Off-target effects of the septin drug forchlorfenuron on nonplant eukaryotes

Abstract

The septins are a family of GTP-binding proteins that form cytoskeletal filaments. Septins are highly conserved and evolutionarily ancient but are absent from land plants. The synthetic plant cytokinin forchlorfenuron (FCF) was shown previously to inhibit budding yeast cell division and induce ectopic septin structures (M. Iwase, S. Okada, T. Oguchi, and A. Toh-e, Genes Genet. Syst. 79:199-206, 2004, http://dx.doi.org/10.1266/ggs.79.199). Subsequent studies in a wide range of eukaryotes have concluded that FCF exclusively inhibits septin function, yet the mechanism of FCF action in nonplant cells remains poorly understood. Here, we report that the cellular effects of FCF are far more complex than previously described. The reported growth arrest of budding yeast cells treated with 1 mM FCF partly reflects sensitization caused by a bud4 mutation present in the W303 strain background. In wild-type (BUD4(+)) budding yeast, growth was inhibited at FCF concentrations that had no detectable effect on septin structure or function. Moreover, FCF severely inhibited the proliferation of fission yeast cells, in which septin function is nonessential. FCF induced fragmentation of budding yeast mitochondrial reticula and the loss of mitochondrial membrane potential. Mitochondria also fragmented in cultured mammalian cells treated with concentrations of FCF that previously were assumed to target septins only. Finally, FCF potently inhibited ciliation and motility and induced mitochondrial disorganization in Tetrahymena thermophila without apparent alterations in septin structure. None of these effects was consistent with the inhibition of septin function. Our findings point to nonseptin targets as major concerns when using FCF.

FIG 1

Loss of Bud4 function sensitizes Saccharomyces cerevisiae cells to growth inhibition by FCF. (A) Tenfold serial dilutions of the indicated strains were spotted on YPD plates containing solvent alone (0.4% ethanol) or 1 mM FCF and incubated at 22°C for 3 days or 5 days, respectively. (B) Duplicate (numbers 1 and 2) 200-μl liquid YPD cultures with 0.4% ethanol (solvent) or FCF at the indicated concentrations were inoculated at an optical density at 600 nm (OD₆₀₀) of 0.1 and monitored at 5-min intervals over the course of 28 h at 25°C. Dashed lines indicate fits of the indicated growth curves from the 7- to 28-h time points; see the text for details on curve fits.

FIG 2

Ectopic septin structures in Saccharomyces cerevisiae are not responsible for growth inhibition by FCF. (A) Cells of the indicated strains carrying the Cdc10-mCherry-expressing plasmid pZL02 were grown to mid-log phase at 22°C in selective synthetic medium, loaded into microfluidic chambers, and exposed to a constant flow (85 μl/h) of the same medium containing 0.4% ethanol (solvent). After 1 h, the cells were imaged, and medium containing 1 mM FCF was introduced at the same flow rate. The cells were imaged again after 2 h. Shown are overlays of transmitted light images (to show cell outlines) and epifluorescence micrographs of mCherry fluorescence. Arrowheads indicate ectopic septin structures. (B) MMY0132 cells shown in panel A were imaged at 15-min intervals over a 3.5-h time course of exposure to 1 mM FCF. Inset numbers indicate time in min. Arrowheads indicate ectopic septin structures. (C) BY4741 cells expressing Cdc11-YFP from plasmid pML43 and overexpressing Bud4 from plasmid pP_GAL-BUD4 were exposed to solvent alone (0.4% ethanol) or 1 mM FCF for 18 h. Arrowheads indicate ectopic septin structures. Lines indicate elongated buds, a hallmark of septin dysfunction. (D) Cells taken from 1.0 mM FCF cultures shown in panel B were visualized by microscopy to assess elongated cellular morphology as an indicator of dysfunction. (E) A YPD culture of JTY3992 was split into four cultures to which no FCF (but 0.4% ethanol; indicated as the zero baseline) or the indicated concentration of FCF was added. After overnight growth at 22°C, the densities of the cultures were measured (OD₆₀₀; filled columns), and samples were washed and imaged as described for panel A to score the fraction of cells containing ectopic septin structures (open columns). At least 260 cells were examined for each condition. Scale bars, 5 μm.

FIG 3

FCF inhibits the growth of Schizosaccharomyces pombe cultures and induces aberrant cellular morphologies that do not phenocopy septin mutation. (A) A YE5S culture of S. pombe strain JCF109 was split into five cultures to which various concentrations of FCF were added prior to overnight growth at 22°C, as described for Fig. 2E. (B) Samples from the cultures shown in panel A were imaged by transmitted light. Note that the 1 mM FCF image is a montage of images of smaller fields due to low cell density. Scale bars, 5 μm.

FIG 4

FCF induces mitochondrial fragmentation in S. cerevisiae. Shown are overlays of transmitted light images (to show cell outlines) and epifluorescence micrographs. (A) Cells of wild-type strain BY4741 carrying the Cdc11-YFP plasmid pML43 and the plasmid pOli1-RFP, which encodes the red-emitting protein HcRed fused to the mitochondrial targeting sequence of Oli1, were loaded into a microfluidic chamber and exposed to a constant flow (85 μl/h) of FCF-free medium before exposure to FCF-containing medium for the indicated times, as described for Fig. 2A. After 12 h, FCF-free medium was provided, and cells were imaged after an additional 2 h. (B, left) BY4741 cells expressing plasmid-encoded GFP fused to the mitochondrial targeting signal of Cit1 were grown overnight at 22°C in the presence of 0.4% ethanol (solvent). (Right) BY4741 cells expressing Cit1-GFP and coexpressing Cdc10-mCherry from plasmid pZL02 were grown overnight at 22°C in selective medium containing 1 mM FCF. (C) Wild-type cells of strain JTY3992 were grown for 18 h in the presence of 0.4% ethanol (solvent) or 1 mM FCF prior to staining with the mitochondrial membrane potential-sensitive dye DiOC₆ for 2 min. Images were captured with two different exposure times, as indicated, to demonstrate the difference in staining intensity. Scale bars, 5 μm.

FIG 5

FCF induces mitochondrial fragmentation in cultured human cells. Human RPE-1 cells were exposed to 0.02% ethanol (solvent) or 50 μM FCF for 2 h prior to staining with MitoTracker red to visualize mitochondria. Scale bar, 100 μm.

FIG 6

FCF causes loss of mitochondrial organization without ectopic septin structures in Tetrahymena thermophila. T. thermophila cells were exposed to 0.4% ethanol (solvent) or the indicated concentrations of FCF. (A) Cells expressing Sep1-GFP or Sep2-GFP, as indicated, exposed to solvent or FCF for 1 h. (B) Cells with untagged septins exposed to FCF for 20 min prior to MitoTracker staining. Images were converted to grayscale and inverted to improve visibility. Scale bars, 20 μm.