Quantitative analysis of protein translocations by microfluidic total internal reflection fluorescence flow cytometry

Area of Science:

• Cellular Biology
• Biophysics
• Molecular Medicine

Background:

• Protein translocation is vital for cellular function and its dysregulation is linked to diseases like cancer.
• Understanding protein movement dynamics is crucial for deciphering cellular mechanisms and disease pathology.

Purpose of the Study:

• To introduce and validate microfluidic total internal reflection fluorescence flow cytometry (TIRF-FC) as a novel single-cell tool.
• To demonstrate TIRF-FC's capability in detecting protein translocation from cytosol to plasma membrane and cytosol to nucleus.
• To quantify translocation dynamics in large cell populations with single-cell resolution.

Main Methods:

• Development of a microfluidic total internal reflection fluorescence flow cytometry (TIRF-FC) system.
• Utilizing fluorescently labeled Syk and NF-κB proteins as model systems for translocation studies.
• Measuring fluorescence density changes in the evanescent field at the plasma membrane and membrane-proximal cytosol.

Main Results:

• Successfully detected and quantitatively analyzed cytosol to plasma membrane translocations (Syk).
• Successfully detected and quantitatively analyzed cytosol to nucleus translocations (NF-κB).
• Achieved single-cell resolution for large cell population analysis of protein translocation events.

Conclusions:

• TIRF-FC is a powerful new tool for studying protein translocation dynamics at the single-cell level.
• This technique offers a quantitative approach to investigate the molecular biology and clinical relevance of protein translocations.
• TIRF-FC has the potential to advance research in various diseases linked to aberrant protein localization.