Real-time imaging of the axonal transport of granules containing a tissue plasminogen activator/green fluorescent protein hybrid.
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.
Lochner
J E
JE
Department of Chemistry, Lewis & Clark College, Portland, Oregon 97219, USA.
Kingma
M
M
Kuhn
S
S
Meliza
C D
CD
Cutler
B
B
Scalettar
B A
BA
D000818:Animals; D001370:Axonal Transport / Q000502:physiology*; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010959:Tissue Plasminogen Activator