Localization and function of septins are susceptible to epitope tagging

Area of Science:

• Cell Biology
• Cytoskeletal Dynamics
• Molecular Genetics

Background:

• Septins are essential cytoskeletal proteins involved in cell division and morphogenesis.
• Epitope tagging is a common method to study protein localization and function, including septins.
• Previous studies suggested interphase septin structures are crucial for protein localization and function.

Purpose of the Study:

• To rigorously test the functionality of epitope-tagged septins in fission yeast.
• To re-evaluate the proposed roles of interphase septin structures in protein localization and function.
• To highlight the importance of functional validation for tagged proteins.

Main Methods:

• Utilizing fission yeast as a model genetic system.
• Analyzing cell morphology and cytokinesis/septation defects.
• Performing genetic interaction studies with arrestin (art1Δ) and anillin (mid2Δ) mutants.

Main Results:

• Septins tagged with tdTomato or 3HA were found to be non-functional.
• Interphase septin structures observed with these tags do not reflect true septin function.
• Sid2 kinase localization to the division site was delayed in septin mutants, contradicting prior findings.

Conclusions:

• Epitope tags like tdTomato and 3HA can perturb septin functionality, leading to misinterpretation of localization and function data.
• Rigorous functional assays are critical for validating results obtained from tagged proteins.
• Septin polymers are susceptible to perturbations, necessitating careful consideration in experimental design and data interpretation.