Journal: Scientific Reports

Article Title: Drosophila Dullard functions as a Mad phosphatase to terminate BMP signaling

doi: 10.1038/srep32269

Figure Lengend Snippet: ( a ) pMad Cter in wild type wing imaginal disc, anterior compartment (A) to the left, posterior compartment (P) to the right in all wing discs shown (males, n = 15). ( b ) Increased pMad Cter levels in Dullard hypomorph (ddd P , a P-element is inserted in the 5′UTR of Dullard) wing imaginal disc (males, n = 31). ( c–c’ ) Wild type male adult wing showing normal wing venation (n = 100). Boxed area enlarged in panel c’ showing normal wild type venation surrounding the anterior cross vein (arrow). ( d–d’ ) Increased wing venation in hypomorphic Dullard male wings (n = 34/94). Boxed area enlarged in panel d’ shows three ectopic cross veins and one vein spur (arrows) in Dullard hypomorphic wings. ( e,f ) UAS-Dullard driven in the posterior wing compartment using Engrailed-gal4, UAS-GFP results in loss of pMad Cter , GFP marker used to highlight cells expressing UAS-Dullard shown in panel f (n = 25). ( g ) Omb protein expression in wild type wing imaginal disc (n = 18). ( h ) UAS-Dullard driven by Engrailed-Gal4 (right of the dashed line) results in a significant decrease in Omb protein expression and reduction in the size of the posterior wing compartment (n = 17) compared to the wild type disc in panel g. ( i ) Dullard overexpression decreases pMad Cter levels S2R+ cells were co-transfected with Flag-Mad, +/− Dullard and +/− Activated-Tkv (to activate BMP signaling). Lysed cells were subjected to western blotting and probed with pMad Cter and Flag (loading control) antibodies (statistical significance when comparing lanes 2 and 3: P < 0.001 using the t-test, n = 3 independent blots measured). ( j ) Drosophila S2R+ cells were transfected as in panel i, but cells were treated with +/− Dullard dsRNA. Dullard knockdown results in increased pMad Cter levels (statistical significance when comparing lanes 1 and 2, P = 0.0067; comparing lanes 3 and 4, P = 0.0067, using the t-test, n = 3 independent blots measured). ( k ) Phosphatase inactive Dullard mutants (Dul-D66E and Dul-D68E) failed to dephosphorylate the Mad C-terminal domain compared to wild type Dullard. Mad C-terminal phosphorylation was induced by co-transfecting S2R+ cells with an activated-Tkv receptor in all cases.

Article Snippet: Primary antibodies were used at the following concentrations, pMad Cter (Cell Signaling), 1:1000; pMad 212 (E. De Robertis), 1:1000; pMad 204/208 (E. De Robertis), 1:1000; anti-Flag (Sigma), 1:1000, anti-β-Tubulin (E7-c, Hybridoma bank), 1:1000; anti-Dullard (Sigma), 1:1000; anti-Mad (Newfeld) 1:1000.

Techniques: Marker, Expressing, Over Expression, Transfection, Western Blot, Control, Knockdown, Phospho-proteomics

Journal: Scientific Reports

Article Title: Drosophila Dullard functions as a Mad phosphatase to terminate BMP signaling

doi: 10.1038/srep32269

Figure Lengend Snippet: In all western blots shown Drosophila S2R+ cells were co-transfected with Flag-Mad, +/− Dullard, or +/− Dullard dsRNA and +/− activated-Tkv. ( a ) Dullard overexpression resulted in a decrease in Mad linker phosphorylation levels at serines 212, 208 and 204 (statistical significance when comparing lanes 1 and 2, pMad S212 P < 0.001 and pMad S204/08 P = 0.004; comparing lanes 3 and 4, pMad S212 P < 0.001 and pMad S204/08 P < 0.001, using the t-test, n = 3 independent blots measured for each). ( b ) Dullard knockdown resulted in increased levels of Mad linker phosphorylations (statistical significance when comparing lanes 1 and 2, pMad S212 P = 0.014 and pMad S204/08 P < 0.001; comparing lanes 3 and 4, pMad S212 P = 0.014 and pMad S204/08 P < 0.001; using the t-test, n = 3 independent blots measured). ( c ) C-terminally mutated Mad proteins (Mad-AVA) are linker phosphorylated. These blots demonstrated that Mad-AVA can be linker phosphorylated in the absence of C-terminal activation by the BMP pathway. ( d ) Phosphatase inactive Dullard mutants (Dul-D66E and Dul-D68E) failed to dephosphorylate the Mad linker domain compared to wild type Dullard.

Article Snippet: Primary antibodies were used at the following concentrations, pMad Cter (Cell Signaling), 1:1000; pMad 212 (E. De Robertis), 1:1000; pMad 204/208 (E. De Robertis), 1:1000; anti-Flag (Sigma), 1:1000, anti-β-Tubulin (E7-c, Hybridoma bank), 1:1000; anti-Dullard (Sigma), 1:1000; anti-Mad (Newfeld) 1:1000.

