Sgg is concentrated on the presynaptic side of the NMJ and negatively regulates NMJ growth ( Franco et al., 2004 ). A role for exosomes in transporting Wnt proteins was first reported at the Drosophila NMJ ( Korkut et al., 2009 ). At the NMJ, muscle-specific knockdown of Sfl reduced muscle size (Kamimura et al., 2013).
Knockdown of LAR resulted in a significant increase in phosphorylation of the substrate LAR Enabled (Ena; Johnson et al., 2006). Dlp expression at the NMJ was recently shown to be activity dependent (Dear et al., 2017). Neuralized (Neur) is a short-range target that requires high Wg signaling, whereas Distalless (Dll) is a long-range target that requires low Wg signaling (Zecca et al., 1996).
Overexpression of Dlp resulted in the binding and sequestration of Wg ligand ( Baeg et al., 2001 ). Notum is also depleted from the surface of dally dlp double mutants ( Kakugawa et al., 2015 ). Finally, Notum has been shown to be able to directly deacylate Wnt proteins, including Wg ( Kakugawa et al., 2015 ).
In the postsynaptic muscle, Wg binding to Fz2 activates the Frizzled Nuclear Import (FNI) signaling pathway, which involves Fz2 endocytosis, followed by Fz2 cleavage and Fz2 C-terminus (Fz2-C) nuclear import (Mathew et al., 2005) . Disruption of this mechanism is the cause of synaptogenic defects of fragile X syndrome (FXS) (Friedman et al., 2013). Wg trans-synaptic signaling regulates NMJ growth, synaptic bouton formation, and ultrastructural assembly ( Packard et al., 2002 ).
Using a fine brush (size 2), remove a third stray star from the feed bottle and place it on the elastomer covered glass. Fill the tip of the glass micropipette with a small volume of glue using the negative air pressure created by the attachment mouth (step 1.5). Place the larva back side up with forceps and attach the head to the elastomer covered coverslip with a small dot of glue using positive air pressure from the mouth.
Repeat this process with the back of the larva, making sure the animal is tensioned between the two glue sticks. Repeat the gluing process for the four case wall tabs, making sure to gently stretch the case wall horizontally and vertically. Lift the ventral nerve cord (VNC) with forceps, carefully cut the motor nerves with scissors, and then completely remove the VNC.
Replace the dissecting salt with Ca2+-free saline (same as CaCl2-free dissecting saline above) to stop SV cycling. Immediately start a digital timer for the predetermined duration of the high [K+] depolarizing stimulation period (eg, 5 min; Figure 16). To confirm a healthy larval preparation, note strong muscle contractions for the duration of the high [K+] depolarization period.
When the timer period ends, quickly remove the high [K+] FM dye solution and replace it with Ca2+-free saline to stop SV cycling. Wash sequentially with Ca2+-free saline (5x for 1 min) to ensure that the high [K+] FM dye solution is completely removed. Keep the larval preparation in fresh Ca2+-free saline for immediate confocal microscopy imaging.
Imaging: Confocal Microscopy
High [K + ] Stimulation: FM Dye Unloading
Immediately start a digital timer for the predetermined duration of the high [K+] stimulation period (e.g., 2 minutes; Figure 16). When the timer period expires, immediately remove the high [K+] saline and replace it with Ca2+-free saline to stop the SV cycle. Wash in rapid succession with Ca2+-free saline (5x for 1 min) to ensure complete removal of the high [K+] saline.
Be sure to image FM1-43 dye fluorescence at the same NMJ mentioned above using the same confocal settings.
Option 2: Electrical Stimulation FM Dye Loading
Test the performance of the suction electrode with a short burst of stimulation while visually monitoring muscle contraction in the selected hemisegment. Stimulate the motor nerve with selected parameters (step 5.4) to induce SV endocytosis and FM1-43 dye uptake (Figure 16). Rinse in rapid succession with Ca2+-free saline (5x 1 minute each) to ensure complete removal of the FM1-43 dye solution.
Store the larval preparation in fresh Ca2+-free saline for immediate imaging using the top-down confocal imaging protocol. Pay close attention to the depicted NMJ (segment, lateral, and muscle) to ensure access to the exact same NMJ after unloading the FM dye.
