, 1989). This transport inhibition is due to the action of alkylating sulfhydryl groups on motors that profoundly alters their interactions with ATP and microtubules, stalling vesicle-motor complexes (Pfister et al., 1989). Without knowledge of the specific anterograde and retrograde motor(s) moving the assortment of cytosolic proteins, NEM provides a useful tool to examine the role of molecular motors in generating the intensity-center shift of www.selleckchem.com/products/cx-5461.html synapsin and CamKIIa in our imaging experiments. Upon the addition of 0.5 μM NEM, axonal transport of APP vesicles

was gradually perturbed and vesicular transport completely ceased at 20 min (Figure 3A). Accordingly, for our experiments we CT99021 visualized synapsin and CamKIIa transport after 10 min of NEM treatment. We found that such NEM

treatment resulted in the accumulation of stationary puncta within axons with a dramatic inhibition of the anterogradely biased motion (Figure 3B). NEM treatment did not lead to any discernible changes in the diffusion kinetics of untagged PAGFP (Figures S2C and S2D). Addition of nocodazole, a microtubule-depolymerizing agent, also resulted in an inhibition of the anterograde bias (Figure 3C and Figure S3A) as did the cellular metabolic poison (oxidative phosphorylation uncoupler) 2,4-dinitrophenol (2,4-DNP) (Figure S3B). Finally, coexpression of headless Farnesyltransferase kinesins 1A, 1B, and 1C (also called Kif-5A, Kif-5B, and Kif-5C) known to act in a dominant negative fashion

to inhibit kinesin-1 mediated transport (Kozielski et al., 1997 and Uchida et al., 2009) also inhibited the anterograde bias of synapsin (Figure S3C), further suggesting that the bias is dependent on the activity of motors. The above experiments show that populations of synapsin and CamKIIa move along axons with a motor-dependent anterograde bias. This movement seems different from fast transport, where individual vesicles are stochastically transported by molecular motors. What is the underlying molecular basis for this unconventional motion of cytosolic protein populations? We reasoned that the overall biased transit of these proteins is ultimately mediated by the movement of particles that are transport competent. First, when neurons are stained for endogenous cytosolic synaptic proteins, particulate structures are seen within naive axons (Figure S1A, and also see Fletcher et al., 1991, Roy et al., 2007 and Withers et al., 1997), suggesting that these are the native structural form of cytosolic proteins within axons. Second, particulate structures are also clearly present within photoactivated zones in our experiments (note vertical lines in kymographs, Figure 1A and elsewhere). Third, when the anterograde bias of the photoactivated pool was inhibited with NEM or nocodazole, stalled particles are seen in axons (Figure 3B and Figure S3A).