Time-lapse confocal laser scanning microscopy analysis of FOOD formation

Area of Science:

• Cell Biology
• Extracellular Vesicles
• Apoptosis Research

Background:

• Apoptotic cells exhibit diverse morphological changes during programmed cell death.
• A novel mechanism of apoptotic adherent cell disassembly involves substrate retraction, forming membrane remnants.
• These remnants, termed 'FOotprint Of Death' (FOOD), mature into extracellular vesicles called 'FOOD-derived Apoptotic cell derived extracellular vesicles' (F-ApoEVs).

Purpose of the Study:

• To detail an imaging approach for monitoring the morphological transformations of apoptotic cells.
• To visualize the formation of FOOD and F-ApoEVs during the late stages of apoptosis.
• To establish a versatile method applicable to various cell types and apoptosis induction methods.

Main Methods:

• Utilized fluorescently conjugated annexin V to label phosphatidylserine on apoptotic cell membranes.
• Employed actin cytoskeletal stains to visualize F-actin dynamics during cell retraction.
• Developed an imaging protocol to track substrate-anchored membrane remnants and their conversion into extracellular vesicles.

Main Results:

• Successfully monitored the retraction of apoptotic adherent cells from the substrate.
• Visualized the formation of phosphatidylserine and F-actin-rich membrane remnants (FOOD).
• Observed the subsequent rounding of FOOD into F-ApoEVs.

Conclusions:

• The described imaging approach effectively visualizes the novel apoptosis disassembly pathway.
• This method allows for the detection and characterization of FOOD and F-ApoEV formation.
• The technique is adaptable for studying apoptosis in various cellular contexts and experimental conditions.