Quantitative Super-Resolution Imaging of ER-Phagy Initiation in Cells

Area of Science:

• Cell Biology
• Molecular Biology
• Microscopy

Background:

• Selective autophagy of the endoplasmic reticulum (ER-phagy) degrades damaged ER components, preventing cellular stress.
• ER-phagy receptors form protein assemblies to orchestrate this degradation process.
• Understanding ER-phagy receptor assembly is key to comprehending cellular homeostasis.

Purpose of the Study:

• To establish a protocol for super-resolution imaging of ER-phagy receptors.
• To visualize and analyze the nanoscale assembly of ER-phagy receptors in fixed cells.
• To enable molecular quantification of ER-phagy receptor clusters.

Main Methods:

• Utilizing DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) for super-resolution microscopy.
• Applying DNA-PAINT to fixed cells for high-resolution imaging of ER-phagy receptors.
• Developing methods for image generation, cluster analysis, and molecular quantification.

Main Results:

• Demonstrated the feasibility of using DNA-PAINT for visualizing ER-phagy receptor clusters.
• Provided a detailed protocol for super-resolution imaging and analysis.
• Enabled the study of receptor assembly at the nanoscale.

Conclusions:

• DNA-PAINT is a powerful technique for resolving ER-phagy receptor assemblies.
• This protocol facilitates the investigation of molecular building principles in ER-phagy.
• Enhanced understanding of ER-phagy mechanisms through nanoscale visualization.