Techniques: Western Blot, Transfection, Over Expression, Phospho-proteomics, Knockdown, Activation Assay

Journal: Scientific Reports

Article Title: Drosophila Dullard functions as a Mad phosphatase to terminate BMP signaling

doi: 10.1038/srep32269

Figure Lengend Snippet: ( a ) S2R+ cells were co-transfected with Tkv-Flag and Dullard. Tkv-Flag was immunoprecipitated (IP) with anti-flag beads and then subjected to western blotting to evaluate if Tkv and Dullard interact. Results demonstrate Tkv and Dullard do not physically interact. ( b ) S2R+ cells were co-transfected with Flag-Mad and Dullard. Flag-Mad was immunoprecipitated (IP) with anti-flag beads and then subjected to western blotting to evaluate if Mad and Dullard interact. Results demonstrate Dullard and Mad proteins do physically interact. ( c ) Treatment of Flag-Mad lysates with calf intestinal phosphatase dephosphorylated Mad (shown by using pMad S212 antibody) compared to untreated lysates, compare lanes 3 and 4. Dephosphorylated Mad failed to bind Dullard compared to phosphorylated Mad, compare lanes 3 and 4. ( d ) S2R+ cells were co-transfected with Flag-Mad and Dullard-D66E. Flag-Mad was immunoprecipitated (IP) with anti-flag beads and then subjected to western blotting to investigate if both proteins interact. Results demonstrate Dullard-D66E and Flag-Mad proteins do physically interact. All western blots presented were repeated at least 2 times.

Article Snippet: Primary antibodies were used at the following concentrations, pMad Cter (Cell Signaling), 1:1000; pMad 212 (E. De Robertis), 1:1000; pMad 204/208 (E. De Robertis), 1:1000; anti-Flag (Sigma), 1:1000, anti-β-Tubulin (E7-c, Hybridoma bank), 1:1000; anti-Dullard (Sigma), 1:1000; anti-Mad (Newfeld) 1:1000.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Cyclo(Pro-Tyr) elicits conserved cellular damage in fungi by targeting the [H + ]ATPase Pma1 in plasma membrane domains

doi: 10.1101/2023.12.29.573620

Figure Lengend Snippet: Effect of cyclo(Pro-Tyr) on membrane microdomains. a . Representative fluorescence microscopy images of Pma1 immunodetection in B. cinerea cells treated with cyclo(Pro-Tyr) in comparison with untreated cells. Immunocytochemistry using an anti-Pma1 mouse monoclonal primary antibody and an anti-mouse GFP-conjugated secondary antibody showed that Pma1 formed foci in B. cinerea cells that colocalized with endocytic vesicles stained with FM4-64 upon the addition of cyclo(Pro-Tyr). No Pma1 signal was observed in control cells. Scale bar 5 μm. b . Representative fluorescence microscopy images of GFP-tagged Pma1 localization in C. albicans . In control cells, Pma1 localization was restricted to the plasma membrane, and no internal vesicles stained with FM4-64 were observed. In cyclodipeptide-treated cells, Pma1 localized in the plasma membrane as well as intracellular foci that colocalized with endocytic vesicles (arrowheads). Scale bar, 10 μm. c . Structural model of the Pma1 monomer subunit depicted as hydrophobic potential showing the insertion within the plasma membrane and the putative interaction with cyclo(Pro-Tyr). The putative binding site was formed by the cavity between alpha helices M5, M7, M8 and M10 located at the C-terminal domain. d . Molecular docking between the cyclo(Pro-Tyr) molecule and Pma1. The proposed binding site is formed by the residues K749, A814, Y875, and Q879. The measured distances between the cyclo(Pro-Tyr) and side chains of these residues were 2.44 Å, 1.88 Å, 2.13 Å, and 2.25 Å, respectively.

Article Snippet: Finally, the cells were stained for immunofluorescence using a 1:50 mouse monoclonal anti-Pma1 primary antibody (Thermo Fisher) followed by a 1:200 GFP-conjugated rabbit anti-mouse secondary antibody (Jackson ImmunoResearch).

Techniques: Membrane, Fluorescence, Microscopy, Immunodetection, Comparison, Immunocytochemistry, Staining, Binding Assay

Journal: bioRxiv

Article Title: Cyclo(Pro-Tyr) elicits conserved cellular damage in fungi by targeting the [H + ]ATPase Pma1 in plasma membrane domains

doi: 10.1101/2023.12.29.573620

Figure Lengend Snippet: Overall scheme of the proposed mechanism for cyclo(Pro-Tyr). Left: In the dynamic environment of the rhizosphere, B. velezensis engages in intricate interactions with a variety of microorganisms, forging connections that influence the ecosystem’s delicate balance. This bacterium employs a multifaceted approach to interact with its neighboring organisms, which includes fungi and nematodes. One of the strategies employed by B. velezensis involves the secretion of bioactive small molecules, including cyclo(Pro-Tyr), most likely as part of vesicles. Right: Cyclo(Pro-Tyr) binds to Pma1, affecting folding or hexamerization. This impairment triggers a membrane damage alert signal, leading to the synthesis of ROS and prompting the removal of damaged membrane regions by endocytosis. The internalization and degradation of these regions, accompanied by the production of ROS, result in the dislocation of key proteins involved in membrane polarity maintenance, such as Pma1, and disruption of the organism’s homeostasis. Simultaneously, the decrease in phospholipids per unit volume increases membrane fluidity, becoming another source of stress contributing to the synthesis of ROS. Due to the increased membrane fluidity, the region is more permeable to that cyclodipeptides are released in vesicles, specialized carriers that can deliver bioactive molecules to their intended targets. Created with BioRender.com.

