| Autor: |
Hanes CM; Department of Biology, Iowa Neuroscience Institute, The University of Iowa, Iowa City, IA 52242., Mah KM; Department of Biology, Iowa Neuroscience Institute, The University of Iowa, Iowa City, IA 52242., Steffen DM; Department of Biology, Iowa Neuroscience Institute, The University of Iowa, Iowa City, IA 52242., Marcucci CG; Department of Biology, Iowa Neuroscience Institute, The University of Iowa, Iowa City, IA 52242., Fuller LC; Department of Biology, Iowa Neuroscience Institute, The University of Iowa, Iowa City, IA 52242., Burgess RW; The Jackson Laboratory, Bar Harbor, ME 04609., Garrett AM; Department of Pharmacology and Department of Ophthalmology, Visual, and Anatomical Sciences, Wayne State University, Detroit, MI 48202., Weiner JA; Department of Biology, Iowa Neuroscience Institute, The University of Iowa, Iowa City, IA 52242. |
| Abstrakt: |
The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically-interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific vs. shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified PKC phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via MARCKS is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation ( Pcdhg S/A ) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon ( Pcdhg CTD ). Both lines are viable and fertile, and the density and maturation of dendritic spines remains unchanged in both Pcdhg S/A and Pcdhg CTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the Pcdhg CTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point, and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains. |
