Pw. Vanderklish et al., Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer, P NAS US, 97(5), 2000, pp. 2253-2258
Citations number
43
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Excitatory synaptic activity can evoke transient and substantial elevations
of postsynaptic calcium. Downstream effects of elevated calcium include th
e activation of the calcium-dependent protease calpain, We have developed a
reagent that identifies dendritic spines in which calpain has been activat
ed. A fusion protein was expressed that contained enhanced yellow and enhan
ced cyan fluorescent protein (EYFP and ECFP, respectively) linked by a pept
ide that included the mu-calpain cleavage site from alpha-spectrin. A PDZ-b
inding site fused to ECFP anchored this protein to postsynaptic densities.
The fusion protein exhibited fluorescence resonance energy transfer (FRET),
and diminution of FRET by proteolysis was used to localize calpain activit
y in situ by fluorescence microscopy. Incubation of the fusion protein with
calpain in the presence of calcium resulted in the separation of EYFP and
ECFP into monomeric fluorophores. In transiently transfected cell lines and
dissociated hippocampal neurons, FRET was diminished by raising intracellu
lar calcium levels with an ionophore or with glutamatergic agonists, Calpai
n inhibitors blocked these changes. Under control conditions, FRET levels i
n different dendritic spines of cultured neurons and in hippocampal slices
were heterogeneous but showed robust decreases upon treatment with glutamat
ergic agonists. Immunostaining of cultured neurons with antibodies to a spe
ctrin epitope produced by calpain-mediated digestion revealed an inverse co
rrelation between the amount of FRET present at postsynaptic elements and t
he concentration of spectrin breakdown products. These results suggest that
the FRET methodology identifies sites of synaptically induced calpain acti
vity and that it may be useful in analyzing synapses undergoing changes in
efficacy.