title: Visualizing of signaling proteins on endosomes utilizing knockdown and reconstitution approach.
abstract: Spatial distribution of intracellular signaling molecules and assembly of signaling complexes are yet to be fully understood. Studies of signaling events in time or space present a particular challenge due to the adverse effects that overexpression of signaling proteins may have on their functions and localization. To follow the distribution of signaling proteins in living cells we developed a methodology named knockdown and reconstitution (KDAR) that allows one to visualize proteins at levels of expression that are close to physiological. This methodology provides a stable expression of "endogenous" shRNA for long-term silencing of the targeted gene and simultaneous expression of a DNA cassette coding for a fluorescently labeled protein, which is insensitive to the targeting shRNA. In this chapter we discuss the needed reagents and outline two experimental approaches to generate KDAR stable cell lines. First, we demonstrate how the plasmid-mediated KDAR approach is successfully utilized to visualize spatial distribution of the GFP-labeled MEK2 in living cells. We then show how the lentivirus-mediated KDAR approach is used to reconstitute and visualize expression of the ERK1/2 scaffold protein Shoc2.
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, USA.
authors.forename: Myoungkun
authors.initials: M
authors.lastname: Jeoung
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, USA. Electronic address: emilia.galperin@uky.edu.
authors.forename: Emilia
authors.initials: E
authors.lastname: Galperin
mesh_terms: D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics; D048370:MAP Kinase Kinase 2 / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; C112913:SHOC2 protein, human; D049452:Green Fluorescent Proteins; C482128:MAP2K2 protein, human; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048370:MAP Kinase Kinase 2
keywords: ERK1/2; Endocytosis; Fluorescence microscopy; MEK1/2; siRNA