High-Throughput Microplate-Based Assay to Monitor Plasma Membrane Wounding and Repair.

Sarika Pathak-Sharma,Xiaoli Zhang,Jonathan G T Lam,Stephanie M Seveau,Noah Weisleder

doi:10.3389/fcimb.2017.00305

Abstract

The plasma membrane of mammalian cells is susceptible to disruption by mechanical and biochemical damages that frequently occur within tissues. Therefore, efficient and rapid repair of the plasma membrane is essential for maintaining cellular homeostasis and survival. Excessive damage of the plasma membrane and defects in its repair are associated with pathological conditions such as infections, muscular dystrophy, heart failure, diabetes, and lung and neurodegenerative diseases. The molecular events that remodel the plasma membrane during its repair remain poorly understood. In the present work, we report the development of a quantitative high-throughput assay that monitors the efficiency of the plasma membrane repair in real time using a sensitive microplate reader. In this assay, the plasma membrane of living cells is perforated by the bacterial pore-forming toxin listeriolysin O and the integrity and recovery of the membrane are monitored at 37°C by measuring the fluorescence intensity of the membrane impermeant dye propidium iodide. We demonstrate that listeriolysin O causes dose-dependent plasma membrane wounding and activation of the cell repair machinery. This assay was successfully applied to cell types from different origins including epithelial and muscle cells. In conclusion, this high-throughput assay provides a novel opportunity for the discovery of membrane repair effectors and the development of new therapeutic compounds that could target membrane repair in various pathological processes, from degenerative to infectious diseases.

Highlights

IntroductionAND LIMITATIONS OF CURRENT MODELSThe repair of the plasma membrane is a fundamental process that maintains cell homeostasis, prevents the loss of difficult to replace cells (e.g., cardiac myocytes or neurons) and eliminates the need for replacing frequently injured cells
AND LIMITATIONS OF CURRENT MODELSThe repair of the plasma membrane is a fundamental process that maintains cell homeostasis, prevents the loss of difficult to replace cells and eliminates the need for replacing frequently injured cells
For each listeriolysin O (LLO) concentration, higher fluorescence intensities were recorded in M2 compared to M1, which is evidence of plasma membrane repair in M1

Summary

Introduction

AND LIMITATIONS OF CURRENT MODELSThe repair of the plasma membrane is a fundamental process that maintains cell homeostasis, prevents the loss of difficult to replace cells (e.g., cardiac myocytes or neurons) and eliminates the need for replacing frequently injured cells. High-Throughput Plasma Membrane Repair Assay and immune system effectors severely compromise the integrity of the plasma membrane during infection and inflammation (Morgan et al, 1986; Geeraerts et al, 1991; Gonzalez et al, 2008). In response to these various sources of injuries, eukaryotic cells rapidly repair their plasma membrane (McNeil and Steinhardt, 1997, 2003; Blazek et al, 2015; Demonbreun and McNally, 2016). Excessive plasma membrane damage and defects in its repair are associated with diverse pathological conditions such as muscular dystrophy, heart failure, diabetes, lung and neurodegenerative diseases (Clarke et al, 1995; Bansal et al, 2003; Han et al, 2007; Idone et al, 2008; Cai et al, 2009; Howard et al, 2011a; Blazek et al, 2015; Fernandez-Perez et al, 2016; Peters et al, 2016; Cong et al, 2017)

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