title: Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis.
abstract: Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.
authors.affiliation: Department of Biological Sciences, Rutgers, The State University of New Jersey, Newark, New Jersey, USA.
authors.forename: Pavan
authors.initials: P
authors.lastname: Vedula
authors.affiliation: Department of Biological Sciences, Rutgers, The State University of New Jersey, Newark, New Jersey, USA.
authors.forename: Lissette A
authors.initials: LA
authors.lastname: Cruz
authors.affiliation: Department of Biological Sciences, Rutgers, The State University of New Jersey, Newark, New Jersey, USA.
authors.forename: Natasha
authors.initials: N
authors.lastname: Gutierrez
authors.affiliation: Department of Biological Sciences, Rutgers, The State University of New Jersey, Newark, New Jersey, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Davis
authors.affiliation: Department of Biological Sciences, Rutgers, The State University of New Jersey, Newark, New Jersey, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Ayee
authors.affiliation: Department of Biological Sciences, Rutgers, The State University of New Jersey, Newark, New Jersey, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Abramczyk
authors.affiliation: Department of Biological Sciences, Rutgers, The State University of New Jersey, Newark, New Jersey, USA.
authors.forename: Alexis J
authors.initials: AJ
authors.lastname: Rodriguez
mesh_terms: D022005:Adherens Junctions / Q000737:chemistry / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D015820:Cadherins / Q000032:analysis / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer; D019084:Fluorescent Antibody Technique, Indirect; D007091:Image Processing, Computer-Assisted; D061985:Madin Darby Canine Kidney Cells; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism*
chemical_list: D015820:Cadherins; D051176:beta Catenin; D009218:Myosins; D002118:Calcium