Journal: bioRxiv

Article Title: Neuronal LRP4 directs the development, maturation, and cytoskeletal organization of peripheral synapses

doi: 10.1101/2023.11.03.564481

Figure Lengend Snippet: (A) Schematic of Drosophila third instar larva. Motor axons descend from the central nervous system (blue) to synapse with muscle fibers (tan). Muscles are organized in repeating segments along the length of the larva. (B) Representative confocal image (left) of a wild-type NMJ stained for HRP (cyan) to visualize neuronal membranes, and Dlg (magenta) to mark postsynapses. A diagram (right) depicting presynaptic boutons (blue) surrounded by the postsynaptic region (pink). Inset depicting the active zone (site of neurotransmitter release) and highlighting the active zone cytomatrix protein Bruchpilot (green). The active zone rests apposite a cluster of glutamate receptors (gray) located at the postsynaptic membrane. (C) Diagram of the lrp4 genomic region indicating the locus of a 3xHA tag, inserted at the C-terminus. (D) Representative confocal image of an NMJ expressing endogenous LRP4-HA, stained with antibodies to HA (red) and HRP (blue). LRP4-HA expression is visible within the motor neuron. Scale bars = 10 μm (E-H) Representative confocal image of an NMJ expressing endogenous LRP4-HA, stained with antibodies to HA (red), HRP (blue), and active zone marker Brp (green). Insets represent high magnification. LRP4-HA staining is visible in a punctate formation near active zones. Note the ring-like structure of HA staining surrounding Brp puncta. Scale bars = 5μm, 2.5μm (insets)

Article Snippet: The following primary antibodies were used: mouse anti-Brp (DSHB, cat. no. mAbnc82, 1:250) , mouse anti-Dlg (DSHB, cat. no. mAb4F3, 1:500) , rabbit anti-GluRIIC (custom, 1:2500) , mouse anti-Futsch (DSHB, mAb22C10, 1:50) , rabbit anti-β Spectrin (custom, 1:1000) , rabbit anti-HA RM305 (RevMab Biosciences, Burlingame, CA, cat. no. 31-1190-00, 1:500).

Techniques: Staining, Expressing, Marker

Journal: bioRxiv

Article Title: Neuronal LRP4 directs the development, maturation, and cytoskeletal organization of peripheral synapses

doi: 10.1101/2023.11.03.564481

Figure Lengend Snippet: (A-B) Representative confocal images of control (A) and lrp4 mutant (B) NMJs stained with antibodies to Brp (green), GluRIIC (red), and HRP (blue). Arrowheads in insets indicate unapposed puncta. Scale bars = 5μm, 2μm (insets) (C) Quantification of Brp puncta density. (D) Quantification of GluRIIC density. (E) Quantification of the ratio of Brp to GluRIIC puncta. (F) Quantification of the number of unapposed puncta per NMJ. Unapposed puncta increase significantly following loss of lrp4. (G-H) Representative images of control (G) and lrp4 mutant (H) NMJs stained with antibodies to HRP (blue) and Brp (green) visualized with STED microscopy. Insets show an example of a donut-shaped puncta. Scale bars = 1μm, 200 nm (inset) (I) Quantification of percent of donut-shaped Brp puncta (with a center hole). The percent of donut-shaped puncta decreases following loss of lrp4. (J) Diagram of experimental setup for electrophysiological analyses. An electrode records from muscle fiber, either in the presence (to record EJPs) or absence (to record mEJPs) of external stimulation of the motor axon. (K-L) Representative EJP and mEJP traces recorded from wild-type (H) and lrp4Del / y (I) muscle. (M) Quantification of EJP amplitude. Loss of lrp4 results in an over 50% decrease in EJP amplitude. (N) Quantification of mEJP amplitude. mEJP amplitude is unchanged in lrp4 mutants. (O) Quantification of quantal content. Quantal content is significantly decreased following loss of lrp4. For all experiments, data are shown as mean ± SEM. p** < 0.01, p*** < 0.001, p**** < 0.0001, ns = not significant. Significance was determined using a two-tailed Student’s t-test. n (C-F) ≥ 12 NMJs, 6 larvae. n (G-H) ≥ 47 boutons, 8 larvae. n (M-O) ≥ 12 NMJs, 5 larvae.

Article Snippet: The following primary antibodies were used: mouse anti-Brp (DSHB, cat. no. mAbnc82, 1:250) , mouse anti-Dlg (DSHB, cat. no. mAb4F3, 1:500) , rabbit anti-GluRIIC (custom, 1:2500) , mouse anti-Futsch (DSHB, mAb22C10, 1:50) , rabbit anti-β Spectrin (custom, 1:1000) , rabbit anti-HA RM305 (RevMab Biosciences, Burlingame, CA, cat. no. 31-1190-00, 1:500).

Techniques: Mutagenesis, Staining, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Neuronal LRP4 directs the development, maturation, and cytoskeletal organization of peripheral synapses

doi: 10.1101/2023.11.03.564481

Figure Lengend Snippet: (A-B) Representative confocal images from control (A) and srpk79D mutant (B) NMJs stained with antibodies to Brp (green), GluRIIC (red), and HRP (blue). Arrowheads in (B) indicate unapposed puncta. Scale bars = 5μm, 2μm (insets) (C) Quantification of Brp density. (D) Quantification of GluRIIC density. (E) Quantification of the number of unapposed puncta per NMJ. Unapposed puncta number is significantly increased following loss of srpk79D. For all experiments, data are shown as mean ± SEM. p* < 0.05, ns = not significant. Significance was determined using a two-tailed Student’s t-test. n ≥ 14 NMJs, 8 larvae.

Article Snippet: The following primary antibodies were used: mouse anti-Brp (DSHB, cat. no. mAbnc82, 1:250) , mouse anti-Dlg (DSHB, cat. no. mAb4F3, 1:500) , rabbit anti-GluRIIC (custom, 1:2500) , mouse anti-Futsch (DSHB, mAb22C10, 1:50) , rabbit anti-β Spectrin (custom, 1:1000) , rabbit anti-HA RM305 (RevMab Biosciences, Burlingame, CA, cat. no. 31-1190-00, 1:500).

Techniques: Mutagenesis, Staining, Two Tailed Test