Heasley Lydia R, Garcia Galo, McMurray Michael A
Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA.
Eukaryot Cell. 2014 Nov;13(11):1411-20. doi: 10.1128/EC.00191-14. Epub 2014 Sep 12.
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.
Septins是一类形成细胞骨架细丝的GTP结合蛋白家族。Septins高度保守且在进化上很古老，但在陆地植物中不存在。先前已表明，合成植物细胞分裂素氯吡脲（FCF）可抑制出芽酵母细胞分裂并诱导异位septin结构（M. Iwase、S. Okada、T. Oguchi和A. Toh-e，《基因与遗传系统》79:199 - 206，2004，http://dx.doi.org/10.1266/ggs.79.199）。随后在多种真核生物中的研究得出结论，FCF专门抑制septin功能，然而FCF在非植物细胞中的作用机制仍知之甚少。在此，我们报告FCF的细胞效应比先前描述的要复杂得多。报道称用1 mM FCF处理的出芽酵母细胞的生长停滞部分反映了W303菌株背景中存在的bud4突变引起的敏感性。在野生型（BUD4(+)）出芽酵母中，FCF浓度对septin结构或功能没有可检测到的影响时，生长就受到了抑制。此外，FCF严重抑制裂殖酵母细胞的增殖，而在裂殖酵母细胞中septin功能并非必需。FCF诱导出芽酵母线粒体网的碎片化和线粒体膜电位的丧失。在用先前假定仅靶向septin的FCF浓度处理的培养哺乳动物细胞中，线粒体也发生了碎片化。最后，FCF强烈抑制嗜热四膜虫的纤毛形成和运动，并诱导线粒体紊乱，而septin结构没有明显改变。这些效应均与septin功能的抑制不一致。我们的研究结果表明，在使用FCF时，非septin靶点是主要关注点。
