Fig. 2.

The presynaptic localization of NRX1β-SEP depends on Ca²⁺ binding and postsynaptic ligands. (A) A PV neuron was labeled with Nrx1β-SEP and DsRed and imaged at EP20. SEP signals along axon segments are shown as heat maps under control condition (A1) or after 5 min of 5 mM EGTA treatment (A2) with the same gain and look-up table. Warmer colors represent higher fluorescent levels. Arrows indicate the presynaptic boutons; dotted lines delineate the axon shaft. (B) Immediate diffusion of NRX1β-SEP signals into the axon shaft (arrowhead) and filopodia (open arrow) within 5 min of EGTA treatment. (Scale bar: A and B, 2 μm.) (C) PV neurons were labeled with NRX1β(D137A)-SEP and DsRed. The D137A mutation, which abolishes binding to Ca²⁺ and NL, resulted in diffuse distribution of NRX1β(D137A)-SEP along axons. (D) PV cells were labeled with NRX1β(+SS4)-SEP and DsRed. This splice site 4-containing (+SS4) NRX1β variant with decreased binding affinity with NL2 showed significant presynaptic localization but was more diffusive along the axon shaft compared with NRX1β–SEP (Fig. 1). (Scale bar: C and D, 10 μm.) Arrows indicate boutons, and arrowheads indicate the axon shaft. (E) Quantification of the relative enrichment of SEP signals on bouton vs. adjacent axon shaft for WT 1β-SEP, β(D137A)-SEP, and 1β(+SS4)-SEP; 10–15 boutons and nearby axon shafts from three to five neurons for each group were analyzed. *P < 0.05 compared with WT value.