Article Snippet: Finally, the cells were stained for immunofluorescence using a 1:50 mouse monoclonal anti-Pma1 primary antibody (Thermo Fisher) followed by a 1:200 GFP-conjugated rabbit anti-mouse secondary antibody (Jackson ImmunoResearch).

Techniques: Membrane, Disruption

Journal: bioRxiv

Article Title: Two transcriptional cascades orchestrate cockroach leg regeneration

doi: 10.1101/2023.12.09.570905

Figure Lengend Snippet: zfh-2 contributes to blastema proliferation and morphogenesis under the control of the BMP and JAK-STAT signaling. (A). Phenotypes of regenerated legs after ds zfh-2 treatments at three different time points. The legs in bottom line are zoom magnified from up line. n=3. (B). EdU and PH3 staining of proliferation positive cells. dsRNAs were injected at 0 dpa and the samples were harvested at 3 dpa, the EdU was injected at 6 h before harvest. n=3. (C). qRT‒PCR detection of the relative expression of zfh-2 under the dsRNA treatments of Mad , Stat and Sd . n=3. (D). Utilizing a dual luciferase assay to detect the regulatory impact of proteins on the zfh-2 promoter. n=3. (E). Immunohistochemistry staining of pMad and zfh-2 protein. (F). IGV snapshot of zfh-2 displaying chromatin accessibility on its TSS region during unamputated (CL) and amputated (AM) conditions. (G). CUT&Tag qPCR detection of the relative Mad protein binding affinity on zfh-2 BS in both ds Mock and ds Mad groups, two replicates were used. Data are mean±sd, the differences were analyzed by two-tailed Student’s t -test. (H). EMSA using overexpressed Mad(2SD)/Med proteins from KC cells incubated with FAM-labelled WT probe and additional unlabeled probes (cold and mutant). All the probes containing a putative binding site (in blue) were derived from the region −634—595 nt distance to ATG, and the mutant nucleotides are marked in red. (I). Volcano plot analysis of the DEGs under ds zfh-2 treatment. The violet dots indicate downregulated genes, and the red dots indicate upregulated genes. (J). Relative down regulation of B-H2 , Lim1 , and bab1 under ds zfh-2 treatment. (K-K’). Phenotypes and relative regenerative length of regenerated legs when B-H2 , Lim1 , and bab1 were knocked down simultaneously. n=3. The significance of differences was analyzed by two-tailed Student’s t test . *: P <0.05, **: P <0.01, ***: P <0.001, “ns” stands for no significant difference.

Article Snippet: To detail the distribution of pMad and zfh-2 protein at the regenerating site, serial section staining was performed because both the primary anti-pMad antibody (Abcam, ab52903) and anti-zfh-2 antibody (rabbit polyclonal antibody, customized from ABclonal) were from rabbit.

Techniques: Staining, Injection, Expressing, Luciferase, Immunohistochemistry, Protein Binding, Two Tailed Test, Incubation, Mutagenesis, Binding Assay, Derivative Assay

Journal: Communications Biology

Article Title: Cyclo(Pro-Tyr) elicits conserved cellular damage in fungi by targeting the [H + ]ATPase Pma1 in plasma membrane domains

doi: 10.1038/s42003-024-06947-3

Figure Lengend Snippet: a Representative fluorescence microscopy images of Pma1 immunodetection in B. cinerea cells treated with cyclo(Pro-Tyr) in comparison with untreated cells. Immunocytochemistry using an anti-Pma1 mouse monoclonal primary antibody and an anti-mouse GFP-conjugated secondary antibody showed that Pma1 formed foci in B. cinerea cells that colocalized with endocytic vesicles stained with FM4-64 upon the addition of cyclo(Pro-Tyr). No Pma1 signal was observed in control cells. Scale bar 5 μm. b Representative fluorescence microscopy images of GFP-tagged Pma1 localization in C. albicans . In control cells, Pma1 localization was restricted to the plasma membrane, and no internal vesicles stained with FM4-64 were observed. In cyclodipeptide-treated cells, Pma1 localized in the plasma membrane as well as intracellular foci that colocalized with endocytic vesicles (arrowheads). Scale bar, 10 μm. c Structural model of the Pma1 monomer subunit depicted as hydrophobic potential showing the insertion within the plasma membrane and the putative interaction with cyclo(Pro-Tyr). The putative binding site was formed by the cavity between alpha helices M5, M7, M8 and M10 located at the C-terminal domain. d Molecular docking between the cyclo(Pro-Tyr) molecule and Pma1. The proposed binding site is formed by the residues K749, A814, Y875, and Q879. The measured distances between the cyclo(Pro-Tyr) and side chains of these residues were 2.44 Å, 1.88 Å, 2.13 Å, and 2.25 Å, respectively.

Article Snippet: Finally, the cells were stained for immunofluorescence using a 1:50 mouse monoclonal anti-Pma1 primary antibody (Thermo Fisher) followed by a 1:200 GFP-conjugated goat anti-mouse secondary antibody (Thermo Fisher).

