Calpain mobilizes Atg9/Bif-1 vesicles from Golgi stacks upon autophagy induction by thapsigargin

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Calpain, a calcium-dependent protease, plays roles in various cellular processes.
• Autophagy is a fundamental cellular process for degrading damaged components.
• CAPNS1 is a regulatory subunit essential for calpain stability and function.

Purpose of the Study:

• To investigate the role of CAPNS1 and calpain in autophagy activation.
• To elucidate the mechanism by which thapsigargin induces autophagy.
• To determine the involvement of Atg9 and Bif-1 trafficking in CAPNS1-dependent autophagy.

Main Methods:

• Human U2OS osteosarcoma cells were used.
• Endoplasmic reticulum Ca2+ ATPase was inhibited using thapsigargin.
• CAPNS1 depletion was achieved via genetic manipulation.
• Immunofluorescence microscopy was employed to track protein localization (Atg9, Bif-1, LC3, GM130, Rab5, transferrin receptor).
• Western blotting was used to assess protein levels (p62, LC3-II).

Main Results:

• Thapsigargin treatment activated micro-calpain and autophagy in a CAPNS1-dependent manner.
• CAPNS1 depletion led to accumulation of LC3 bodies and early endosomes.
• Atg9 and Bif-1 failed to traffic from the Golgi to the endocytic route in CAPNS1-depleted cells.
• Calpain processing of Bif-1 appeared necessary for autophagic flux.

Conclusions:

• CAPNS1-dependent calpain activity is essential for the dynamic trafficking of Atg9/Bif-1 vesicles.
• Calpain facilitates the movement of these vesicles from the Golgi towards autophagosome formation.
• This study reveals a novel role for calpain in regulating autophagic vesicle trafficking.