Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell...

Area of Science:

• Cell Biology
• Biophysics
• Physiology

Background:

• The pulmonary artery endothelium maintains a semipermeable barrier via intercellular junctions.
• Dynamic membrane interactions at these junctions are crucial but incompletely quantified.
• Understanding these dynamics is key to pulmonary vascular health.

Purpose of the Study:

• To introduce a novel technique for quantifying paracellular morphological fluctuations (PMFs).
• To investigate the impact of substrate stiffness on PMFs in pulmonary artery endothelial cells.
• To elucidate the role of calcium signaling in regulating PMFs.

Main Methods:

• Developed a new method to quantify peripheral membrane motion (PMFs).
• Cultured pulmonary artery endothelial cells on hydrogels of varying stiffness (e.g., 1.25 kPa, 30 kPa).
• Utilized thapsigargin to activate store-operated calcium channels and induced hypocalcemic conditions.

Main Results:

• Substrate stiffness significantly affected cell size and movement speed, with optimal movement at 1.25 kPa.
• Baseline PMFs were largely independent of substrate stiffness.
• PMFs increased upon activation of store-operated calcium channels.
• Hypocalcemia abolished PMF increase on healthy stiffness (1.25 kPa) but not on hypertensive stiffness (30 kPa).

Conclusions:

• PMFs occur in endothelial cell clusters regardless of substrate stiffness.
• Calcium influx via store-operated channels transiently increases PMFs.
• Stiffer substrates enhance PMFs independently of calcium influx, suggesting distinct regulatory pathways.