Techniques: Fluorescence, Microscopy, Immunodetection, Comparison, Immunocytochemistry, Staining, Control, Clinical Proteomics, Membrane, Binding Assay

Journal: Communications Biology

Article Title: Cyclo(Pro-Tyr) elicits conserved cellular damage in fungi by targeting the [H + ]ATPase Pma1 in plasma membrane domains

doi: 10.1038/s42003-024-06947-3

Figure Lengend Snippet: a Immunocytochemistry using an anti-Pma1 mouse monoclonal primary antibody and an anti-mouse GFP-conjugated secondary antibody showed that Pma1 concentrated in foci in B. cinerea cells, as observed previously in this work. Concurrently, the rhodamine-labeled cyclodipeptide exhibited red fluorescence, also forming foci. The overlay of these fluorescent signals revealed a significant degree of colocalization, evidenced by the appearance of yellow signals in the merged images. This colocalization suggests that Pma1 and the cyclodipeptide occupy the same intracellular compartments, potentially within endosomes, indicating a direct or closely associated interaction between them. Scale bar = 5 μm. b The graph illustrates the effect of the cyclo(Pro-Tyr) dipeptide on the ATPase activity of Pma1 2 μM, as measured by the concentration of free ATP. As the concentration of cyclo(Pro-Tyr) increases, there is a corresponding increase in the concentration of free ATP. This suggests that cyclo(Pro-Tyr) inhibits the ATPase activity of Pma1, preventing it from hydrolyzing ATP into ADP and inorganic phosphate. The inhibition is indicated by the accumulation of free ATP in the presence of higher concentrations of cyclo(Pro-Tyr), highlighting a dose-dependent relationship. c The graph presents a Lineweaver-Burk plot (double reciprocal plot) to analyze the effect of the cyclo(Pro-Tyr) dipeptide on the kinetic behavior of the Pma1 enzyme. Data points are plotted for Pma1 in the absence (red circles) and presence (black circles) of cyclo(Pro-Tyr). In the absence of cyclo(Pro-Tyr), the plot shows a linear relationship between 1/[ATP] and 1/V, indicative of typical Michaelis-Menten kinetics for Pma1. However, in the presence of cyclo(Pro-Tyr), the plot exhibits a steeper slope and a higher y-intercept, suggesting significant changes in the enzyme kinetics. The increase in slope (Km/Vmax) indicates that cyclo(Pro-Tyr) causes an increase in the apparent Km value, reflecting a lower affinity for ATP. Additionally, the elevated y-intercept implies a decrease in the Vmax of the enzyme. These changes are characteristic of a mixed-type inhibition, where cyclo(Pro-Tyr) can bind to both the free enzyme and the enzyme-substrate complex, altering both the affinity for the substrate and the maximum reaction rate. d Top: ATR-FTIR spectra of Pma1 alone (red) and cyclodipeptide-treated Pma1 (black) in the fingerprint region (1500–800 cm -1 ). Bottom: second derivative of the amine C-N stretch band (1350–1200 cm -1 ) where a clear shift was observed in the presence of cyclodipeptide. Individual data points for the graphs have been provided in the Supplementary Data file .

Article Snippet: Finally, the cells were stained for immunofluorescence using a 1:50 mouse monoclonal anti-Pma1 primary antibody (Thermo Fisher) followed by a 1:200 GFP-conjugated goat anti-mouse secondary antibody (Thermo Fisher).

Techniques: Immunocytochemistry, Labeling, Fluorescence, Activity Assay, Concentration Assay, Inhibition

Journal: Communications Biology

Article Title: Cyclo(Pro-Tyr) elicits conserved cellular damage in fungi by targeting the [H + ]ATPase Pma1 in plasma membrane domains

doi: 10.1038/s42003-024-06947-3

Figure Lengend Snippet: Left: In the dynamic environment of the rhizosphere, B. velezensis engages in intricate interactions with a variety of microorganisms, forging connections that influence the ecosystem’s delicate balance. This bacterium employs a multifaceted approach to interact with its neighboring organisms, which includes fungi and nematodes. One of the strategies employed by B. velezensis involves the secretion of bioactive small molecules, including cyclo(Pro-Tyr), most likely as part of vesicles. Right: Cyclo(Pro-Tyr) binds to Pma1, affecting folding or hexamerization. This impairment triggers a membrane damage alert signal, leading to the synthesis of ROS and prompting the removal of damaged membrane regions by endocytosis. The internalization and degradation of these regions, accompanied by the production of ROS, result in the dislocation of key proteins involved in membrane polarity maintenance, such as Pma1, and disruption of the organism’s homeostasis. Simultaneously, the decrease in phospholipids per unit volume increases membrane fluidity, becoming another source of stress contributing to the synthesis of ROS. Due to the increased membrane fluidity, the region is more permeable to that cyclodipeptides are released in vesicles, specialized carriers that can deliver bioactive molecules to their intended targets. Created with BioRender.com.

Article Snippet: Finally, the cells were stained for immunofluorescence using a 1:50 mouse monoclonal anti-Pma1 primary antibody (Thermo Fisher) followed by a 1:200 GFP-conjugated goat anti-mouse secondary antibody (Thermo Fisher).

