergid localisation of HERK1, ANJ, LRE, FER and NTA pre-fertilisation and impaired relocalisation of NTA after pollen tube reception in herk1 anj and lre-5.

A Localisation of HERK1, ANJ, LRE, FER and NTA in the synergid cell of wild-type (Col-0; WT), herk1 anj and lre-5 in unfertilised ovules, as shown by pFER::HERK1-GFP, pANJ::ANJ-GFP, pLRE::LRE-Citrine, pFER::FER-GFP and pMYB98::NTA-GFP. DIC and fluorescence images are shown, left to right, respectively. White and red dotted lines delineate the egg cell and synergid cells, respectively. Scale bars = 25 µm.

B Localisation of NTA in the synergid cell of wild-type and herk1 anj plants before (upper panels) and after (lower panels) pollen tube arrival. In green, NTA localisation as shown by pMYB98::NTA-GFP fluorescence. In magenta, callose of the filiform apparatus and pollen tube stained with SR2200. From left to right, images shown are DIC, merged fluorescence images, and merged images of DIC and fluorescence. White and red dotted lines delineate the pollen tube and synergid cells, respectively. Scale bars = 25 µm. M, micropyle.

C,D Profile of relative fluorescence intensity of NTA-GFP along the synergid cells of wild-type and herk1 anj ovules (C); and wild-type and lre-5 ovules (D) before (virgin) and after (pollinated) pollen arrival. Data shown are means ± SEM, n = 25. *** p<0.001 (Student's t-test). FA, filiform apparatus.
CrRLK1L receptor-like kinases HERK1 and ANJEA are female determinants of pollen tube reception (2019)
Figure 4

Figure 4. herk1 anj mature female gametophytes are morphologically normal and unaffected in ROS production at the micropyle.

A Representative images of ovules from wild-type (Col-0), herk1 anj, lre-5 and fer-4 20 hours after emasculation (HAE) displaying the mature female gametophyte structure. Images presented here are maximum intensity projections from confocal microscopy images across several z-planes of ovules stained as per [70]. Scale bars = 50 µm.

B Quantification of H2DCF-DA staining of ROS in ovules from wild-type, herk1 anj, lre-5 and fer-4 plants at 20 HAE. Categories are listed in the legend (see also Appendix Figure S7A). Ovules dissected from at least five siliques per line. *** p<0.001 (χ-square tests).

C Percentage of pollen tubes with normal reception at the female gametophyte (black bars) and displaying overgrowth (grey bars) in wild-type, herk1 anj, lre-5 and fer-4 plants, manually selfed at 20 HAE. Fertilisation events counted from at least three siliques per line. *** p<0.001 (Student's t-test).
CrRLK1L receptor-like kinases HERK1 and ANJEA are female determinants of pollen tube reception (2019)
Figure 5

Figure 5. HERK1 and ANJ interact with LRE and FER.

A Yeast two hybrid (Y2H) assays of the extracellular juxtamembrane domains of HERK1 and ANJ (HERK1exJM and ANJexJM, respectively) with LRE (residues 23-138; signal peptide and C-terminal domains excluded).

B Y2H assays with the extracellular domains of HERK1, ANJ and FER (HERK1-ECD, ANJ-ECD and FER-ECD, respectively). Ø represents negative controls where no sequence was cloned into the activating domain (AD) or DNA-binding domain (BD) constructs. -L-W-H, growth medium depleted of leucine (-L), tryptophan (-W) and histidine (-H).

C Co-immunoprecipitation of HA-LRE with HERK1-GFP or ANJ-GFP following 2 days of transient expression in N. benthamiana leaves.

D Co-immunoprecipitation of FER with HERK1-GFP in Arabidopsis seedlings expressing pFER::HERK1-GFP. Numbers indicate molecular weight marker sizes in kDa. Assays were performed twice with similar results. CBB refers to Coomassie Brilliant Blue staining of total proteins.
PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation (2014)
Figure 1

Loss of mtm‐3 impairs autophagy

A‐F′ Confocal fluorescent images of wild‐type (A, C, E) and mtm‐3(tm4475) (B, D, F) embryos at the 1.5‐fold stage stained by DAPI (A–F) and anti‐SQST‐1 (A′, B′), anti‐SEPA‐1 (C′, D′) or anti‐LGG‐1 (E′, F′) antibodies. Scale bars: 5 μm.
G Western blot analysis of LGG‐1‐I and LGG‐1–II (lipidated) in wild type, epg‐6, and mtm‐3.
H mtm‐3 and epg‐6 mutant L1 larvae have shortened mean and maximum life span in the absence of food. At least 300 animals were scored each day.