Electrical Stimulation: FM Dye Unloading
Option 3: Channelrhodopsin Stimulation FM Dye Loading
Place the larval preparation in the Plexiglas chamber on a dissecting microscope stage equipped with a camera port. Connect a blue LED (470 nm; Table 2) to a programmable stimulator using a coaxial cable and place the LED in the port of the camera. Focus the blue LED light beam on the dissected larva using the microscope's zoom function.
Replace the Ca 2+ -free saline on the larval preparation with the above FM1-43 saline (4 µM; 1 mM CaCl 2 ) at the optogenetic stage. Start the light stimulation and track with a timer for the predetermined duration of the optogenetic stimulation period (eg, 5 min; Figure 16). When the timer stops, quickly remove the FM dye solution and replace it with Ca2+-free saline to stop the SV cycle.
Wash rapidly in succession with Ca 2+ -free saline (5x for 1 min) to ensure complete removal of the FM dye solution. Maintain the larval preparation in fresh Ca 2+ -free saline for immediate imaging with the confocal microscope using the overhead imaging protocol.
Channelrhodopsin Stimulation: FM Dye Unloading
Start the light stimulation and track with a timer for the predetermined duration of the optogenetic stimulation period (eg, 2 min; Figure 16). When the timer period ends, quickly remove the FM staining solution and replace it with Ca 2+ -free saline to stop SV cycling. Wash sequentially with Ca2+-free saline (5 times for 1 min) to ensure that the extrinsic dye is completely removed.
Make sure to image the FM1-43 dye fluorescence at the same NMJ as mentioned above using the same confocal settings.
Fluorescence Quantification
AZs are directly connected to glutamate receptor (GluR) clusters in the postsynaptic muscle membrane (Schuster et al., 1991). Notum, a secreted Wg deacylase, was recently shown to also restrict Wg signaling to the NMJ (Kopke et al., 2017). MHC-CD8-GCaMP6f-Sh Ca2+ reporter (SynapGCaMP6f; Newman et al., 2017) was obtained from Ehud Isacoff (University of California, Berkeley, CA, USA).
Null notumKO (4)(w+) ( Kakugawa et al., 2015 ) was obtained from Jean-Paul Vincent (Francis Crick Institute, London, UK). Anti-Discs Large (DLG) has been used to label the postsynaptic scaffold in the subsynaptic reticulum (Lahey et al., 1994; Parnas et al., 2001). For quantal imaging, the SynapGCaMP reporter (MHC-CD8-GCaMP6f-Sh) contains a myosin heavy chain (MHC) promoter for muscle targeting, CD8 transmembrane domain for membrane targeting, and Shaker (Sh) K+ channel C-terminal tail for postsynaptic targeting (Newman et al., 2017).
Each AZ directly hosts a cluster of postsynaptic glutamate receptors (GluRs) to mediate fast neurotransmission ( Schuster et al., 1991 ). Brp AZ punctae are directly opposite GluR clusters in a functional synapse ( Menon et al., 2013 ). It has been proposed that Dally/Dlp HSPGs are involved in the movement of extracellular Wg to form a morphogenic gradient ( Han et al., 2005 ).
Presynaptically, we find increased synaptic bouton number in cow null mutants that phenocopy the Wg OE state (Kopke et al., 2017), consistent with this hypothesis. These results indicate that Cow normally inhibits NMJ bouton formation, consistent with the effects of inhibiting presynaptic Wg signaling ( Packard et al., 2002 ). Increasing Wg signaling increases the evoked transmission strength and functional synapse number (Kopke et al., 2017), which is phenocopied in cow null mutants.
SynapGCaMP is an exciting new tool to test function at individual synapses (Newman et al., 2017). Consistently, genetic correction of Wg levels at the synapse in notum null alleviates synaptogenic phenotypes (Kopke et al., 2017). In addition to cow, Perlecan (Trol) is another secreted HSPG reported to regulate bidirectional Wg signaling at the Drosophila NMJ ( Kamimura et al., 2013 ).
In the absence of Trol, Wg accumulates presynaptically, resulting in excessive formation of satellite boutons (Kamimura et al., 2013). Ghost boutons are increased in trol mutants due to reduced postsynaptic Wg signaling (Kamimura et al., 2013).
2004) Functions of heparan sulfate proteoglycans in cell signaling during development
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