Techniques: Membrane, Disruption

Journal: bioRxiv

Article Title: Genomic structure of Hstx2 modifier of Prdm9 -dependent hybrid male sterility in mice

doi: 10.1101/670422

Figure Lengend Snippet: (A) Transcript variants of Fmr1nb are shown, comprising six, five and four exons. Deletion mutants of B6 and PWD alleles of Fmr1nb were generated by TALEN nuclease pair constructs targeted to the ATG start codon of Fmr1nb in C57Bl/6N (B6N) laboratory strain and C57BL/6J-ChrX.1s PWD/Ph (B6.DX.1s) subconsomic strain, respectively. (B) FMR1NB protein levels in the testes of males of indicated genotypes were assessed by western blot. None of the three isoforms of FMR1NB was detectable in the Fmr1nb -deficient strain. Loading control was beta-TUBULIN. (C) Immunolabeling of FMR1NB and SYCP3 in histological sections of testis of B6.DX.1s and B6.DX.1s. Fmr1nb . FMR1NB , green; SYCP3 , violet; DAPI, blue. Scale bar, 50 μm.

Article Snippet: Primary antibodies against FMR1NB (Santa Cruz Biotechnology, sc-246953, Goat polyclonal) and ◻-tubulin (Proteintech, 66031-1-Ig, Mouse monoclonal) were used at the 1:1000 and 1:2000 dilutions, respectively.

Techniques: Generated, Construct, Western Blot, Immunolabeling

Journal: bioRxiv

Article Title: Genomic structure of Hstx2 modifier of Prdm9 -dependent hybrid male sterility in mice

doi: 10.1101/670422

Figure Lengend Snippet: (A) Average numbers of apoptotic cells per one tubule were plotted for individual males (N=3) of both genotypes (n, total number of tubules analyzed; p<0.01). (B) Apoptotic cells were visualized by FITC fluorescence using TUNEL assay in histological sections of testis of B6.DX.1s wild type and B6.DX.1s. Fmr1nb -null mutant genotypes.

Article Snippet: Primary antibodies against FMR1NB (Santa Cruz Biotechnology, sc-246953, Goat polyclonal) and ◻-tubulin (Proteintech, 66031-1-Ig, Mouse monoclonal) were used at the 1:1000 and 1:2000 dilutions, respectively.

Techniques: Fluorescence, TUNEL Assay, Mutagenesis

Journal: PLoS Genetics

Article Title: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

doi: 10.1371/journal.pgen.1005810

Figure Lengend Snippet: (A) Diagram of BMP signaling complexes that control the accumulation of nuclear and synaptic pMad. Extracellular BMPs bind to a complex composed of Type I and Type II BMP receptors. The BMP/BMPR complexes are endocytosed and transported to the neuron soma, where they phosphorylate Mad and allow for translocation and accumulation of pMad in the motor neuron nuclei. Synaptic pMad mirrors the active postsynaptic GluRIIA and likely reflects local accumulation of BMP/BMPR complexes. (B-D) 3D-SIM images of NMJ12 boutons from third instar larvae labeled for Brp (green), pMad (red), and Neto (blue). SIM z stack maximum projections are shown in (A–B) and a single z plane is shown in (C). See also and Movies. (E) High magnification view of a single synapse profile (from panel C). The line indicates the position used for the linescan plotted in panel F. (F) Side view of a surface rendered volume of the synapse shown in panel D. (G) Intensity profile of Neto, pMad and Brp signal along the line drawn in panel E. Linescans like this were performed across many synapses to measure the distance of pMad and Neto from Brp. (H) High magnification view of a z series through a single synapse imaged en face . The z interval was 110nm. Both merged and individual channels are shown. See also . Scale bars: 2 μm (B), 1 μm (C), 500 nm (C), 200 nm (E and H).

Article Snippet: Other primary antibodies were as follows: rabbit anti-phosphorylated Mothers against decapentaplegic (pMad), 1:500, (a gift from Carl Heldin) [ ]; rabbit anti-pSmad3, 1:500, (Epitomics, [ ]); FITC-, rhodamine-, and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.); rabbit anti-GFP, 1:250 (Abcam); rat anti-Neto, 1:1000 [ ]; Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Control, Translocation Assay, Labeling

Journal: PLoS Genetics

Article Title: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

doi: 10.1371/journal.pgen.1005810

Figure Lengend Snippet: (A-D) Confocal images of NMJ4 boutons (A) or ventral ganglia (B) (quantified in C-D) from third instar larvae immunostained for pMad (red), GFP (green), and HRP (blue). (A) Lack of synaptic pMad at mad null mutants is restored by expression of Mad-GFP in motor neurons ( mad; N>Mad-GFP ). Muscle expression of Mad-GFP ( mad; M>Mad-GFP ) does not rescue synaptic pMad, even though Mad-GFP accumulates around synaptic boutons. (B) Expression of Mad-GFP in motor neurons of mad mutants leads to elevated nuclear pMad levels. Muscle expression of Mad-GFP does not restore nuclear pMad in mad mutants, except for a small subset of neurons expressing Mad-GFP, which were excluded from quantification. Genotypes: control ( w 1118 ), mad ( mad 12/Df ), mad; N>Mad-GFP (380-Gal4/+; mad 12/Df ; UAS-Mad-GFP/+ ), mad; M>Mad-GFP ( mad 12/Df ; 24B-Gal4/UAS-Mad-GFP) . Error bars indicate SEM. ***; p<0.001. Scale bars: 10 μm.

