Local pH mapping in the cell adhesion nano-interfaces on a pH-responsive fluorescence-dye-immobilized substrate

Area of Science:

• Cell Biology
• Biophysics
• Nanotechnology

Background:

• Cell-substrate adhesion involves complex nano-interfaces.
• The glycocalyx, a cell surface layer, influences the local microenvironment.
• Previous methods lacked the ability to image local pH at these interfaces.

Purpose of the Study:

• To develop and apply a novel methodology for visualizing local pH distribution at the cell-adhesion interface.
• To investigate the relationship between focal adhesions, glycocalyx compression, and local pH.
• To compare pH profiles between fibroblast (NIH/3T3) and cancer (HeLa) cells.

Main Methods:

• Chemically modifying glass substrates with a pH-responsive fluorescent dye (fluorescein isothiocyanate, FITC).
• Observing FITC fluorescence intensity at the adhesion interface of cultured cells.
• Correlating FITC images with focal adhesion distribution and converting fluorescence to pH mapping.

Main Results:

• Localized regions of lowered pH (6.8-7.0) were observed at the cell-adhesion interface, distinct from the external pH (7.4).
• HeLa cells exhibited larger lowered pH regions at their thicker periphery compared to NIH/3T3 cells.
• Lowered pH regions significantly overlapped with focal adhesions, with larger adhesions showing more pronounced acidic sites.

Conclusions:

• Glycocalyx compression, driven by integrin-ligand interactions, leads to reduced local pH at cell-substrate interfaces.
• This pH reduction is more pronounced in HeLa cancer cells than in NIH/3T3 fibroblasts.
• The findings highlight the functional role of local pH modulation in cell adhesion dynamics.