Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

Area of Science:

• Cell Biology
• Microscopy Techniques

Background:

• Electron microscopy (EM) provides ultrastructural details of autophagy but lacks molecular information.
• Correlating EM ultrastructure with fluorescence microscopy localization of autophagy proteins is challenging.
• Autophagosomes are rare in cells, limiting EM investigation due to small fields of view.

Purpose of the Study:

• To develop and validate an on-section correlative light-electron microscopy (CLEM) method.
• To correlate the autophagosomal marker LC3 with ultrastructure.
• To study autophagy in native conditions without drug treatments or genetic alterations.

Main Methods:

• Applied on-section CLEM using fluorescently labeled LC3.
• Screened cells using fluorescence microscopy for LC3 labeling.
• Identified ultrastructural features of LC3-labeled spots using CLEM on starved cells.

Main Results:

• Successfully correlated LC3 localization with EM ultrastructure.
• LC3 was predominantly found on autophagosomes and rarely in autolysosomes in starved cells.
• Demonstrated the feasibility and sensitivity of CLEM for studying autophagy.

Conclusions:

• CLEM is a valuable tool for ultrastructural localization of autophagy proteins like LC3.
• This method bridges light microscopy and EM data for autophagy research.
• Enables ultrastructural insights into autophagy in native cellular conditions.