The existence of protein complexes is supported by the following data: (1) Imaging experiments directly reveal assembly and vectorial Z-VAD-FMK ic50 transport of punctate fluorescent speckles containing these proteins (Figure S4). (2) Such particles are also seen natively by immunostaining (Figure S1). (3) Upon motor

inhibition, we saw clusters of stalled synapsin and CamKIIa particles clearly in axons (Figure 3B), suggesting that their mobility was interrupted by these manipulations. (4) Finally, biochemical experiments show that subsets of these cytosolic proteins are present in high-speed pellets from synaptosome-depleted (P100) as well as axon-enriched corpus callosum brain fractions that are detergent resistant, indicating the existence of higher order macromolecular structures within axons in vivo (Figure 5). We posit that the vast majority of these complexes transiently engage with motors (directly or indirectly) within axons, leading to a slow overall movement of the synapsin/CamKIIa population. Early nerve ligation/crushing

studies showed that synapsin was associated with vesicles accumulating proximally in ligated/crushed sites (Bööj et al., 1986). More detailed pulse-chase radiolabeling studies showed that while a small population LY2157299 concentration of newly synthesized synapsin (≈15%) departed the soma immediately afterwards, the vast majority (≈85%) was released from the cell body only after several days, and this pool moved much more slowly, at rates consistent with slow axonal transport (Baitinger

and Willard, 1987 and Petrucci et al., 1991). More recent studies have shown that in cultured neurons, synapsin is associated with mobile synaptic vesicular precursors (transport packets) probably conveyed in fast axonal transport (Ahmari et al., 2000). Synapsin is also an established component of synaptic vesicles (Takamori et al., 2006). Our data (Figure 4A) directly show that photoactivated somatic synapsin is transported into proximal axons both as punctate particles that are highly persistent and as a slow wave that departs the Idoxuridine soma with a transport behavior consistent with slow axonal transport. The persistent punctate synapsin particles colocalize with synaptophysin, a vesicular protein conveyed in fast axonal transport (Figure 4B). Collectively, the data indicate that a small fraction of newly synthesized synapsin is associated with vesicles and conveyed in fast axonal transport, while the remainder is conveyed in slow axonal transport with intricate particle kinetics. Though the biological basis for this bimodal transport behavior is unclear, it may have some evolutionary significance, in which cytosolic proteins in higher organisms may have acquired novel roles at synapses that require them to quickly localize to boutons, necessitating rapid transport in the fast component as well.