Axonal membrane proteins are transported in distinct carriers: A two-colorvideo microscopy study in cultured hippocampal neurons

Neurons transport newly synthesized membrane proteins along axons by microt ubule-mediated fast axonal transport. Membrane proteins destined fur differ ent axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagg ed the amyloid precursor protein (APP) and synaptophysin (p38) with green f luorescent protein (GFP) variants. The resulting fusion proteins, APP-yello w fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluore scent protein, were expressed in hippocampal neurons, and the cells were im aged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 mu m/s) and over long distances. Ln c ontrast, p38-enhanced GFP-transporting structures were more vesicular and m oved four times slower (0.9 mu m/s) and over shorter distances only. Two-co lor video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly tr ansfected neurons. Antisense treatment using oligonucleotides against the k inesin heavy chain slowed down the long, continuous movement of APP-YFP tub ules and increased frequency of directional changes. These results demonstr ate for the first time directly the sorting and transport of two axonal mem brane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier.