Neuronal dendrites are the initial post-synaptic interpreters and integrators of presynapfic information. Over the last several decades, data has been generated from both in vitro and in vivo experiments that show that dendrites increase in number and size in response to various behavioral and pharmacological manipulations. More recently mRNAs have been localized in dendrites. Further data shows that mRNAs can be translated locally, in the dendrite, in response to various types of modulators. We have recently shown that dendritic translation occurs in immobile hotspots along the length of the dendrite through the monitoring of fluorescence from GFP that was synthesized from GFP that had been transfected into isolated dendrites. Further these hotspots were heterogeneous in their translational rate in response to DHPG, a mGluR1 agonist. Most of the hotspots exhibited exponential translation rates (EXP) while the remaining hotspots were linear (LIN) in their response. The existence of these translational hotspots suggests a subdendritic specificity to post-translational responsiveness. We propose to further characterize these translational hotspots to determine 1) do the EXP hotspots always respond in an EXP manner or can they also exhibit LIN translation rates and vice-versa, 2) whether known modulators of dendritic translation alter the ratio of linear exponential translation hotspots 3) what translational machinery component co-localize with each type of hotspot, 4) do dendritic spine associated proteins colocalize with either type of hotspot preferentially and finally, 5) do changes in mRNA structure alter the distribution or number of EXP and LIN hotspots. In addition, we will go one step further and determine the protein profile of dendrites using a novel proteomics method recently developed in our lab called Immuno-Detectlon Amplified by T7 RNA Polymerase (IDAT). We propose to utilize two different phage display libraries that are enriched (panned) for dendritic protein detection in conjunction with IDAT to determine the identity of proteins in the dendritic compartment and, using a differential screening procedure, changes in the abundance of these proteins after treatment with pharmacological modulators of dendritic function. These data will be compared with the mRNA expression profiles that have been generated to distinguish between the potential for somatic or dendritically synthesized proteins to contribute to the predominant protein profile of dendritic responsiveness to modulation. Included in our analysis is the possibility of looking at post-translational modification of dendritically localized proteins. These data will likely have significant impact upon how we think about post-synaptic involvement in regulation of the Hebbian synapse
| Zeng, Fanyi; Peritz, Tiina; Kannanayakal, Theresa J et al. (2006) A protocol for PAIR: PNA-assisted identification of RNA binding proteins in living cells. Nat Protoc 1:920-7 |
| Peritz, Tiina; Zeng, Fanyi; Kannanayakal, Theresa J et al. (2006) Immunoprecipitation of mRNA-protein complexes. Nat Protoc 1:577-80 |