Article Snippet: Other primary antibodies were as follows: rabbit anti-phosphorylated Mothers against decapentaplegic (pMad), 1:500, (a gift from Carl Heldin) [ ]; rabbit anti-pSmad3, 1:500, (Epitomics, [ ]); FITC-, rhodamine-, and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.); rabbit anti-GFP, 1:250 (Abcam); rat anti-Neto, 1:1000 [ ]; Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Expressing, Control

Journal: PLoS Genetics

Article Title: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

doi: 10.1371/journal.pgen.1005810

Figure Lengend Snippet: (A-D) Confocal images of NMJ4 boutons (A and D) (quantified in B-C) from larvae of indicated genotypes immunostained for pMad (red), HRP (blue), which labels the neuronal surface, and GluRIIA or Brp (green). (A) pMad localizes as discrete puncta at control and nwk or hiw mutant NMJs, but is absent in GluRIIA; nwk and hiw; GluRIIA double mutants. Loss of synaptic pMad caused by mutations in GluRIIA does not rescue the aberrant morphology of nwk mutant NMJs, and not prevent the NMJ overgrowth of hiw mutants. (D) Local pMad levels are reduced at imp mutant NMJs and elevated in nwk mutants. Loss of nwk restores the synaptic pMad at imp NMJs ( imp; nwk ), but morphology remains similar to nwk alone. Muscle overexpression of GluRIIA restores the synaptic pMad, but not the NMJ growth to imp mutants ( imp; M>IIA ). The number of NMJs examined is indicated in each bar. Genotypes: control ( w 1118 ), nwk ( nwk 1/γ3 ), IIA; nwk ( GluRIIA SP16/Df ; nwk 1/γ3 ), hiw ( hiw ND8 ), hiw; IIA ( hiw ND8 ; GluRIIA SP16/Df ), imp ( imp 24/70 ), imp; nwk ( imp 24/70 ; nwk 1/γ3 ), imp; M>IIA ( UAS-GluRIIA / + ; imp 24/70 ; 24B-Gal4/+) . Error bars indicate SEM. ***; p<0.001, **; p<0.01, *; p<0.05. Scale bars: 10 μm and 1 μm (details).

Article Snippet: Other primary antibodies were as follows: rabbit anti-phosphorylated Mothers against decapentaplegic (pMad), 1:500, (a gift from Carl Heldin) [ ]; rabbit anti-pSmad3, 1:500, (Epitomics, [ ]); FITC-, rhodamine-, and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.); rabbit anti-GFP, 1:250 (Abcam); rat anti-Neto, 1:1000 [ ]; Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Control, Mutagenesis, Over Expression

Journal: PLoS Genetics

Article Title: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

doi: 10.1371/journal.pgen.1005810

Figure Lengend Snippet: (A) Confocal images of ventral ganglia of third instar larvae of indicated genotypes labeled for pMad (red) and Elav (blue), which marks the motor neuron nuclei. Nuclear pMad is greatly increased when Mad-GFP is expressed in neurons ( N>Mad-GFP ) but not when expressed in muscle ( M>Mad-GFP ). (B) None of these manipulations affect the accumulation of pMad at synaptic sites, as indicated in confocal images of NMJ4 boutons (quantified in (C)). (D) Neuronal expression of Twit-GFP (green), a MiMIC-generated chimera, provides a read-out for the retrograde BMP signaling. Twit-GFP levels are not detectable in wit mutants, and are elevated when Mad is overexpressed in motor neurons. (E-F) Confocal images of NMJ4 boutons from larvae of indicated genotypes labeled for pMad, GluRIIA and HRP (E), or GFP (F). Local pMad is lost at GluRIIA mutant NMJs even when Mad-GFP is overexpressed and abundantly present at synaptic terminals. Genotypes: control ( w 1118 ), N>Mad-GFP ( 380-Gal4/+; +; UAS-Mad-GFP/+ ), M>Mad-GFP ( 24B-Gal4/UAS-Mad-GFP ), M , N>Mad-GFP ( elav-Gal4 , 24B-Gal4/UAS-Mad-GFP ), Twit-GFP ( Mi(MIC)twit MI06552 /+ ), Twit-GFP;wit A12/Df ( Mi(MIC)twit MI06552 /+ ; wit A12/Df ), Twit-GFP;N>Mad-Myc ( 380-Gal4/+; Mi(MIC)twit MI06552 /+; UAS-Mad-Myc ). Error bars indicate SEM. ***; p<0.001. Scale bars: 20 μm (A and D) and 5 μm (B, E and F).