Source data are available online for this figure.
PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation (2014)
Figure 2

GFP::MTM‐3(C459S) associates with autophagic structures

A–C Fluorescent images of wild‐type embryos (1.5‐fold stage) expressing GFP::MTM‐3 (A), GFP::MTM‐3(C459S) (B), or GFP::MTM‐3(C459S, del PH‐G) (C).
D‐G″ Confocal fluorescent images of wild‐type (D‐D″), mtm‐3 (E‐E″), atg‐2 (F‐F″), and vps‐34 (G‐G″) embryos at the 200‐cell stage expressing GFP::MTM‐3(C459S) and stained by anti‐LGG‐1 antibody. Insets show magnified views. Arrows indicate overlapping GFP and LGG‐1 puncta. Scale bars: 5 μm.
H, I Number of GFP::MTM‐3(C459S)‐positive puncta and co‐localization of GFP::MTM‐3(C459S)‐ and LGG‐1‐positive puncta in various strains [mtm‐3(tm4475), atg‐2(bp576), lgg‐1(bp500), vps‐34(h797)]. At least 10 (I) and 15 (H) embryos were scored per strain. Data are shown as mean ± SD. Data from different mutant backgrounds were compared with wild type. **P 0.0001; N.S.: not statistically different.
J MTM‐3 but not MTM‐3(C459S) exhibits phosphatase activity toward PtdIns3P and PtdIns(3,5)P2 in vitro.

Source data are available online for this figure.
PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation (2014)
Figure 3

MTM‐3 acts downstream of EPG‐8, ATG‐9, ATG‐18, and ATG‐2 in the aggrephagy pathway

A–K Confocal fluorescent images of 200‐cell‐stage embryos from the indicated strains stained by both anti‐SEPA‐1 and anti‐LGG‐1 antibodies. Insets show magnified views. Arrows indicate overlapping SEPA‐1 and LGG‐1 puncta. The wild type contains no visible SEPA‐1 aggregates at this stage and is therefore not shown. Scale bars: 5 μm.
L The percentage of SEPA‐1 puncta co‐localizing with LGG‐1 in each strain. At least 10 embryos were scored per strain. Data are shown as mean ± SD. **P 0.0001; N.S.: not statistically different.

Source data are available online for this figure.
PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation (2014)
Figure 4

Loss of mtm‐3 impairs autolysosome formation

A–G Confocal fluorescent images of 1.5‐fold‐stage embryos from the indicated strains expressing both SQST‐1::GFP and NUC‐1::mCHERRY. Insets show magnified views. Arrows indicate overlapping or closely associated GFP and mCHERRY. Scale bars: 5 μm. The wild type contains no visible SQST‐1::GFP puncta at this stage and is therefore not shown.
H, I The percentage of SQST‐1::GFP‐positive puncta that overlap with (H) or are close to (I) NUC‐1::mCHERRY‐positive structures in each strain. At least 10 embryos were scored per strain. Data are shown as mean ± SD. **P 0.0001; N.S.: not statistically different.
J–L TEM analysis of embryos in wild type (I) and mtm‐3(tm4475) (J, K). Double‐membrane autophagosomes (arrows) were observed in mtm‐3 but not wild‐type embryos. Scale bars: 500 nm.

Source data are available online for this figure.
PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation (2014)
Figure 5

Loss of mtm‐3 causes increased autophagic association of ATG‐18

A–H Fluorescent images of embryos at the 4‐fold stage in the indicated strains expressing ATG‐18::GFP (A–D), ATG‐18(FKKG)::GFP (E, F), or ATG‐18(FTTG)::GFP (G, H).
I Confocal fluorescent images of 4‐fold mtm‐3 embryos expressing ATG‐18::GFP (I′) and stained by DAPI (I) and anti‐LGG‐1 (I″) antibody. Insets show magnified views. Arrows indicate ATG‐18 puncta labeled by anti‐LGG‐1 antibody. Scale bars: 5 μm.
J Number of wild‐type or mutated ATG‐18::GFP puncta in each strain. At least 15 embryos were scored per strain (mean ± SD). **P 0.0001; N.S: not statistically different.

Source data is available online for this figure.
Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations (2020)
Figure 1

Figure 1: IPSCs generated from patient fibroblasts carrying homozygous and heterozygous POLG mutations.

A: Morphology on phase contrast microscopy for parental fibroblast lines (upper panel) and iPSCs (lower panel) from control Detroit 551, WS5A and CP2A POLG patients (scale bars, 50 µm).

B: Immunofluorescence staining of stem cell markers POU5F1 (green) and SSEA4 (red): upper panel - Detroit 551 control iPSCs, middle panel - WS5A iPSCs and lower panel - CP2A iPSCs (Scale bar, 100 µm). Nuclei are stained with DAPI (blue).

