Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons

Abstract

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

Axonal Membrane Proteins Are Transported in Distinct Carriers: A Two-Color Video Microscopy Study in Cultured Hippocampal Neurons

“…The construct was sequenced, and a point mutation was found, changing codon 185 from C to R, apparently without observable effects, because a construct with a reversed mutation behaved very similar (our unpublished results). Moreover, the dynamics are very similar to another p38-GFP construct (Nakata et al, 1998).…”

“…p38-EGFP moving structures could be tracked on average only 6.6 m, eight times shorter than APP-YFP-containing tubules. A comparable p38-GFP has been shown to move in a similar manner in dorsal root ganglion neurons (Nakata et al, 1998). Because all p38-EGFP-labeled organelles, endosomes and transport vesicles, move with dynamics strikingly different from those labeled with APP-YFP, these data suggest that the two proteins are transported in different carriers.…”

“…Shorter and slower moving tubulovesicular post-Golgi structures have been described in neurons (Nakata et al, 1998) and unpolarized cells (Hirschberg et al, 1998;Toomre et al, 1999). Axonal transport in different systems was reported to be between 1 and 3 m/s on average, sometimes with maximal velocities of up to 5 m/s (Allen et al, 1982;Kreis et al, 1989;Bloom et al, 1993;Lochner et al, 1998;Nakata et al, 1998;Zakharenko and Popov, 1998;Bridgman, 1999). The velocity measured for APP-YFP in our system is in the range of the fast component of fast axonal transport in mammals in vivo, which has been calculated to have a velocity of 410 mm/d (Ochs, 1972), corresponding to 4.7 m/s.…”

“…ND, not determined. Data on p38-GFP were taken from Nakata et al (1998). proteins is visualized simultaneously. The observed difference in velocities between the two types of carriers in the same neuron is consistent with the idea that the transport is mediated by different motor proteins (Hirokawa, 1998).…”

“…Therefore, a net anterograde transport of newly synthesized protein would be expected, which is not observed in our system. Because both endosomes and transport carriers show movement in both directions (Nakata et al, 1998;Prekeris et al, 1999), and because p38-EGFP is present in both types of organelles (Nakata et al, 1998; this study), it is possible that a net anterograde movement of p38-EGFP transport vesicles is masked. Therefore, even a slight net anterograde movement that is not detectable in our system could still account for the distribution of p38-EGFP-labeled organelles along the axon, because there is sufficient time for equilibration between transfection and microscopy.…”