Preparing recombinant yeast septins and their analysis by electron microscopy.

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• Septins are crucial eukaryotic cytoskeletal proteins interacting with the plasma membrane.
• They play vital roles in cell division and morphogenesis, and are implicated in diseases.
• Septin gene expression varies by organism and tissue, with 2-13 genes typically found.

Purpose of the Study:

• To present detailed methods for analyzing the molecular organization of septin complexes.
• To describe techniques applicable to budding yeast septins and generalizable to other organisms.
• To outline procedures for purification, electron microscopy sample preparation, and image processing.

Main Methods:

• Purification of septin complexes from budding yeast.
• Sample preparation techniques for electron microscopy (EM).
• Single-particle EM methodology and image processing for subunit localization using genetic tags (e.g., MBP, GFP).

Main Results:

• Established methods for visualizing and analyzing septin rod and filament structures.
• Demonstrated the localization of specific septin subunits within the complex.
• Provided a detailed protocol for EM-based septin structural analysis.

Conclusions:

• The presented methods enable detailed molecular analysis of septin organization.
• These techniques are adaptable for studying septins from diverse organisms.
• High-resolution structural insights into septin complexes can be achieved via EM.