Article Snippet: Other primary antibodies were as follows: rabbit anti-phosphorylated Mothers against decapentaplegic (pMad), 1:500, (a gift from Carl Heldin) [ ]; rabbit anti-pSmad3, 1:500, (Epitomics, [ ]); FITC-, rhodamine-, and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.); rabbit anti-GFP, 1:250 (Abcam); rat anti-Neto, 1:1000 [ ]; Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Labeling, Expressing, Generated, Mutagenesis, Control

Journal: PLoS Genetics

Article Title: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

doi: 10.1371/journal.pgen.1005810

Figure Lengend Snippet: (A-B) Confocal images of ventral ganglia (A) and NMJ4 boutons (B) from larvae of indicated genotypes labeled for pMad (red) and Elav (blue) (A), or Brp (green) and HRP (blue) (B). Nuclear pMad is greatly reduced in both gbb and wit mutants, but synaptic pMad appears normal in all gbb null alleles tested. (C) Maximum intensity projection of 3D-SIM images of NMJ12 boutons from third instar gbb mutant larvae labeled for Brp (green), pMad (red), and Neto (blue). Arrows, enlarged active zones; arrowheads, multiple T-bars. (D) A single z plane of the top right bouton in panel (C) magnified. See also . SIM z stack maximum projections are shown in (C) and single z plane in (D). (E) High magnification view of a 3D-SIM z series through of an individual gbb mutant synapse imaged en face . See also . Scale bars: 20 μm (A), 5 μm (B), 1 μm (C), 500 nm (D), 100 nm (E).

Article Snippet: Other primary antibodies were as follows: rabbit anti-phosphorylated Mothers against decapentaplegic (pMad), 1:500, (a gift from Carl Heldin) [ ]; rabbit anti-pSmad3, 1:500, (Epitomics, [ ]); FITC-, rhodamine-, and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.); rabbit anti-GFP, 1:250 (Abcam); rat anti-Neto, 1:1000 [ ]; Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Labeling, Mutagenesis

Journal: PLoS Genetics

Article Title: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

doi: 10.1371/journal.pgen.1005810

Figure Lengend Snippet: (A-C) Confocal images of NMJ4 boutons from larvae of indicated genotypes labeled for pMad (red), HRP (blue) and GluRIIA (green). The accumulation of pMad at gbb mutant NMJs is reduced by postsynaptic GluRIIA knockdown (A) or loss of Wit (B). Knockdown of Mav in the glia or Put in the striated muscle (C) diminished the synaptic pMad accumulation (quantified in D). Genotypes: control ( w 1118 ), gbb ( gbb 1/2 ), gbb; IIA RNAi ( gbb 1/2 ; UAS-GluRIIA RNAi / 24B-Gal4 ), gbb; wit ( gbb 1/2 ; wit A12/Df ), G>mav RNAi (repo-Gal4/UAS-mav RNAi ) , gbb; G>mav RNAi (gbb 1/2 ; repo-Gal4/UAS-mav RNAi , M>put RNAi (G14-Gal4/UAS-put RNAi ) . Error bars indicate SEM. ***; p<0.001, *; p<0.05. Scale bars: 5 μm.

Article Snippet: Other primary antibodies were as follows: rabbit anti-phosphorylated Mothers against decapentaplegic (pMad), 1:500, (a gift from Carl Heldin) [ ]; rabbit anti-pSmad3, 1:500, (Epitomics, [ ]); FITC-, rhodamine-, and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.); rabbit anti-GFP, 1:250 (Abcam); rat anti-Neto, 1:1000 [ ]; Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Labeling, Mutagenesis, Knockdown, Control

Journal: PLoS Genetics

Article Title: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

doi: 10.1371/journal.pgen.1005810

Figure Lengend Snippet: (A-E) Confocal images of NMJ4 boutons (A, D) and ventral ganglia (B) from larvae of indicated genotypes labeled for pMad (red), Elav (green) and HRP (blue). (A-C) Synaptic and nuclear pMad require the type I BMP receptor Sax (quantified in C). (D-E) Synaptic pMad does not require the LIMK1 binding domain of Wit (quantified in E). Genotypes: control ( w 1118 ), sax ( sax 4/Df ), wit ( wit A12/Df ), gbb ( gbb 1/2 ), gbb; IIA RNAi ( gbb 1/2 ; UAS-GluRIIA RNAi / 24B-Gal4 ), gbb; wit ( gbb 1/2 ; wit A12/Df ), wit ΔC ( wit ΔC . genomic ; wit A12/Df ), wit wt ( wit genomic ; wit A12/Df ). Error bars indicate SEM. ***; p<0.001. Scale bars: 5 μm (A, D), 10 μm (B).

Article Snippet: Other primary antibodies were as follows: rabbit anti-phosphorylated Mothers against decapentaplegic (pMad), 1:500, (a gift from Carl Heldin) [ ]; rabbit anti-pSmad3, 1:500, (Epitomics, [ ]); FITC-, rhodamine-, and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.); rabbit anti-GFP, 1:250 (Abcam); rat anti-Neto, 1:1000 [ ]; Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Labeling, Binding Assay, Control

Journal: PLoS Genetics

Article Title: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

doi: 10.1371/journal.pgen.1005810

Figure Lengend Snippet: (A-D) Confocal images of NMJ4 boutons from third instar larvae of indicated genotypes labeled for GluRIIA (green), GluRIIB (red), and HRP (blue) (quantified in B-C). The relative intensity of postsynaptic GluRIIA and GluRIIB signals decreases unequally in mutants lacking synaptic pMad, and induces a reduction in IIA/IIB ratio, except for gbb mutants (A-B). Equal reduction of GluRIIA and GluRIIB signals (and thus normal IIA/IIB ratio) is found in larvae with Mav-depleted glia and Put-depleted muscle (C-D). Genotypes: control ( w 1118 ), gbb ( gbb 1/Df ), wit ( wit A12/Df ), mad ( mad 12/Df ), imp ( imp 24/70 ), G>mav RNAi (repo-Gal4/UAS-mav RNAi ) , M>put RNAi (G14-Gal4/UAS-put RNAi ) . Error bars indicate SEM. ***; p<0.001, **; p<0.01, *; p<0.05. Scale bars: 5 μm and 1 μm (details).

