Visualizing Reversible Cisternal Stacking in Budding Yeast Pichia pastoris.

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Golgi apparatus cisternal stacking is a dynamic, reversible process.
• Trans-Golgi Network (TGN) peeling is a key feature of this reversible stacking.
• Visualizing rapid TGN peeling events in live cells is methodologically challenging.

Purpose of the Study:

• To develop and describe a quantitative live microscopic methodology for visualizing TGN peeling.
• To investigate the frequency and dynamics of TGN peeling in Pichia pastoris.
• To study the role of specific proteins in reversible cisternal stacking and TGN dynamics.

Main Methods:

• Quantitative live microscopy in Pichia pastoris.
• Analysis of TGN peeling frequency in wild-type and mutant cells.
• Focus on mutants affecting reversible cisternal stacking (e.g., PpImh1, Arl3, Arl1 GTPase).

Main Results:

• TGN peeling occurs rapidly and at low frequency in wild-type cells, making it difficult to observe.
• TGN peeling becomes significantly more frequent in mutants affecting proteins like PpImh1, Arl3, and Arl1 GTPase.
• The developed methodology enables the visualization and quantification of TGN peeling events.

Conclusions:

• The described live microscopic method effectively visualizes the challenging TGN peeling phenomenon.
• Mutations in key Golgi-related proteins increase the frequency of TGN peeling, providing insights into cisternal stacking regulation.
• This methodology facilitates further research into the mechanisms of reversible Golgi stacking and TGN dynamics.