Characteristics of weak base-induced vacuoles formed around individual acidic organelles

Area of Science:

• Cell Biology
• Molecular Biology
• Toxicology

Background:

• Previous research linked 4-aminopyridine to organelle traffic disorders via vacuole formation.
• Weak bases are known to affect cellular processes, but the precise mechanisms of vacuole induction require further elucidation.

Purpose of the Study:

• To investigate the characteristics of vacuoles induced by various weak bases (NH(4)Cl, aminopyridines, chloroquine) in mouse cells.
• To understand the cellular mechanisms underlying weak base-induced vacuolization and its consequences.

Main Methods:

• Utilized mouse cells and fluorescent protein expression to identify acidic organelles within vacuoles.
• Employed amine-reactive fluorescence and immunochemical staining to analyze vacuole and intravacuolar content.
• Assessed vacuole acidity using LysoTracker and LysoSensor, and evaluated the effects of vacuolar H(+)-ATPase inhibitors and chloride-free medium.

Main Results:

• Vacuoles formed around acidic organelles, with mitochondria and actin filaments at the vacuole rim.
• Vacuoles lacked protein/amino acid content, suggesting cytosol partitioning, while weak bases were found within organelles, not vacuoles.
• Vacuolization was reversible in early stages but led to cell death with prolonged exposure; this process was inhibited by blocking vacuolar H(+)-ATPase or removing chloride.
• Intravacuolar organelles were highly acidic, while vacuoles exhibited slight acidity.

Conclusions:

• Weak base-induced vacuolization involves the accumulation of weak bases and protons in acidic organelles, facilitated by vacuolar H(+)-ATPase and chloride influx.
• The process likely involves the extrusion of protons and water into the cytoplasm, leading to organelle dysfunction and potential cell death.
• Understanding these mechanisms is crucial for evaluating the cellular toxicity of weak bases and developing potential interventions.