high-throughput-measurement-plasma-membrane-resealing-efficiency

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

The Ohio State University

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.

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

Host cell invasion is an indispensable step for a successful infection by intracellular pathogens. Recent studies identified pathogen-induced host cell plasma membrane perforation as a novel mechanism used by diverse pathogens (, , and adenovirus) to promote their internalization into target cells. It was concluded that  and adenovirus damage the host cell plasma membrane to hijack the endocytic-dependent membrane resealing machinery, thereby invading the host cell. We studied  and its secreted pore-forming toxin listeriolysin O (LLO) to identify key signaling events activated upon plasma membrane perforation that lead to bacterial internalization. Using various approaches, including fluorescence resonance energy transfer imaging, we found that the influx of extracellular Ca subsequent to LLO-mediated plasma membrane perforation is required for the activation of a conventional protein kinase C (cPKC). cPKC is positioned upstream of Rac1 and the Arp2/3 complex, which activation leads to F-actin--dependent bacterial internalization. Inhibition of this pathway did not prevent membrane resealing, revealing that perforation-dependent  endocytosis is distinct from the resealing machinery. These studies identified the LLO-dependent endocytic pathway of  and support a novel model for pathogen uptake promoted by plasma membrane injury that is independent of membrane resealing.

Host cell perforation by listeriolysin O (LLO) activates a Ca-dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes  internalization independently of membrane resealing.

Jonathan G.T. Lam

Department of Microbial Infection and Immunity,
Department of Microbiology,
Infectious Diseases Institute

The Ohio State University

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Jonathan G.T. Lam

Department of Microbial Infection and Immunity,Department of Microbiology,Infectious Diseases Institute

The Ohio State University

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Department of Microbial Infection and Immunity,
Department of Microbiology,
Infectious Diseases Institute