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Vol. 14, Issue 4, 1570-1582, April 2003
Max-Planck-Institute for Developmental Biology,
Tübingen, Germany
An inducible fluorescent system based on GFP is presented that
allows for the uncoupling of dendritic mRNA transport from subsequent
protein synthesis at the single cell level. The iron-responsive element
(IRE) derived from ferritin mRNA in the 5'-UTR of the GFP reporter mRNA renders translation of its mRNA dependent on iron.
The addition of the full-length 3'-UTR of the
Ca2+/calmodulin-dependent protein kinase II alpha
(CaMKII
) after the stop codon of the GFP reading frame targets the
reporter mRNA to dendrites of transfected fully polarized hippocampal
neurons. As we show by time-lapse videomicroscopy, iron specifically
turns on GFP reporter protein synthesis in a single transfected
hippocampal neuron. We investigate whether GFP expression is
affected
in addition to ironby synaptic activity. Interestingly,
synaptic activity has a clear stimulatory effect. Most importantly,
however, this activity-dependent protein synthesis is critically
dependent on the presence of the full-length 3'-UTR of CaMKII
confirming that this sequence contains translational activation
signals. The IRE-based system represents a new convenient tool to study
local protein synthesis in mammalian cells where mRNA localization to a
specific intracellular compartment occurs.
Online version
of this article contains video materials. Online version of this
article is available at www.molbiolcell.org.
Corresponding author. E-mail address:
michael.kiebler{at}tuebingen.mpg.de.
*
Both authors contributed equally to this work.
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