Figure 2.

Myo6 is partially localized to synapses and associated with the PSD in an actin-independent manner. (A) Hippocampal neurons (at 20 d) were costained for Myo6 and the synapse marker PSD-95, revealing Myo6 localization throughout the cell body and dendrite shaft and in clusters that partially overlap with PSD-95 puncta (arrows). Insets show enlarged regions of dendrite. Arrowheads indicate areas of colocalization. Bar, 10 μm. (B) Western blots of homogenate (H), synaptosome (Syn), and PSD fractions of whole brain, cortex, cerebellum, or hippocampus (equal protein loading) show that Myo6 is enriched in the PSD fraction throughout the brain. Controls for both preparation purity and gel loading included postsynaptically enriched NMDA-R1 and PSD-95, as well as presynaptically enriched synaptophysin (Synphys). Lanes on the same blot are shown separated for easier comparison. (C) Isolated PSD and TX-permeabilized synaptosome (Syn) fractions were incubated in the presence or absence of ATP, and the pellet (P) and supernatant (S) fractions were equal volume loaded. The majority of Myo6 in the PSD fraction, but not the synaptosome fraction, was pelleted despite ATP treatment.