Fig. 3. Presynaptic overexpression of GFP-tagged wild-type or mutant helSyn at C1-B2 synapses. (A) Phase-contrast micrograph of a C1-B2 soma-to-soma synapse in culture. (B) Epifluorescence micrograph of the same cell pair shown in A, 24 hours after the intracellular injection of mRNA encoding for GFP in the C1 neuron. GFP is expressed at high levels in the C1 neuron cytoplasm. At the focal plane of the picture, the dark profile of the C1 nucleus is visible (arrowheads), as well as the initial part of small neurites projecting from C1 onto the B2 surface (arrow). (C-H) Confocal stacks encompassing the whole volume of different C1-B2 cocultures overexpressing GFP (C,D), GFP-tagged wild-type helSyn (E,F) or GFP-tagged helSyn phosphorylation mutant (G,H). (C) GFP is distributed quite uniformly in the C1 cytoplasm and in the neurites growing onto the B2 surface. (D) Detail of the area of contact between the same C1 and B2 neurons shown in C. The arrow indicates a C1 neurite growing onto the B2 surface. (E) helSyn-GFP is more concentrated in discrete spots of increased fluorescence, which are particularly evident in the area of contact between the two cell bodies (arrowheads) and in varicose structures along the neurites (arrows). (F) Detail of the area of contact between the same C1 and B2 neurons shown in E. The arrow and arrowhead indicate sites of helSyn-GFP accumulation in the C1-B2 contact area and in neurites, respectively. (G) helSynALA9-GFP is strongly concentrated in numerous clusters widely distributed in the C1 cell body (double arrowhead) as well as in the C1-B2 contact area (arrowheads) and along C1 neurites growing onto B2 (arrow). (H) Detail of the area of contact between the same C1 and B2 neurons shown in G. The arrow and arrowhead indicate sites of helSynALA9-GFP accumulation in the C1-B2 contact area and in neurites, respectively. Note the higher fluorescence intensity of these helSynALA9-GFP puncta, as compared with the background fluorescence, with respect to the helSyn-GFP puncta shown in E and F. (I) Electron micrograph of a soma-to-soma C1-B2 synapse section encompassing the two cell bodies. The inset shows at higher magnification the meshwork of cellular processes in the contact area between the two somata. (L) Electron micrograph of a C1 neurite (asterisk) growing onto the surface of the postsynaptic B2 soma and containing a cluster of synaptic vesicles (arrowheads) at a putative synaptic site. (M) Detail of the synaptic site shown in L. Bars, 50 µm (A,B); 20 µm (C,E,G); 20 µm (D,F,H); 50 µm (I); 0.5 µm (L); 0.5 µm (M).