Membrane dynamics are slowed for Alexa594-labeled membrane proteins due to substrate interactions

Area of Science:

• Cell biology
• Biophysics
• Molecular imaging

Background:

• Fluorescent dyes are crucial for tracking biological molecules.
• Dye-substrate interactions can non-specifically alter molecular dynamics and function.
• Understanding these interactions is vital for accurate biophysical measurements.

Purpose of the Study:

• To investigate the impact of fluorescent dye-substrate interactions on protein diffusion.
• To determine the role of dye labeling on the mobility of transmembrane protein Syntaxin1a (Syx1a-EGFP).
• To compare the effects of fibronectin (Fn) and poly-L-lysine (PLL) cell culture substrates on protein diffusion.

Main Methods:

• Fluorescence Recovery After Photobleaching (FRAP) was employed to measure protein diffusion.
• Syntaxin1a (Syx1a-EGFP) was expressed on the cell surface and labeled with an EGFP nanobody.
• Cell cultures were grown on either fibronectin (Fn) or poly-L-lysine (PLL) coated substrates.

Main Results:

• Alexa594-labeled nanobodies significantly reduced Syx1a-EGFP mobility on PLL substrates.
• Unlabeled nanobodies did not affect Syx1a-EGFP mobility on either substrate.
• Protein mobility was restored on Fn substrates, even with the Alexa594-labeled nanobody.
• Single molecule tracking measurements corroborated FRAP findings.

Conclusions:

• The choice of cell culture substrate critically influences protein diffusion measurements when using fluorescently labeled probes.
• Fibronectin (Fn) is a more suitable substrate than poly-L-lysine (PLL) for FRAP and single molecule tracking of Syx1a-EGFP with Alexa594.
• This highlights the importance of considering dye-substrate interactions for accurate interpretation of molecular dynamics studies.