C: RT-qPCR quantification of gene expression for LIN28A, NANOG, and POU5F1 for all iPSCs from control Detroit 551, WS5A and CP2A POLG patients (n=7, technical replicates per line for ESCs; n=4, technical replicates per clone for control, WS5A and CP2A iPSCs). The gene expression of the individual clones is assessed with fold change using the comparative ΔΔCt method by normalizing iPSCs to ESC1.

D, E: Flow cytometric quantification of expression level of SSEA4 (D, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) and POU5F1 (E, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) for ESCs and iPSCs for both ESC control lines and iPSCs generated from Detroit 551 control, WS5A and CP2A fibroblasts.

Data information: The data presented in C-E were generated from 2 distinct ESC lines, 2 iPSC clones from Detroit 551 control, 3 different clones from WS5A patient and 2 different clones from CP2A patient iPSCs. Data in D and E are presented as individual (a) and combination as a group (b). Data are presented as mean ± SEM for the number of samples. Mann-Whitney U test was used for the data presented. Significance is denoted for P values of less than 0.05. *** P<0.001.
Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations (2020)
Figure 2

Figure 2: POLG iPSCs manifested a partial phenotype presenting with energy depletion. A: Representative confocal images of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN (red) and HNF4A (green) (a) (scale bar, 100 µm), mesodermal derived cardiomyocytes with positive expression of TNNT2 (red) (b) (scale bar, 100 µm) and ectodermal derived dopaminergic neurons with positive expression of TH (green) and MAP2 (red) (c) (scale bar, 10 µm). Nuclei are stained with DAPI (blue).

B: Confocal images of mitochondrial morphology for iPSC lines with co-staining of MTG (upper panel) and TMRE (lower panel) (scale bars, 25 µm). Nuclei are stained with DAPI (blue).

C-E: Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for mitochondrial volume (MTG) (C, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A), total MMP (TMRE) (D, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) and specific MMP (E, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG.

F: Intracellular ATP production in iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts (n=3, technical replicates per clone for control and WS5A; n=4, technical replicates per clone for CP2A).

G-H: Flow cytometric analysis of TOMM20 expression level (G, n=4, technical replicates per clone) and specific TFAM level (total TFAM/TOMM20) (H, n=4, technical replicates per clone).

I: Relative mtDNA copy number in Detroit 551, WS5A and CP2A iPSCs by RT-qPCR analysis using ND1 and APP (n=5, technical replicates per clone for control; n=5, technical replicates per clone for WS5A and CP2A). Values are presented as Log2 of the ratio between the expression values of ND1 in relation to APP.

Data information: The data presented in C-I were generated from 2 different iPSC clones from Detroit 551 control, 3 different clones from WS5A patient iPSCs and 2 different clones from CP2A patient iPSCs. Data are presented as mean ± SEM for the number of samples. Mann-Whitney U test was used for the data presented in C and E. Two-sided Student's t-test was used for the data presented in D and F-I. Significance is denoted for P values of less than 0.05. *** P<0.001.
Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations (2020)
Figure 3

Figure 3: Validation of patient specific NSCs.

A: Schematic of dual differentiation and seeding paradigm for NSCs via the dual SMAD protocol.

B: Representative phase contrast images (upper panel) and immunostaining for specific stages during neural induction from iPSCs to NSCs. Upper panel displays the morphology in culture of different cell types during neural induction to NSCs from iPSCs including iPSCs (a); neuroepithelium with rosette-like structures (b); neurospheres with defined round shapes in suspension culture (c) and NSCs in monolayers (d) (scale bars, 50 µm). Lower panel demonstrates the representative phase contrast images for the immunostaining corresponding to the specific stages in the upper panel: iPSCs with positive staining of SSEA4 (red) and POU5F1 (green) (e) (scale bar, 50 µm); neuroepithelium with rosette-like structures with positive staining of PAX6 (green) and NESTIN (red) (f) (scale bar, 50 µm); neurospheres with positive staining of NESTIN (red) (g) (scale bar, 100 µm); NSCs with positive staining of PAX6 (green) (h) (scale bar, 50 µm).

C: Flow cytometric assessment of stemness marker POU5F1 and NSC markers NESTIN and PAX6 during neural induction from day 0 to day 5 (n=3, technical replicates).

D: Representative images of the immunofluorescent labeling for NSC markers SOX2 (green) and NESTIN (red) (scale bar, 25 µm) from Detroit 551 control iPSC-derived NSCs. Nuclei are stained with DAPI (blue).

Data information: The data presented in C were generated from one iPSC clones (clone 1) from Detroit 551 control. Data are presented as mean ± SEM for the number of samples. Mann-Whitney U test was used to test the significant difference in the expressions of day 1-5 compared to day 0. Significance is denoted for P values of less than 0.05. * P<0.05; ** P<0.01.