d systems cat  (R&D Systems)

Journal: Nature Communications

Article Title: Siah2 antagonism of Pard3/JamC modulates Ntn1-Dcc signaling to regulate cerebellar granule neuron germinal zone exit

doi: 10.1038/s41467-024-55400-w

Figure Lengend Snippet: A Western blot of HEK293T cell lysates transfected with Dcc-pHluorin (Dcc-pH) and JamC-HALO. Immunoprecipitation was performed using GFP or negative control IgG antibodies. Blots were probed for Dcc and HALO-tag, with the yellow arrowhead indicating the expected JamC-HALO band size. B Schematic depicting interactions between Dcc, JamC, and the polarity protein Pard3. Pard3 recruits JamC to the membrane through its PDZ1 domain, which binds the Class 2 PDZ motif at JamC’s C-terminus. JamC interacts with Dcc’s extracellular domain, while Pard3’s PDZ3 the PDZ 3 domain is predicted to interact with a Class 1 PDZ binding motif (X-S/T-X-ϕ COOH ) , , present on Dcc intracellular domain. C , D Airyscan confocal imaging of CGNs nucleofected with Dcc-pHluorin (cyan), JamC-SNAP (yellow), and Halo-Pard3 (magenta). Phluorin and the SNAP dye used here are both pH sensitive highlighting membrane-bound proteins. C Single focal plane showing overlap of Dcc with JamC and Pard3 at the proximal dilation of a CGN (white arrowheads). D Maximum projection of two CGNs forming an adhesion, showing Dcc clustering at the adhesion site before and after Ntn1 addition (200 ng/L). Dcc co-localized with JamC/Pard3 at the adhesion (white arrowhead) and accumulated at the adhesion periphery (hollow arrowhead). Five minutes after the addition of Ntn1 at 200 ng/L, the number of bright Dcc clusters (blue arrowhead) at the membrane surface increased and some newly formed clusters were recruited to the periphery of the JamC/Pard3/Dcc-positive adhesion (white arrowhead). Proximity Labelling Assay (PLA) using Duolink™ fluorescence protocol on fixed dissociated granule neurons plated on laminin and cultured for 24 h, using 2 pairs of primary antibodies: Rabbit against Dcc extracellular domain (ECD) and a Goat against JamC ECD ( E ), and Mouse against Dcc intracellular domain (ICD) and a Rabbit against Pard3 ( F ). Duolink™ staining with no primary, only one or both primaries were compared. Bar graphs represent the ratio of PLA staining intensity (Gray) against Dapi (Cyan) intensity normalized to the negative control without primary antibody (E: n = 4, F: n = 4), replicated 4 (E) and 3 (F) times with similar results. Scale bars: (C, D) 5 µm, (E-F) 10 µm. Error bars represent SEM. See Source Data File.

Article Snippet: Two pairs of primary antibodies were used: Rabbit against Dcc (ECD) (ab273570, Abcam 1:200 dil) and Goat against JamC (ECD) (AF1213, R&D systems, 1:50 dil), and Mouse against Dcc (ICD) (A-1, Santa Cruz 1:100 dil) and Rabbit against Pard3 (07-330, Sigma-Aldrich 1:200 dil).

Techniques: Western Blot, Transfection, Immunoprecipitation, Negative Control, Membrane, Binding Assay, Imaging, Fluorescence, Cell Culture, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Adhesion-clutch between DCC and netrin-1 mediates netrin-1–induced axonal haptotaxis

