Transsynaptic Coordination of Synaptic Growth, Function, and Stability by the L1-Type CAM Neuroglian
Figure 3
Mutations in the FIGQY–Ankyrin binding motif alter the Nrg–Ank2 interaction and increase Nrg mobility in vivo.
(A) Co-immunoprecipition (Co-IP) of Ank2-L from larval brain by Nrg180. IP of Nrg180 using the neuronal-specific antibody NrgBP104 co-precipitates the large Ank2-L isoform (450 kDa) from larval brain extracts. No Ank2-L signal is observed when using empty beads. (B) IP of Nrg180-HA proteins using Ank2-S–GFP from co-transfected S2-cells. Ank2-S pulled down wild-type Nrg180-HA efficiently. Mutations in the FIGQY domain differentially affected binding efficiency. Western blots show IPs and input controls. (C) Quantification of four independent IP experiments demonstrated reduced Ank2 binding due to the specific mutations within the FIGQY motif. (D–G) Fluorescence recovery after photobleaching (FRAP) experiments using GFP-tagged versions of wild-type and mutant forms of Nrg180 at the NMJ. (D) Representative recoveries of FRAP in motoneurons for Nrg180wt, Nrg180Y-F, and Nrg180ΔFIGQY. (E) Equal levels of all GFP-tagged constructs were expressed in motoneurons (ok371–Gal4) in wild-type animals as demonstrated by Western blot analysis. The Nrg180cyto antibody recognizes both wild-type Nrg180 and Nrg180–GFP. In contrast, Nrg180BP104 recognizes endogenous Nrg180 but only the wild-type version of Nrg180–GFP. This indicates that NrgBP104 binds specifically to the Nrg180 FIGQY motif and any alteration of the tyrosine will abolish protein recognition. (F and G) Recovery curves of multiple independent FRAP experiments were fitted to a double exponential curve and used to calculate the mobile fraction of Nrg180. Wild-type Nrg180 recovered to about 40% within the 200 s time frame. The mobility of Nrg180 was significantly increased (more than 1.5×) when the FIGQY motif was mutated (Nrg180Y-F, Nrg180Y-A, Nrg180Y-D). An almost 2-fold increase in mobility was observed after deletion of the Ankyrin-binding motif (Nrg180ΔFIGQY). Numbers in F represent number of independent experiments analyzed. Scale bar in (D) represents 5 µm. Error bars represent SEM.