Article Snippet: Other primary antibodies were as follows: rabbit anti-phosphorylated Mothers against decapentaplegic (pMad), 1:500, (a gift from Carl Heldin) [ ]; rabbit anti-pSmad3, 1:500, (Epitomics, [ ]); FITC-, rhodamine-, and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.); rabbit anti-GFP, 1:250 (Abcam); rat anti-Neto, 1:1000 [ ]; Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Labeling, Control

Journal: PLoS Genetics

Article Title: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

doi: 10.1371/journal.pgen.1005810

Figure Lengend Snippet: (A) Confocal images of NMJ4 boutons from third instar larvae labeled for GluRIIA (green), GluRIIB (red), and HRP (blue) (quantified in (B)). The Nrx-depleted NMJs have larger boutons, with increased GluRIIA and GluRIIB signals, and 25% increase in the IIA/IIB ratio. (C-D) pMad levels are increased at nrx mutant NMJs (C), but not in the motor neuron nuclei (D). (E-F) Confocal images of NMJ4 boutons from larvae of indicated genotypes labeled for pMad (red), HRP (blue) and Brp (green). The increased accumulation of pMad at nrx mutant NMJs is suppressed by loss of GluRIIA or Imp. Some synaptic pMad is observed in imp; nrx double mutants, but levels remain greatly reduced compared to control, matching the levels seen in imp mutants (quantified in F). Genotypes: control ( w 1118 ), nrx ( nrx 273/Df ), IIA; nrx ( GluRIIA SP16/Df ; nrx 273/Df ), imp ( imp 24/70 ), imp; nrx ( imp 24/70 ; nrx 273/Df ). Error bars indicate SEM. ***; p<0.001, *; p<0.05. Scale bars: 10 μm (A, C and E), 20 μm (D) and 1 μm (details).

Article Snippet: Other primary antibodies were as follows: rabbit anti-phosphorylated Mothers against decapentaplegic (pMad), 1:500, (a gift from Carl Heldin) [ ]; rabbit anti-pSmad3, 1:500, (Epitomics, [ ]); FITC-, rhodamine-, and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.); rabbit anti-GFP, 1:250 (Abcam); rat anti-Neto, 1:1000 [ ]; Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Labeling, Mutagenesis, Control

Journal: PLoS Genetics

Article Title: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

doi: 10.1371/journal.pgen.1005810

Figure Lengend Snippet: (A-D) Confocal images of NMJ4 boutons (A, D) and ventral ganglia (B) (quantified in C) from control and third instar larvae with a phosphomimetic Mad variant overexpressed in motor neurons ( N>Mad S25D ). Neuronal expression of Mad S25D greatly reduces the accumulation of synaptic pMad (A), but does not affect the nuclear pMad levels (B). Excess presynaptic Mad S25D induces a reduction of GluRIIA synaptic signals (green) and an increase of GluRIIB (red) relative to HRP (blue) (D). Scale bars: 10 μm (A and D) and 20 μm (B). (E-I) TEVC recordings from muscle 6, segment A3, of control and third instar larvae with excess presynaptic Mad S25D ( N>Mad S25D ) or Mad S25A ( N>Mad S25A ). (E) Representative traces of spontaneous junction currents recorded at 0.5 mM Ca 2+ . Summary graphs showing the mean amplitude (F) cumulative probability (G) mean frequency (H) and decay time constant (I) of mEJCs. The mEJC amplitude and decay constant were reduced when Mad S25D was overexpressed in the motor neurons. Overexpressing Mad S25A did not affect mEJC amplitude or decay constant but showed a reduction in mEJC frequency. Scale bars: 0.5 nA/500 ms (E) and 0.2 nA/25 ms (I). Genotypes: control ( 380-Gal4/Y ), N>Mad S25D ( 380-Gal4/Y; +; UAS-Mad S25D /+ ), N>Mad S25A ( 380-Gal4/Y; +; UAS-Mad S25A /+ ). Error bars indicate SEM. ***; p<0.001, **; p<0.01, *; p<0.05.

Article Snippet: Other primary antibodies were as follows: rabbit anti-phosphorylated Mothers against decapentaplegic (pMad), 1:500, (a gift from Carl Heldin) [ ]; rabbit anti-pSmad3, 1:500, (Epitomics, [ ]); FITC-, rhodamine-, and Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.); rabbit anti-GFP, 1:250 (Abcam); rat anti-Neto, 1:1000 [ ]; Cy5- conjugated goat anti-HRP, 1:1000 (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Control, Variant Assay, Expressing