RRC ID 67775
Author Kinoshita N, Huang AJY, McHugh TJ, Miyawaki A, Shimogori T.
Title Diffusible GRAPHIC to visualize morphology of cells after specific cell-cell contact.
Journal Sci Rep
Abstract The ability to identify specific cell-cell contact in the highly heterogeneous mammalian body is crucial to revealing precise control of the body plan and correct function. To visualize local connections, we previously developed a genetically encoded fluorescent indicator, GRAPHIC, which labels cell-cell contacts by restricting the reconstituted green fluorescent protein (GFP) signal to the contact site. Here, we modify GRAPHIC to give the reconstituted GFP motility within the membrane, to detect cells that make contact with other specific cells. Removal of leucine zipper domains, located between the split GFP fragment and glycophosphatidylinositol anchor domain, allowed GFP reconstituted at the contact site to diffuse throughout the entire plasma membrane, revealing cell morphology. Further, depending on the structural spacers employed, the reconstituted GFP could be selectively targeted to N terminal (NT)- or C terminal (CT)-probe-expressing cells. Using these novel constructs, we demonstrated that we can specifically label NT-probe-expressing cells that made contact with CT-probe-expressing cells in an epithelial cell culture and in Xenopus 8-cell-stage blastomeres. Moreover, we showed that diffusible GRAPHIC (dGRAPHIC) can be used in neuronal circuits to trace neurons that make contact to reveal a connection map. Finally, application in the developing brain demonstrated that the dGRAPHIC signal remained on neurons that had transient contacts during circuit development to reveal the contact history. Altogether, dGRAPHIC is a unique probe that can visualize cells that made specific cell-cell contact.
Volume 10(1)
Pages 14437
Published 2020-9-2
DOI 10.1038/s41598-020-71474-0
PII 10.1038/s41598-020-71474-0
PMID 32879377
PMC PMC7468259
MeSH Animals Blastomeres / cytology Cells, Cultured GPI-Linked Proteins / genetics GPI-Linked Proteins / metabolism* Green Fluorescent Proteins / genetics Green Fluorescent Proteins / metabolism HEK293 Cells Humans Intercellular Junctions / ultrastructure* Leucine Zippers Mice Mice, Inbred ICR Microscopy, Fluorescence / methods Neurons / cytology Protein Engineering / methods* Rats Rats, Sprague-Dawley Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / metabolism Swine Xenopus
IF 3.998
DNA material AAV:NT-probe-nuc-mCherry (RDB19166) AAV:CT-probe-cyto-mCherry (RDB19167)
Human and Animal Cells 293T(RCB2202)