Localization and function of septins are susceptible to epitope tagging.

Septins are hetero-oligomeric cytoskeletal proteins that assemble into filaments and scaffolds on the plasma membrane to aid cytokinesis, morphogenesis, and other cellular processes. Epitope tagging is widely used to study septin localization and function. However, functionality testing of tagged septins is often insufficient because of technical challenges. Fission yeast provides an ideal genetic system to test functionalities and localizations of tagged septins. mEGFP/mYFP tagged septins Spn1 and Spn4 localize exclusively to the division site as double rings during cytokinesis, but tdTomato tagged septins also localize to puncta or short linear structures across the plasma membrane. It was proposed that these additional septin structures serve as diffusion barriers and are important for the localizations and functions of several proteins, including the NDR-kinase Sid2 and active Cdc42 GTPase. By analyzing cell morphology, cytokinesis defects, and genetic interactions between tagged septins and three mutations, we find that septins are less functional with tdTomato or 3HA than other tags. Additionally, Sid2 appearance at the division site is after septins and delayed in septin deletions, contrary to previous reports. Our data re-emphasize the need for rigorous functional tests of tagged septins and for caution in interpreting function and localization data when using epitope tagged septins. [Media: see text].