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and
Departments of
*Chemistry and
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.
Physics, Lewis & Clark
College, Portland, Oregon 97219
Corresponding author. E-mail address:
bethe{at}l.clark.edu.
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