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Updated: Jun 17, 2025

Study of Protein-protein Interactions in Autophagy Research
14:08

Study of Protein-protein Interactions in Autophagy Research

Published on: September 9, 2017

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Studying Selective Autophagy of Protein Aggregates Using Particles Induced by Multimerization (PIMs)

Giel Korsten1, Lukas C Kapitein2,3

  • 1Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.

Methods in Molecular Biology (Clifton, N.J.)
|August 8, 2024
PubMed

Insights

This study presents a protocol for the PIM assay, enabling controlled formation and visualization of protein aggregates for studying aggrephagy and cellular homeostasis.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Autophagy Research

Background:

  • Selective autophagy, or aggrephagy, is crucial for cellular homeostasis by clearing protein aggregates.
  • Studying aggrephagy is difficult due to infrequent events and lack of control over aggregate formation.
  • Previous work introduced the PIM (particles induced by multimerization) assay for chemically induced aggregate formation.

Purpose of the Study:

  • To provide a detailed protocol for utilizing the PIM assay.
  • To enable controlled and specific induction of protein aggregates for aggrephagy studies.
  • To facilitate the visualization of aggregate clearance in live and fixed cells.

Main Methods:

  • Utilizing two variants of the PIM assay for aggregate induction.
  • Employing chemically induced, fluorescently labeled protein aggregates.
  • Using pH-sensitive fluorescent proteins (e.g., GFP, mKeima) for visualization.
  • Applying the protocol to both live and fixed cell imaging.

Main Results:

  • The PIM assay allows for controlled formation of protein aggregates.
  • Aggregates formed via the PIM assay are recognized and degraded by the selective autophagy machinery.
  • The use of pH-sensitive fluorescent proteins enables direct visualization of aggregate clearance.
  • The protocol is effective in both live and fixed cell imaging.

Conclusions:

  • The PIM assay provides a robust method for studying aggrephagy.
  • This protocol enhances the ability to investigate cellular mechanisms of protein aggregate clearance.
  • The PIM assay is a valuable tool for research in cellular homeostasis and autophagy.
Keywords:
AggrephagyInducible dimerizationLive-cell imaging

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