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Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

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Published on: December 19, 2020

A Peptide-Based Fluorescent Sensor for Anionic Phospholipids.

Amitava Chandra¹, Ankona Datta¹

¹Department of Chemical Sciences, Tata Institute of Fundamental Research, 1 Homi Bhabha Road, Colaba, Mumbai 400005, India.

|April 6, 2022

Summary

This summary is machine-generated.

Researchers developed a new fluorescent sensor to track anionic phospholipids, crucial for cell signaling. This cell-permeable probe allows visualization of these lipids inside cells and during cell death.

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Area of Science:

Cell Biology
Biochemistry
Molecular Biology

Background:

Anionic phospholipids are vital mediators of cellular signaling pathways.
Their precise localization on cell and organelle membranes regulates protein recruitment and cellular functions.
Detecting anionic phospholipids within living cells is crucial for understanding lipid-mediated signal regulation.

Purpose of the Study:

To develop a cell-permeable, ratiometric fluorescent sensor for detecting anionic phospholipids in living cells.
To overcome challenges in visualizing anionic phospholipids located in inner membrane leaflets and intracellular organelles.
To enable rapid detection and tracking of intracellular anionic phospholipid pools.

Main Methods:

Development of a novel peptide-based, water-soluble, ratiometric fluorescent sensor.
Cellular uptake studies via endosomal machinery and lipofection.
Validation using colocalization studies with fluorescent lipids and protein-based sensors.
Imaging of anionic phospholipid externalization during programmed cell death.

Main Results:

The sensor demonstrated rapid (15 min) cell entry via endosomes, showing cytoplasmic punctate labeling.
Lipofection enabled imaging of anionic phospholipids on the cell membrane, bypassing endosomal uptake.
Successful colocalization confirmed the sensor's specificity for intracellular anionic phospholipids.
The sensor effectively visualized externalization of anionic phospholipids during programmed cell death.

Conclusions:

The developed fluorescent sensor is effective for detecting both intracellular and extracellular anionic phospholipids.
This tool provides a valuable method for studying lipid-mediated signaling and cellular processes.
The sensor's cell permeability and ratiometric nature offer advantages for live-cell imaging applications.