(A–E) Defective dendrites of Tor, Pten and chico mutant Dm8 neurons exhibit altered membrane contacts with R7 photoreceptors, revealed by the ‘receptor-based’ version of GFP Reconstitution Across Synaptic Partners technique (R-GRASP). (A) A schematic illustration of R-GRASP is shown. Membrane tethered spGFP1-10 (CD4-GFPsp1-10) (grey) is expressed in presynaptic neurons (R7s), while the other GFP fragment (blue), spGFP11 fused to histamine receptor Ort (Ort-GFPsp11), is expressed at postsynaptic neurons (Dm8). Functional reconstitution of the two GFP fragments generates fluorescent GFP (green), indicating neuronal contact sites. (B–E) The R-GRASP technique was applied to monitor neuronal contacts between R7 photoreceptors and their postsynaptic Dm8 partners in single wild-type (B, B'), Tor mutant (C, C'), Pten mutant (D, D') and chico mutant (E, E') Dm8 neurons. The photoreceptor R7 axons were labeled with anti-24B10 (blue), and the Dm8 MARCM clones were labeled by mCD8Cherry (red). The R-GRASP signal between R7 and single Dm8 cells is shown in green. Tor (C, C') and chico (E, E') mutations in Dm8 neurons diminished the number and area of contact with R7 cells, while Pten (D, D') mutation in Dm8 neurons significantly increased the contact number. (F) A schematic illustration of the neurotransmitter ‘receptor-based’ version of synaptobrevin GRASP (R-synGRASP). Upon neuronal firing, activity-dependent synaptobrevin-fused spGFP1-10 (Syb-GFPsp1-10) in synaptic vesicles become localized to the presynaptic sites and exposed to the Ort-GFPsp11 expressed at postsynaptic sites. Reconstitution of the two GFP fragments in the synaptic cleft generates functional GFP to indicate active synapses. (G–O) Dendritic defects in Tor, Pten and chico mutant Dm8 neurons caused synaptic defects, as revealed by R-synGRASP under a 12 hr light-dark cycle (L:D). R-synGRASP between R7 and Dm8 neurons was monitored in 6-day-old (G, G') and 12-day-old (L, L') wild-type flies, 6-day-old (H, H') and 12-day-old (M, M') Tor mutants, 6-day-old (I, I') and 12-day-old (N, N') Pten mutants, and 6-day-old (J, J') and 12-day-old (O, O') chico mutant Dm8 neurons. (G, G', L, L') 6-day-old and 12-day-old wild-type flies exhibited the most prominent GRASP signal in the central R7 column. (H, H', M, M') Tor mutation in Dm8 neurons caused a complete loss of active synapses with R7 photoreceptors. (I, I', N, N') Pten mutation in Dm8 neurons resulted in aberrant synapse formation with R7 photoreceptors. (J, J', O, O') chico mutant Dm8 neurons exhibited significantly decreased number of active synapses with R7 photoreceptors. (K) Quantification of R-synGRASP in 0- to 12-day-old flies and quantification of R-GRASP in 3-day-old flies under a 12 hr light-dark cycle (L:D cycle). Pten mutant Dm8 neurons displayed R-synGRASP signal in consistently more R7 columns, while chico and Tor mutants exhibited R-synGRASP signal in fewer R7 columns. The R-synGRASP signals in Pten mutant and wild-type 12-day-old flies were comparable to R-GRASP in 3-day-old flies. (P–T) Pten mutant Dm8 neurons form synapses with R7 photoreceptors under constant darkness (D:D cycle). (P) R-synGRASP signal was quantified in wild-type and Pten mutant Dm8 neurons of flies raised in constant darkness. R-synGRASP signal was nearly absent in 12-day-old wild-type neurons (Q, Q'). In contrast, GRASP signal appeared in 3-day-old Pten mutant Dm8 neurons (R, R') and became stronger in 6-day-old (S, S') and 12-day-old (T, T') adult flies. (B’–E’, G’–J’, L’–O’, Q’–T’) GRASP signals shown in inverted black-and-white images for (B–E, G–J, L–O, Q–T), respectively.