Real-Time Imaging of the Axonal Transport of Granules Containing a Tissue Plasminogen Activator/Green Fluorescent Protein Hybrid

Abstract

A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.

Induction of the Plasminogen Activator System Accompanies Peripheral Nerve Regeneration after Sciatic Nerve Crush

“…The staining pattern at day 7 showed areas of punctate reactivity indicative of vesicular packaging. This was in agreement with Lochner et al (1998) who showed, using a green fluorescent protein-tPA, that vesicular packaged tPA was transported within axons of differentiated PC12 cells and was found at the growth cones. Furthermore, induced tPA protein is shown here in the more distal regions of the regenerating axon (Fig.…”

“…At day 7 (Fig. 9H ), several axons displayed a punctate pattern of tPA staining within axons of regenerating sciatic nerve, suggesting vesicular packaging of the protein (Lochner et al, 1998). At day 7, tPA was also seen in regenerating axons ϳ125 m distal to the crush site (Fig.…”

“…The tPA immunoreactivity within the surrounding areas of the sensory neuron cell bodies may also reflect tPA protein secretion from the neurons. Within the neurons of the crushed sensory ganglia, tPA immunoreactivity was seen as distinct points, suggesting a vesicular packaging of the protein (Lochner et al, 1998). Sham ganglia displayed less tPA immunoreactivity, and most of the protein appeared to be localized with the supportive cells of the sensory ganglia (Fig.…”

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

“…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.…”

Localization and Regulation of the Tissue Plasminogen Activator–Plasmin System in the Hippocampus

“…Previous experiments suggest the following model for tPA expression in adult neurons: tPA is synthesized under basal conditions and is stored in vesicles (Baranes et al, 1996;Gualandris et al, 1996;Parmer et al, 1997;Lochner et al, 1998). On depolarization, tPA is released into the extracellular space (Gualandris et al, 1996;Parmer et al, 1997), and tPA mRNA is upregulated (Qian et al, 1993;Carroll et al, 1994).…”