Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: C. elegans BRC-1 and BRD-1. A , Structures of BRC-1 and BRD-1. Schematic diagrams of BRC-1, BRD-1, and their mammalian counterparts, BRCA1 and BARD1, are shown. RING finger domain is shown in red and BRCT domains in yellow. The bold lines underneath denote the extent of the deleted regions in the tm1145 , dw1 , and gk297 mutants. An asterisk indicates a premature stop codon caused by the km88 mutation. B , Isolation of brc-1 mutants. Genomic structure of the brc-1 gene is shown. Exons are indicated by boxes, and introns and untranslated regions are indicated by bars. Small and capital letters indicate nucleotides and the corresponding amino acids, respectively. The brc-1(km88) mutation is a 2-bp deletion, causing a frameshift (bold amino acids) and premature stop codon (*) in exon 2. C , Ring finger domain. Sequence alignment in the RING finger domain between BRCA1 and BRC-1 is shown. Identical and similar residues are highlighted with black and gray shading, respectively. The black arrow indicates the conserved isoleucine residue required for E3-Ub ligase activity.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques: Mutagenesis, Isolation, Sequencing, Activity Assay

Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: BRC-1 and BRD-1 are required for axon regeneration of D-type motor neurons. A , Representative D-type motor neurons in wild-type and brc-1(tm1145) brd-1(dw1) mutant animals 24 h after laser surgery. In wild-type animals, severed axons exhibited regenerated growth cones (yellow arrowheads). In brc-1(tm1145) brd-1(dw1) mutants, the proximal ends of axons failed to regenerate (white arrowheads). Scale bar: 10 µm. B , Percentages of axons that initiated regeneration 24 h after laser surgery at the young adult stage. The number of axons examined is shown. Error bars indicate 95% confidence intervals; ** p < 0.01, *** p < 0.001, as determined by Fisher's exact test. NS, not significant. C , Length of regenerating axons 24 h after laser surgery. Data are presented as a box-plot representing median (thick line within the box) and interquartile range (edge of box) with individual data points. The number ( n ) of axons examined is shown. Statistical significance was determined by Wilcoxon rank-sum test.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques: Mutagenesis

Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: BRC-1 and BRD-1 are required for axon regeneration of PLM sensory neurons. A , Representative PLM sensory neurons in wild-type and brc-1(tm1145) brd-1(dw1) mutant animals 24 h after laser surgery. Red arrowheads indicate cut sites. Scale bar: 10 µm. B , Length of PLM regrowth 24 h after laser surgery. Data are presented as a box-plot representing median (thick line within the box) and interquartile range (edge of box) with individual data points. The number ( n ) of axons examined is shown. Statistical significance was determined by Wilcoxon rank-sum test.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques: Mutagenesis

Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: BRC-1–BRD-1 functions in the EGL-30 Gqα signaling pathway to regulate axon regeneration. A , Percentages of axons that initiated regeneration 24 h after laser surgery at the L4 or young adult stage. The number of axons examined is shown. Error bars indicate 95% confidence intervals; ** p < 0.01, *** p < 0.001, as determined by Fisher's exact test. NS, not significant. B , Percentages of axons that initiated regeneration 24 h after laser surgery at the young adult stage. The number of axons examined is shown. Error bars indicate 95% confidence intervals; ** p < 0.01, *** p < 0.001, as determined by Fisher's exact test. NS, not significant.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques:

Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: Effects of negative regulators in the EGL-30 pathway on BRC-1–BRD-1-mediated axon regeneration. A , The EGL-30 pathway regulating axon regeneration. EGL-30 Gqα activates EGL-8 PLCβ, which in turn generates DAG from phosphatidylinositol bisphosphate [PI(4, 5)P2]. DAG activates TPA-1 PKC, resulting in activation of the JNK pathway to promote axon regeneration. GOA-1 Goα and EAT-16 RGS antagonize EGL-30 signaling. DGK downregulates the EGL-30 pathway by converting DAG to PA. B , Percentages of axons that initiated regeneration 24 h after laser surgery at the young adult stage. The number of axons examined is shown. Error bars indicate 95% confidence intervals; * p < 0.05, as determined by Fisher's exact test. NS, not significant.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques: Activation Assay

Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: BRC-1–BRD-1 downregulates DGK-3 to promote axon regeneration. A , Isolation of dgk-3 mutants. Genomic structure of the dgk-3 gene is shown. The dgk-3(km89) mutation is a 20-bp insertion (red nucleotides) that contains an in-frame premature stop codon (*), thus terminating translation in the middle of exon 1. The dgk-3(km90) mutation is a 5-bp deletion, causing a frameshift (bold amino acids) and premature stop codon (*) in exon 1. B , Percentages of axons that initiated regeneration 24 h after laser surgery at the young adult stage. The number of axons examined is shown. Error bars indicate 95% confidence intervals; * p < 0.05, ** p < 0.01, as determined by Fisher's exact test. NS, not significant.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques: Isolation, Mutagenesis

Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: BRC-1–BRD-1 mediates poly-ubiquitylation of DGK-3. A , Poly-ubiquitylation of DGK-3 by BRC-1–BRD-1. COS-7 cells were transfected with T7-DGK-3, HA-Ub, GFP-BRC-1, and BRD-1-RFP, as indicated. Cells were incubated with or without MG132. Cell lysates were immunoprecipitated (IP) with anti-T7 antibody and immunoblotted (IB) with anti-HA and anti-T7 antibodies. Total lysates were analyzed by IB with anti-GFP and anti-RFP antibodies. The experiment was done in triplicate with similar results, shown here for trial #2. B , Comparison of DGK-3 poly-ubiquitylation levels. The DGK-3 poly-ubiquitylation experiment was performed three times and each bar represents the result of each trial (#1–#3). Data represent the percentage of normalized poly-ubiquitylated DGK-3 in lane 5 relative to that found in lane 4. The blots in lanes 4 and 5 of A from three trials are shown in the upper part. C , Interaction of DGK-3 with BRC-1. COS-7 cells were co-transfected with T7-DGK-3, GFP-BRC-1, and BRD-1-RFP, as indicated. Cells were then incubated with MG132. Cell lysates were immunoprecipitated (IP) with anti-T7 antibody and immunoblotted (IB) with anti-GFP and anti-T7 antibodies. Total lysates were analyzed by IB with anti-GFP antibody.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques: Transfection, Incubation, Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: BRC-1–BRD-1 promotes axotomy induced degradation of cytoplasmic DGK-3. A , Fluorescent images of wild-type and brc-1 brd-1 mutant animals expressing Punc-47::mcherry (D-type motor neuron, top) and Punc-25::dgk-3::GFP (bottom) are shown. Images were taken at 0 or 8 h after laser surgery. Red arrowheads indicate the tip of the severed axons. Yellow arrows indicate cell bodies corresponding to the severed axons and their magnification is shown in the insets. Scale bar: 10 µm. B , Quantification of DGK-3::GFP fluorescence levels in cytoplasm of D-type neurons. Relative DGK-3 intensity was calculated as a fraction of the relative DGK-3::GFP intensity 8 h after laser surgery divided by the corresponding value at 0 h postaxotomy. Data are presented as a box-plot representing median (thick line within the box) and interquartile range (edge of box) with individual data points. The number ( n ) of axons examined is shown. Statistical significance was determined by Wilcoxon rank-sum test.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques: Mutagenesis, Expressing, Fluorescence

Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: PKA phosphorylates BRC-1. A , A schematic diagram of BRC-1. RING finger domain is shown in red, and BRCT domains in yellow. The amino acid sequences around a PKA phosphorylation consensus site (underline) and a putative nuclear localization signal (red characters) are shown below. The Ser-266 residue is indicated by an asterisk. B , PKA phosphorylates BRC-1 at Ser-266 in vitro . In vitro phosphorylation of BRC-1 by PKA is shown. COS-7 cells were transfected with GFP-BRC-1 (WT) or GFP-BRC-1(S266A), and cell lysates were immunoprecipitated (IP) with anti-GFP antibody. The immunoprecipitates were subjected to in vitro kinase assay using active recombinant PKA. Phosphorylated BRC-1 was detected by immunoblotting (IB) with anti-phospho-PKA substrate rabbit monoclonal antibody. C , Percentages of axons that initiated regeneration 24 h after laser surgery at the young adult stage. The number of axons examined is shown. Error bar indicates 95% confidence interval; *** p < 0.001, as determined by Fisher's exact test.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques: In Vitro, Transfection, Immunoprecipitation, Kinase Assay, Recombinant, Western Blot

Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: PKA phosphorylation induces cytoplasmic localization of BRC-1. A , Localization of BRC-1 in response to PKA activation. Fluorescent images of wild-type animals expressing Punc-47::mcherry (D-type motor neuron, top) and Punc-25::GFP::brc-1 or Punc-25::GFP::brc-1(S266A) (bottom) with or without forskolin treatment are shown. Red and yellow arrowheads indicate cell nucleus. Scale bar: 10 µm. B , Quantification of GFP::BRC-1 fluorescence levels in D-type neurons with or without forskolin treatment. The cytoplasmic-to-nuclear ratio of GFP::BRC-1 signal was calculated as a fraction of the relative GFP::BRC-1 intensity in the cytoplasm divided by the corresponding value in the nucleus. Data are presented as a box-plot representing median (thick line within the box) and interquartile range (edge of box) with individual data points. The number ( n ) of cell bodies examined is shown. Statistical significance was determined by Wilcoxon rank-sum test.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques: Activation Assay, Expressing, Fluorescence

Journal: The Journal of Neuroscience

Article Title: BRCA1–BARD1 Regulates Axon Regeneration in Concert with the Gqα–DAG Signaling Network

doi: 10.1523/JNEUROSCI.1806-20.2021

Figure Lengend Snippet: Schematic model for the regulation of axon regeneration by BRC-1–BRD-1. Under normal conditions, BRC-1–BRD-1 is localized in the nucleus. In response to axon injury, PKA phosphorylation of BRC-1 Ser-266 induces the translocalization of BRC-1–BRD-1 to the cytoplasm. The BRC-1–BRD-1 complex poly-ubiquitylates DGK-3, resulting in its degradation. BRC-1–BRD-1 enhances the EGL-30 signaling pathway to promote axon regeneration.

Article Snippet: GFP-BRC-1 proteins were immunopurified from transfected COS-7 cells using anti-GFP antibody (mouse M048-3; MBL).

Techniques: