Assays for genetic dissection of septin filament assembly in yeast, from de novo folding through polymerization.

In Saccharomyces cerevisiae, septin mutations have severe effects on colony-forming ability, particularly at high temperatures, allowing the full variety of genetic tools available in this model organism to be applied to the study of septin biology. Although many details of septin function remain unknown, one can exploit a small number of easily scored phenotypes-proliferation capacity, cell morphology, septin localization, and septin ring integrity-as sensitive readouts of properly assembled septin filaments. Accordingly, this chapter focuses on genetic approaches targeted toward understanding the molecular mechanisms of de novo septin folding, heterooligomerization, and polymerization into filaments. The same general methods can be used to interrogate septin function, although interpretation of results can be more complicated. As genetic-based methodologies are technically simple but particularly dependent on interpretation, here I focus on the logic underlying the most common interpretations of results using septin mutants.

Small molecule perturbations of septins.

Progress on the study of the molecular and cellular biology of septins would be greatly accelerated by the development of small molecules that directly inhibit higher-order septin assembly in vivo. By comparison, molecules like latrunculin, paclitaxil, benomyl, etc. allow researchers to acutely perturb the actin or tubulin cytoskeletal networks. Two small molecules, forchlorfenuron (FCF; N-(2-chloro-4pyridyl)-N-phenylurea) and 1-ethyl-3-(4-methoxyphenyl)-6-methylpyrimido[5,4-e][1,2,4]triazine-5,7-dione (PubChem CID 906558), have documented effects on septin localization and/or function, although for each molecule there is also strong evidence for off-target effects. In this chapter we provide a summary of ways to utilize FCF to alter higher-order septin assembly properties in living cells.