Quantification of Cellular Proteostasis in Live Cells by Fluorogenic Assay Using the AgHalo Sensor

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Cellular proteostasis is critical for cell health and survival.
• Environmental stress can disrupt proteostasis, leading to protein aggregation.
• Quantifying proteostasis deficiency in live cells is challenging with existing methods.

Purpose of the Study:

• To develop and present protocols for a novel fluorogenic sensor, AgHalo, to quantify stress-induced proteostasis deficiency.
• To enable visualization and quantification of proteome stress in live cells.
• To facilitate two-color imaging experiments for advanced proteostasis analysis.

Main Methods:

• Development of a fluorogenic sensor (AgHalo) using a destabilized HaloTag variant.
• Utilizing fluorogenic probes that signal soluble oligomer or insoluble aggregate formation.
• Employing fluorescence microplate readers and microscopes for direct fluorescence read-out.
• Establishing protocols for single- and two-color imaging experiments.

Main Results:

• The AgHalo sensor effectively visualizes and quantifies proteome stress in live cells.
• Demonstrated protocols for using AgHalo with various probes for detailed analysis.
• Enabled direct fluorescence read-out, overcoming limitations of always-fluorescent methods.

Conclusions:

• The AgHalo sensor provides a robust method for assessing cellular proteostasis under stress.
• The developed protocols allow for dynamic visualization and quantification of protein aggregation.
• This tool advances the study of cellular stress responses and proteostasis maintenance.