Distinct trans-plasma membrane redox pathways reduce cell-impermeable dyes in HeLa cells

Area of Science:

• Cell Biology
• Biochemistry
• Physiology

Background:

• Trans-plasma membrane electron transport (tPMET) is crucial in mammalian cells.
• Previous studies used cell-impermeable dyes to demonstrate tPMET, but pathway distinctions were unclear.
• Understanding these pathways is vital for interpreting cellular redox states and functions.

Purpose of the Study:

• To elucidate the distinct reduction pathways of three common tPMET dyes: WST-1, ferricyanide (FeCN), and dichlorophenolindophenol (DCIP).
• To compare the mechanistic properties underlying the reduction of these dyes in mammalian cells.

Main Methods:

• Comparative analysis of WST-1, FeCN, and DCIP reduction in mammalian cells.
• Investigation of the role of intermediate electron carriers (mPMS, CoQ1) in dye reduction.
• Assessment of the effects of superoxide dismutase (SOD) and aminooxyacetate (AOA) on dye reduction.
• Evaluation of inhibitors like dicoumarol, diphenyleneiodonium, and capsaicin on tPMET pathways.

Main Results:

• WST-1 reduction required an intermediate electron carrier, unlike FeCN and DCIP which were reduced directly.
• FeCN reduction was enhanced by CoQ1, while DCIP was unaffected.
• SOD and AOA inhibited WST-1 and DCIP reduction but not FeCN reduction, suggesting distinct mechanisms involving mitochondrial NADH and possibly superoxide.
• Dicoumarol, diphenyleneiodonium, and capsaicin inhibited the reduction of all three dyes.

Conclusions:

• WST-1, FeCN, and DCIP are reduced through distinct trans-plasma membrane electron transport pathways.
• These findings clarify the specific mechanisms and cellular components involved in the reduction of commonly used tPMET dyes.
• The study highlights the complexity of tPMET and provides a basis for more precise investigations into cellular redox signaling.