Cellular requirements for the small molecule forchlorfenuron to stabilize the septin cytoskeleton

Abstract

The septins are filament-forming, GTP-binding proteins that are conserved from yeast to humans. Septins assemble into higher-order structures such as rings, bars, and gauzes with diverse functions including serving as membrane diffusion barriers and scaffolds for cell signaling. The basis for septin filament polymerization and the rules governing septin polymer dynamics are presently not well understood. Pharmacological agents are essential tools in studying such properties of the actin and microtubule cytoskeletons however there are only limited reports of a drug specific to the septin cytoskeleton. Forchlorfenuron (FCF) is a synthetic plant cytokinin used in agriculture which has been shown to alter septin organization in yeast and mammalian tissue culture cells. Here we assess cellular requirements and properties of septin-based structures induced by FCF. Treatment of the filamentous fungus Ashbya gossypii with FCF leads to assembly of extensive septin fibers throughout hyphae which is rapidly reversed upon removal of the drug. These fibers do not exchange or add septin subunits after assembly, indicating that FCF suppresses normal septin dynamics and stabilizes the polymers. While FCF-induced septin fibers do not co-localize to actin or microtubules, a polarized F-actin cytoskeleton is likely required for the assembly of drug-induced septin fibers. Thus, FCF is a potent inducer of septin polymerization and acts as a reversible stabilizer of extended septin polymers. This drug will be a powerful tool for studying mechanisms of septin polymerization and function, particularly in cell types where molecular analyses are complicated by the presence of multiple isoforms and limited genetics.

Recruitment of septin cytoskeletal proteins by Botulinum toxin A protease determines its remarkable stability

“…This compound has been recently found to affect septin assembly, organization and dynamics, both in yeast and mammalian cells Iwase et al, 2004). FCF impaired normal septin assembly and induced the formation of abnormally large septin structures (DeMay et al, 2010;Hu et al, 2008). The effects of FCF on mitosis, cell migration and cell adhesion were similar to those induced by depletion of septin-2 by siRNA Sidhaye et al, 2011).…”

Septin Dynamics Are Essential for Exocytosis

“…Here, to elucidate the role of septins in exocytosis, we used acute down-regulation of the major ubiquitously expressed septin, septin-2, by transient siRNA. In addition, we used the inhibitor of septin organization, forchlorfenuron (FCF), which specifically impairs assembly and disassembly of septin hetero-oligomers (27)(28)(29) without affecting actin or tubulin polymerization (27,30). Both FCF and knockdown of septin-2 decreased the rate of exocytosis in cultured cells.…”

Off-Target Effects of the Septin Drug Forchlorfenuron on Nonplant Eukaryotes

“…Off-target responses to FCF have been documented in other cell types, most notably A. gossypii, in which 125 M FCF caused, in addition to ectopic septin structures, reversible inhibition of growth at the hyphal tip, a gnarled cellular morphology, and the accumulation of abnormally thick septa, all common symptoms of general stress responses that are not seen in septin mutants of this fungus and occur even in septin mutant cells treated with FCF (25). At higher FCF concentrations (500 M to 2 mM), the actin and tubulin components of the cytoskeleton also were perturbed (25).…”

“…At higher FCF concentrations (500 M to 2 mM), the actin and tubulin components of the cytoskeleton also were perturbed (25). In organisms for which septin loss-of-function mutants are available or there are clear cellular phenotypes associated with septin dysfunction (such as elongated bud morphologies in S. cerevisiae and chained cells in S. pombe), it is possible to determine which are the off-target effects of FCF.…”

“…The ectopic structures contained all of the mitotic septins, suggesting that the structures were composed of polymers of native septin heterooctamers (24). The effects of FCF on the filamentous fungus Ashbya gossypii were subsequently examined and included, at 125 M, reversible inhibition of growth at the hyphal tip and ectopic polymerization of septins into long, stable fibers (25). Striking electron microscopy (EM) images of fixed FCF-treated Ashbya cells revealed paired filaments sharing all of the known properties of septin filaments formed in vitro from purified proteins (26).…”