doi: 10.3389/fnmol.2024.1307755

Figure Lengend Snippet: DCC interacts with shootin1a. (A) Co-immunoprecipitation of DCC-ICD with shootin1a in HEK 293T cells. HEK293T cells were transfected with vectors to express FLAG-shootin1a and EGFP-DCC-ICD; cells were also co-transfected with a vector to express EGFP as a negative control. Cell lysates were prepared and incubated with anti-FLAG antibody for immunoprecipitation. The immunoprecipitants were immunoblotted with anti-FLAG or anti-GFP antibody. Whole immunoblot images are presented in . (B) Co-immunoprecipitation of endogenous shootin1a and DCC in cultured cortical neurons (DIV 2). Cell lysates were incubated with anti-shootin1 antibody or control IgG, then precipitated with protein G-Sepharose 4B. The immunoprecipitants were immunoblotted with anti-shootin1a or anti-DCC antibody. Whole immunoblot images are presented in . (C) Fluorescence images of axonal growth cone of a DIV 2 hippocampal neuron labeled with anti-shootin1a (red) and anti-DCC (green) antibodies. Images below show enlarged views of the lamellipodium in the white square and the filopodium in the yellow square. Arrowheads indicate DCC colocalized with shootin1a. (D) Quantification of shootin1a-DCC colocalization using Pearson’s coefficient correlation. A typical quantified region of axonal growth cone is illustrated by the dotted line (left) . The Pearson’s coefficient correlations of all the growth cones ( n = 11) exceeded 0.5 (0.68 ± 0.03), indicating colocalization of shootin1a with DCC in growth cones. (E) Fluorescence images of axonal growth cone of a DIV 2 hippocampal neuron labeled with anti-shootin1a (red) and anti-paxillin (green) antibodies. Images below show enlarged views of the filopodia in the squares. Yellow and cyan arrowheads indicate shootin1a and paxillin, respectively. (F) Quantification of shootin1a-paxillin colocalization using Pearson’s coefficient correlation. The Pearson’s coefficient correlations of all the growth cones ( n = 11) were below 0.5 (0.33 ± 0.02), indicating weak colocalization between shootin1a and paxillin in growth cones. Scale bars: 5 μm for (C,E) upper figures; 1 μm for (C,E) enlarged figures.

Article Snippet: The following primary antibodies were used in immunoblotting: rabbit anti-shootin1a (1:5000) , and mouse anti-DCC antibodies (1:4000) (Santa Cruz Biotechnology, catalog number sc-515834).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Negative Control, Incubation, Western Blot, Cell Culture, Control, Fluorescence, Labeling

Journal: Frontiers in Molecular Neuroscience

Article Title: Adhesion-clutch between DCC and netrin-1 mediates netrin-1–induced axonal haptotaxis

doi: 10.3389/fnmol.2024.1307755

Figure Lengend Snippet: DCC and shootin1a couple F-actin flow with substrate-bound netrin-1. (A) Fluorescent speckle images of HaloTag-actin in axonal growth cones of WT and shootin1 KO neurons cultured on netrin-1 or polylysine. Time-lapse montages of HaloTag-actin speckles in filopodia (boxed areas) at 5-s intervals are shown below; pink dashed lines indicate the retrograde flow of speckles (see ). (B) Quantification of F-actin flow speeds measured from the time-lapse montage analyses in panel (A) (WT on netrin-1, n = 8 growth cones; KO on netrin-1, n = 8 growth cones; WT on polylysine, n = 6 growth cones; KO on polylysine, n = 8 growth cones). (C) Fluorescence images showing axonal growth cones expressing EGFP (blue) of DIV 2 WT (left) and shootin1 KO (right) neurons cultured on netrin-1–coated polyacrylamide gels with embedded 200-nm fluorescent beads. The pictures show representative images from time-lapse series taken every 3 s for 147 s. White lines indicate the growth cone boundaries (see ). The kymographs along the axis of bead displacement (white dashed arrows) at the indicated areas show movement of beads recorded every 3 s. The beads in areas 2 and 2′ are reference beads. (D) Quantification of the magnitude of the traction forces under axonal growth cones of WT and shootin1 KO neurons cultured on netrin-1–coated polyacrylamide gels in panel (C) (WT, n = 7 growth cones; shootin1 KO, n = 8 growth cones). (E) A diagram showing F-actin-substrate coupling in the axonal growth cone through cortactin, shootin1a, DCC and substrate-bound netrin-1. Shootin1a interacts with the actin binding protein cortactin and DCC . Scale bars: 2 μm for (A,C) . Data represent means ± SEM; *** p < 0.01; ns, not significant.

Article Snippet: The following primary antibodies were used in immunoblotting: rabbit anti-shootin1a (1:5000) , and mouse anti-DCC antibodies (1:4000) (Santa Cruz Biotechnology, catalog number sc-515834).

