Figure 5.

NMDA-induced Pyk2–GFP clustering occurs postsynaptically and independently of Pyk2 kinase activity. Primary hippocampal cultures (15 DIV) were transfected with Pyk2–GFP or GFP-tagged kinase-dead Pyk2 (KD-GFP) (72 h; red in all overlays at higher magnification at bottom), treated with TTX plus either vehicle or NMDA (50 μm, 5 min), fixed, and stained with antibodies against synapsin (green in all dual overlays at the very bottom labeled “1 + 3”) and either MAP2B or PSD-95 (green in the respective overlays in the row labeled “1 + 2”). Scale bars, 10 μm for the corresponding magnifications. Pyk2–GFP underwent a dramatic relocation from a fairly smooth distribution under control conditions in dendritic shafts that was primarily colocalized with MAP2B to a pronounced punctate synaptic distribution away from MAP2B staining in dendritic shafts. Arrows illustrate examples of colocalization of Pyk2 and PSD-95 puncta that are juxtaposed to synapsin puncta. Kinase-dead Pyk2 also clusters during NMDA stimulation (right column). All pictures are representatives from three independent experiments.