Neuroscience 2004 Abstract
| Presentation Number: | 164.16 |
|---|---|
| Abstract Title: | Interaction between Arc and CaM Kinase II in dendrites monitored by fluorescence resonance energy transfer. |
| Authors: |
Okuno, H.*1,2
; Chowdhury, S.2
; Worley, P.2
; Bito, H.1,3
1Dept. of Neurochemistry, Univ. of Tokyo Grad. Sch. of Med., Tokyo, Japan 2MD, 7-3-1 Hongo, Bunkyo-ku, 113-0033, 3USA, 7-3-1 Hongo, Bunkyo-ku, 113-0033, |
| Primary Theme and Topics |
Synaptic Transmission and Excitability - Intracellular Signaling Pathways -- Other |
| Secondary Theme and Topics | Synaptic Transmission and Excitability<br />- Synaptic Plasticity<br />-- Other |
| Session: |
164. Intracellular Signaling Pathways: Other I Poster |
| Presentation Time: | Sunday, October 24, 2004 11:00 AM-12:00 PM |
| Location: | San Diego Convention Center - Hall A-H, Board # I25 |
| Keywords: | POSTSYNAPTIC DENSITY, FRET, SPINES, HIPPOCAMPAL NEURONS |
A neuronal immediate early gene Arc is rapidly induced in response to synaptic stimulation, and its mRNA is transported from the nucleus to the activated regions of the primary dendritic shafts (Layford et al., 1995). Furthermore, Arc protein associates with cytoskeletal proteins and accumulates in the postsynaptic density. We previously showed that Arc directly binds to several postsynaptic proteins including CaMKII and PSD-95 (Chowdhury et al., SFN Annual Meeting 1999, 2002). To further investigate Arc-CaMKII interaction, we measured fluorescence resonance energy transfer (FRET) between CFP-tagged Arc and YFP-tagged CaMKII pairs. A screening system that combined random insertion and yeast 2-hybrid (Y2H) assay was developed to obtain optimal FRET pairs. GFP variants were introduced into Arc and CaMKII using transposon-based gene insertion, and fusion proteins that maintained the integrity of Arc-CaMKII interaction were screened using Y2H. Optimal pairs were then selected based on FRET efficiency in COS cells. The FRET efficiency significantly varied depending on the fluorophore locations. We successfully isolated several Arc-CaMKII pairs that gave strong FRET signals. When expressed in dissociated hippocampal neurons, CFP-tagged Arc and YFP-tagged CaMKII both formed spatially overlapping punctate accumulations in the dendrites. Acceptor photobleaching experiments confirmed genuine FRET between Arc and CaMKII within the punctate signals. These results indicate that potential usefulness of our strategy in monitoring dynamics of Arc-CaMKII interaction in response to synaptic activation in living neurons. This Y2H approach may also be widely applicable to generate optimized pairs of tagged molecules for measuring FRET between interacting protein partners in intact cells.
Supported by HFSP, NIMH, JSPS, MEXT and JST
Sample Citation:
[Authors]. [Abstract Title]. Program No. XXX.XX. 2004 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2004. Online.
Copyright © 2004-2026 Society for Neuroscience; all rights reserved. Permission to republish any abstract or part of any abstract in any form must be obtained in writing by SfN office prior to publication.
