title: Live imaging of endogenous PSD-95 using ENABLED: a conditional strategy to fluorescently label endogenous proteins.
abstract: Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo.
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239.
authors.forename: Dale A
authors.initials: DA
authors.lastname: Fortin
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239.
authors.forename: Shane E
authors.initials: SE
authors.lastname: Tillo
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239.
authors.forename: Guang
authors.initials: G
authors.lastname: Yang
authors.affiliation: Howard Hughes Medical Institute, Janelia Farms Research Campus, Ashburn, Virginia 20147.
authors.forename: Jong-Cheol
authors.initials: JC
authors.lastname: Rah
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239.
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Melander
authors.affiliation: Howard Hughes Medical Institute, Janelia Farms Research Campus, Ashburn, Virginia 20147.
authors.forename: Suxia
authors.initials: S
authors.lastname: Bai
authors.affiliation: Department of Pharmacology and Physiology, Ponce School of Medicine, Ponce, Puerto Rico, 00716, and.
authors.forename: Omar
authors.initials: O
authors.lastname: Soler-Cedeño
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239.
authors.forename: Maozhen
authors.initials: M
authors.lastname: Qin
authors.affiliation: Center for Learning and Memory, The University of Texas at Austin, Austin, Texas 78712.
authors.forename: Boris V
authors.identifier: http://orcid.org/0000-0003-2463-4887
authors.initials: BV
authors.lastname: Zemelman
authors.affiliation: Howard Hughes Medical Institute, Janelia Farms Research Campus, Ashburn, Virginia 20147.
authors.forename: Caiying
authors.initials: C
authors.lastname: Guo
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239, zhong@ohsu.edu mao@ohsu.edu.
authors.forename: Tianyi
authors.identifier: http://orcid.org/0000-0002-3532-8319
authors.initials: T
authors.lastname: Mao
authors.affiliation: Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239, Howard Hughes Medical Institute, Janelia Farms Research Campus, Ashburn, Virginia 20147, zhong@ohsu.edu mao@ohsu.edu.
authors.forename: Haining
authors.identifier: http://orcid.org/0000-0002-7109-4724
authors.initials: H
authors.lastname: Zhong
mesh_terms: D000818:Animals; D056547:CA1 Region, Hippocampal / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D049229:Dendritic Spines / Q000737:chemistry / Q000378:metabolism; D000076122:Disks Large Homolog 4 Protein; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D051528:Guanylate Kinases / Q000032:analysis* / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009924:Organ Culture Techniques
chemical_list: D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D051528:Guanylate Kinases
keywords: PSD-95; cell-type-specific labeling; conditional knock-in; live imaging; protein labeling; sparse labeling