Ashbya gossypii as a model system to study septin organization by single-molecule localization microscopy

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Septins are GTP-binding proteins forming higher-order structures crucial for cell division.
• Existing microscopy techniques have limitations in resolving individual septin complexes within cellular assemblies.
• Understanding septin organization is key to deciphering their role in fundamental cellular processes.

Purpose of the Study:

• To develop and present a protocol for investigating septin complex organization in higher-order structures.
• To leverage single-molecule localization microscopy (SMLM) for high-resolution imaging of septins in intact cells.
• To provide a versatile assay applicable to various cell types for studying septin assembly mechanisms.

Main Methods:

• Utilized the model organism Ashbya gossypii for its advantageous biological features.
• Employed single-molecule localization-based fluorescence superresolution microscopy (SMLM).
• Combined SMLM with cellular assays to achieve nanometer-level resolution and molecular specificity.

Main Results:

• Established a protocol enabling high-resolution visualization of septin complexes within cellular structures.
• Demonstrated the capability to identify individual septin complexes in assemblies using SMLM.
• Showcased the applicability of the assay for studying general septin assembly mechanisms.

Conclusions:

• SMLM offers superior resolution and specificity for studying septin organization compared to traditional methods.
• The developed protocol provides a powerful tool for investigating the dynamics and assembly of septin complexes in vivo.
• This approach facilitates a deeper understanding of the molecular basis of cell division and other septin-dependent processes.