Retrograde BMP Signaling Modulates Rapid Activity-Dependent Synaptic Growth via Presynaptic LIM Kinase Regulation of Cofilin

Ghost bouton budding induced by high K⁺ stimulation is a rapid local signaling event. A, Live imaging of bouton budding (arrowheads) in response to 2 min incubations in high K⁺ spaced 10 min apart. New varicosity formation was visualized by presynaptic expression of membrane-tethered CD8-GFP. Scale bar, 7 μm. B, Quantification of ghost boutons in relation to existing bouton number at the NMJ following high K⁺ stimulation. N = 123 NMJs, 18 animals. C, New bouton budding (arrowheads) in response to high K⁺ stimulation is strongly dependent upon external Ca²⁺ but is not changed when axons are severed from motor neuron cell bodies. Scale bar, 14 μm. D, Quantification of ghost bouton budding detected by live imaging of animals presynaptically expressing membrane-tethered CD8-GFP at the indicated conditions. N (NMJs, animals): control = 38, 7; mock treated = 26, 4; 0 mm Ca²⁺ = 25, 4; 0.5 mm EGTA = 13, 4; axon cut = 21, 4. Error bars indicate SEM.

Ghost bouton budding requires normal synaptic transmission and local retrograde BMP signaling. A, Wandering third instar animals were fixed in formaldehyde after high K⁺ stimulation and were stained with anti-HRP and anti-DLG to identify ghost boutons. Loss of Syt1 and postsynaptic knockdown of GluRIIA and GluRIIB reduce activity-dependent budding. Likewise, loss of the postsynaptic Ca²⁺ sensor Syt4, or postsynaptic knockdown of Gbb with the muscle driver 24B-Gal4, reduces ghost bouton budding. Knockdown of Gbb with the muscle 6-specific H94-Gal4 preferentially reduces budding at muscle 6. Scale bar, 12 μm. Arrowheads indicate ghost boutons. B, Quantification of ghost boutons per NMJ in the indicated genetic backgrounds. N (NMJs, animals): wild-type = 57, 11; syt1^AD4/N13 = 45, 6; 24B, gluRIIA^RNAi = 31, 4; 24B, gluRIIB^RNAi = 29, 4; 24B, gbb^RNAi = 20, 3; syt4^BA1 = 65, 11; 24B, syt4^RNAi = 3.548 ± 2.694, n = 31, 3; wit^A12/B11 = 37, 6. C, Quantification of baseline bouton number in the indicated genetic backgrounds. N, same as in B. D, The average number of ghost boutons that bud onto muscle 6 or muscle 7 is quantified for Gbb knockdown by the muscle 6-specific driver H94-Gal4. N (NMJs, animals): wild-type = 52, 7; H94, gbb^RNAi = 40, 6. **p < 0.01; ***p < 0.001; ANOVA. Error bars indicate SEM.

Ghost bouton budding requires Smad signaling and is modulated by Limk activity. A, Wandering third instar larvae were fixed in formaldehyde after high K⁺ stimulation, and stained with anti-HRP and anti-DLG to identify ghost boutons. The overexpression of the inhibitory Smad dad causes synaptic undergrowth and a reduction in ghost bouton budding frequency. Trio protein levels correlate with ghost bouton budding frequency. Overexpression of full-length UAS-wit causes a reduction in bouton budding, while the overexpression of truncated UAS-wit^dCT does not. Motor neuron rescue with full-length UAS-wit did not completely rescue ghost bouton budding frequency, while rescue with UAS-wit^dCT rescued ghost bouton budding to a significantly greater extent. Scale bar, 12 μm. Arrowheads indicate ghost boutons. B, c164, UAS-trio animals were stimulated, fixed, and stained with anti-Trio antibody. Scale bar, 12 μm. Arrowheads indicate ghost boutons identified by morphology. C, Normalized fluorescence intensity of Trio antisera staining within ghost boutons was normalized to average fluorescence intensity of all other normal boutons at the same NMJ. N (ghost boutons, NMJs) = 77, 9. Solid line indicates mean; dashed line indicates the average normal bouton fluorescence intensity. D, Quantification of ghost boutons per NMJ in the indicated genetic background. N (NMJs, animals): wild-type = 57, 11; c164, dad = 70, 8; trio^S137203 = 37, 5; c164, trio = 27, 4; wit^A12/B11 = 37, 6; wit rescue = 45, 7; wit^dCT rescue = 56, 7; c164, wit = 55, 8; c164 wit^dCT = 55, 8. E, Quantification of baseline bouton number. N, same as in D. F, Wit and Tsr show genetic interactions for defective ghost bouton budding. Quantification of ghost boutons per NMJ in the indicated genetic background is shown. N (NMJs, animals): wild-type = 57, 11; tsr¹/+ = 24, 3; wit^B11/+ = 45, 6; tsr¹/+;wit^B11/+ = 37, 5. *p < 0.05; **p < 0.01; ***p < 0.001; ANOVA. Error bars indicate SEM.

Ghost bouton budding is regulated by Limk and Cofilin activity. A, Presynaptic overexpression of limk strongly reduces activity-dependent bouton budding. Presynaptic overexpression of constitutively inactive tsr^S3E reduces ghost bouton budding, while presynaptic overexpression of constitutively active tsr^S3A increases ghost bouton budding above wild-type levels. Scale bar, 12 μm. Arrowheads indicate ghost boutons. B, Quantification of ghost bouton budding frequency in the indicated genetic background. N (NMJs, animals): wild-type = 57, 11; c164, limk = 40, 5; limk^P1/Y = 36, 5; c164, tsr^S3E = 28, 4; c164, tsr^S3A = 36, 6. C, Quantification of baseline bouton number in the indicated genetic background. D, Live confocal imaging of GMA-GFP at NMJs driven by c164-Gal4. F-actin labeled by GMA appears as dynamic puncta with relatively even size and spacing in wild-type animals. GMA labeling in axons and extended interbouton regions is stable and uniform at wild-type NMJs (double arrow), but is interrupted by puncta and appears less uniform in tsr^S3E and tsr^S3E NMJs (double arrowheads). Boutons lacking discernable F-actin puncta occurred rarely at wild-type NMJs and more frequently at tsr^S3E NMJs (arrows). Large and bright GMA labeling was observed in some boutons in tsr^S3E and tsr^S3A NMJs that was not observed in wild-type (arrowheads). Scale bar, 12 μm. *p < 0.05; **p < 0.01; ***p < 0.001; ANOVA. Error bars indicate SEM.

Ghost bouton budding is accompanied by local rearrangements of the presynaptic actin cytoskeleton. A, Animals presynaptically expressing the membrane marker CD8-RFP and the F-actin marker GMA-GFP were imaged before and after high K⁺ stimulation. New F-actin puncta (arrowheads) are observed at sites of budding (arrows) where newly formed ghost boutons (double arrowheads) attach to the main axonal arbor. Scale bar, 6 μm. B, Application of 10 μm latrunculin A to the bath solution rapidly disperses F-actin puncta, while application of 10 μm jasplakinolide causes formation and stabilization of F-actin puncta. Scale bar, 6 μm. C, Wild-type animals pretreated with latrunculin A or jasplakinolide for 15 min before high K⁺ stimulation display a reduction in ghost bouton budding frequency. N (NMJs, animals): no drug = 30, 4; latrunculin A = 35, 5; jasplakinolide = 30, 4. *p < 0.05; ***p < 0.001; ANOVA. Error bars indicate SEM.