These studies provide evidence for a detailed model that can explain the mechanistic logic behind the axonal transport of these cytosolic cargoes in neurons, providing insights into a long-standing scientific question. To investigate bulk axonal transport of cytosolic protein populations, we transfected cultured hippocampal neurons with synapsin (synapsin-Ia) or CamKIIa tagged to photoactivatable green fluorescent protein (PAGFP), selectively photoactivated discrete protein pools within the primary axon emerging from the soma (away from presynaptic boutons), and tracked the mobility
of photoactivated cytosolic protein populations at various time compressions (Figures 1 and 2). We focused our studies on two cytosolic proteins enriched at synapses—synapsin and CamKIIa—as radiolabeling studies have established the overall transport of these proteins, showing that they are largely conveyed by slow axonal transport GPCR Compound Library solubility dmso (Baitinger and Willard, 1987, Lund and McQuarrie, 2001, Lund and McQuarrie, 2002 and Petrucci et al., 1991). The GFP fusions of these synaptic proteins
have been characterized in previous studies (Gitler et al., 2004 and Sturgill et al., 2009; also see Figure S1, available online). Note that punctate particles are clearly visible both in axons expressing the fluorescent proteins and adjacent naive axons (Figure S1B) suggesting that the fusion proteins generally mimic the behaviors of their in situ counterparts. Figures 1A and 1B show typical results from photoactivation experiments (also see Movie S1. PA:GFP:Synapsin Transport and Movie S2. PAGFP:CamKIIa Transport, DAPT mw Aldehyde dehydrogenase available online). The photoactivated axonal protein pool of synapsin and CamKIIa dispersed as a plume of fluorescence with a distinct anterograde bias, as shown in the representative kymographs (Figures 1A and 1B). This directional bias of fluorescence is unlikely to be
a result of some nonspecific bulk axonal flow that moves all soluble proteins in its wake, as there was no bias in the axonal dispersion of untagged PAGFP, which showed bidirectional rapid diffusion as expected (Figure 1C; also see Movie S3. Untagged Soluble PAGFP Transport and Movie S5. Untagged Soluble PAGFP:GFP Kinetics). Also, the intensity-center analyses (see below) are not likely influenced by photobleaching as similar trends of intensity-center shifts were observed under imaging conditions that greatly minimized photobleaching (Figures S2A and S2B). The transport behavior of cytosolic proteins is also very different from the fast component amyloid precursor protein (APP), where discrete photoactivated vesicles rapidly escaped the activated zone over time (Figure 1D; also see Movie S4), in line with conventional stochastic motor-driven transport (Kaether et al., 2000). Similar results were also obtained with PAGFP:synaptophysin (data not shown).