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.
Highlights
Complex assembly[6,7]
Quantifying fluorescence signal covariance using Pearson’s Correlation Coefficients (PCC) (Equation 2)[6] at cell-cell contacts for E-cadherin and F-actin more accurately measures the extent of their association with the same macromolecular complex
Cells treated with E-cadherin function blocking antibody fail to reach the steady state proportion of high Fluorescence Covariance Index (FCI) values for E-cadherin and F-actin (≈31% at 3 hours following calcium repletion with E-cadherin function blocking antibody compared to ≈49% at steady state), indicating an inability to fully assemble mature adherens junctions (Fig. 1g). These results demonstrate FCI analysis for E-cadherin and F-actin is sensitive to E-cadherin function, and the frequency of high FCI values provides a quantitative measure of adherens junction assembly/disassembly dynamics in epithelial monolayers
Summary
Complex assembly[6,7]. Since PCC is a measure of covariance between two fluorescent signals, it serves as a more reliable tool to quantify the extent to which two proteins are part of a multi-molecular complex than conventional colocalization analysis[8]. Using the calcium switch approach[10] we quantified several aspects of the mechano-transduction apparatus during monolayer assembly: the formation and trafficking of the minimal cadherin-catenin complex, F-actin anchoring of cadherin complexes and, correlation of α-catenin/F-actin interaction to established tissue tension profiles[11]. We show this quantitative approach based on measuring covariance, accurately assesses adherens junction complex assembly dynamics in live cells using inexpensive image acquisition equipment while minimizing false-positives caused by non-specific signal overlap
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