Counting kinetochore protein numbers in budding yeast using genetically encoded fluorescent proteins

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• Genetically encoded fluorescent proteins (FPs) are indispensable tools for visualizing and quantifying cellular components in vivo.
• Accurate measurement of intracellular protein concentrations and numbers is crucial for understanding cellular function.
• Existing methods often lack the precision required for absolute quantification of protein molecules.

Purpose of the Study:

• To detail the methodology for using genetically encoded fluorescent proteins as accurate reporters of in vivo protein numbers.
• To present a case study on quantifying kinetochore proteins in budding yeast.
• To discuss essential considerations for developing robust intracellular fluorescence signal quantification techniques.

Main Methods:

• Selection criteria for optimal fluorescent proteins and microscopy/imaging systems.
• Development of fluorescence signal quantification techniques to minimize measurement errors.
• Utilization of available calibration standards for converting fluorescence intensity to molecular counts.

Main Results:

• Established a framework for accurate evaluation of intracellular fluorescence signals.
• Successfully applied the method to determine the in vivo copy numbers of budding yeast kinetochore proteins.
• Demonstrated the utility of fluorescence-based protein number determination in elucidating cellular structure details.

Conclusions:

• Genetically encoded fluorescent proteins can serve as precise reporters for quantifying intracellular protein numbers.
• The developed methodology provides a reliable approach for molecular counting in live cells.
• Accurate protein quantification using FPs offers valuable insights into cellular organization and function.