title: Recombinant probes for visualizing endogenous synaptic proteins in living neurons.
abstract: The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed Fibronectin intrabodies generated with mRNA display (FingRs), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and that, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices, FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo.
authors.affiliation: Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Garrett G
authors.initials: GG
authors.lastname: Gross
authors.forename: Jason A
authors.initials: JA
authors.lastname: Junge
authors.forename: Rudy J
authors.initials: RJ
authors.lastname: Mora
authors.forename: Hyung-Bae
authors.initials: HB
authors.lastname: Kwon
authors.forename: C Anders
authors.initials: CA
authors.lastname: Olson
authors.forename: Terry T
authors.initials: TT
authors.lastname: Takahashi
authors.forename: Emily R
authors.initials: ER
authors.lastname: Liman
authors.forename: Graham C R
authors.initials: GC
authors.lastname: Ellis-Davies
authors.forename: Aaron W
authors.initials: AW
authors.lastname: McGee
authors.forename: Bernardo L
authors.initials: BL
authors.lastname: Sabatini
authors.forename: Richard W
authors.initials: RW
authors.lastname: Roberts
authors.forename: Don B
authors.initials: DB
authors.lastname: Arnold
mesh_terms: D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics; D002522:Chlorocebus aethiops; D000076122:Disks Large Homolog 4 Protein; D020869:Gene Expression Profiling / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis* / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics; D009474:Neurons / Q000737:chemistry* / Q000502:physiology; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics; D013569:Synapses / Q000737:chemistry / Q000502:physiology
chemical_list: D002352:Carrier Proteins; C501444:DLG4 protein, human; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C073441:gephyrin