Techniques: Cell Culture, Fluorescence, Expressing, Binding Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Adhesion-clutch between DCC and netrin-1 mediates netrin-1–induced axonal haptotaxis

doi: 10.3389/fnmol.2024.1307755

Figure Lengend Snippet: Netrin-1–induced axon outgrowth and haptotaxis require shootin1a-mediated actin-DCC coupling. (A) Fluorescence images of DIV 2 hippocampal neurons prepared from WT or shootin1 KO mouse and cultured on netrin-1 or polylysine. Neurons were stained with anti-Tuj1 antibody. (B) Quantification of axon length obtained by the analyses using neurons in panel (A) (WT on netrin-1, n = 55 growth cones; KO on netrin-1, n = 55 growth cones; WT on polylysine, n = 55 growth cones; KO on polylysine, n = 55 growth cones). (C) Hippocampal neurons cultured on micro-scale patterns of netrin-1 (red) and polylysine (black) till DIV 3. Neurons were stained with anti-Tuj1 antibody (green). (D) Quantification of the percentage of axon length located on netrin-1 (see ) (WT, n = 16 cells; shootin1 KO, n = 14 cells). Scale bars, 25 μm for (A) ; 50 μm for (C) . Data represent means ± SEM; *** p < 0.01; ns, not significant.

Article Snippet: The following primary antibodies were used in immunoblotting: rabbit anti-shootin1a (1:5000) , and mouse anti-DCC antibodies (1:4000) (Santa Cruz Biotechnology, catalog number sc-515834).

Techniques: Fluorescence, Cell Culture, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Adhesion-clutch between DCC and netrin-1 mediates netrin-1–induced axonal haptotaxis

doi: 10.3389/fnmol.2024.1307755

Figure Lengend Snippet: DCC in growth cones undergoes differential grip and slip on the substrates. (A) Fluorescent speckle images of DCC-HaloTag at the plasma membrane of axonal growth cones cultured on polylysine (left) and netrin-1 (right) ; time-lapse montages of DCC-HaloTag speckles in filopodia on netrin-1 and on polylysine (boxed areas) at 5-s intervals are shown (grip and slip phases are indicated by dashed pink and blue lines, respectively) (see ). (B,C) Ratio of the grip and slip states (B) and retrograde flow speed (C) of DCC-HaloTag in filopodia measured from the time-lapse montage analyses in panel (A) (polylysine, n = 106 signals, 7 growth cones; netrin-1, n = 114 signals, 8 growth cones). (D) Grip and slip mechanism for netrin-1–induced axonal haptotaxis. The force of F-actin retrograde flow in the growth cone (yellow arrows) is transmitted to DCC through cortactin and shootin1a, allowing to pull the bond between DCC and the adhesive substrates. DCC molecules undergo grip or frictional slip (black arrows) on the substrates. As the substrate presenting netrin-1 has higher affinity to DCC than the control substrate, a larger number of DCC molecules undergo grip on the netrin-1-presenting substrate compared to the control substrate, leading to more efficient force transmission on the netrin-1 side (blue arrows). This asymmetric grip and slip of DCC within the growth cone generate directional force for axonal haptotaxis toward netrin-1 (green arrow). Scale bars, 2 μm for (A) . Data represent means ± SEM; *** p < 0.01.

Article Snippet: The following primary antibodies were used in immunoblotting: rabbit anti-shootin1a (1:5000) , and mouse anti-DCC antibodies (1:4000) (Santa Cruz Biotechnology, catalog number sc-515834).

Techniques: Clinical Proteomics, Membrane, Cell Culture, Adhesive, Control, Transmission Assay

Journal: iScience

Article Title: Mitochondrial network adaptations of microglia reveal sex-specific stress response after injury and UCP2 knockout

doi: 10.1016/j.isci.2023.107780

Figure Lengend Snippet:

Article Snippet: Mouse: B6;129S- Gt(ROSA)26Sor tm1(CAG-COX8A/Dendra2)Dcc /J , The Jackson Laboratory , JAX: #018385.

Techniques: Purification, Recombinant, cDNA Synthesis, Amplification, Software