Abstract
Septins are filamentous GTPases that associate with cell membranes and the cytoskeleton and play essential roles in cell division and cellular morphogenesis. Septins are implicated in many human diseases including cancer and neuropathies. Small molecules that reversibly perturb septin organization and function would be valuable tools for dissecting septin functions and could be used for therapeutic treatment of septin-related diseases. Forchlorfenuron (FCF) is a plant cytokinin previously shown to disrupt septin localization in budding yeast. However, it is unknown whether FCF directly targets septins and whether it affects septin organization and functions in mammalian cells. Here, we show that FCF alters septin assembly in vitro without affecting either actin or tubulin polymerization. In live mammalian cells, FCF dampens septin dynamics and induces the assembly of abnormally large septin structures. FCF has a low level of cytotoxicity, and these effects are reversed upon FCF washout. Significantly, FCF treatment induces mitotic and cell migration defects that phenocopy the effects of septin depletion by small interfering RNA. We conclude that FCF is a promising tool to study mammalian septin organization and functions.
Highlights
Septins are guanine nucleotide-binding proteins that form filaments associated with membranes and the cytoskeleton [22, 23]
Our studies show that FCF directly alters septin assembly in vitro and affects septin organization in mammalian cells by stabilizing septin filaments; these effects of FCF in cells are reversible upon FCF washout
Septin 2 (SEPT2)/6/7 complexes were dialyzed in the presence of FCF or DMSO in low salt buffer to induce septin self-assembly into higher-order structures, which were analyzed by immunofluorescence microscopy (Fig. 1B) or transmission electron microscopy (Fig. 1F)
Summary
HeLa S3 cells were maintained in high glucose DMEM supplemented with 10% fetal bovine serum (FBS). Staining of MDCK cells for centromere-associated protein E (CENP-E) and CREST serum was performed as described previously [18]. Cytotoxicity Assays—HeLa or MDCK cells were plated overnight on collagen-coated 35-mm tissue culture dishes and treated with DMSO or FCF for 4 h. Live Cell Imaging and FRAP—Cells were grown on collagencoated coverslips for h at subconfluent density and imaged in phenol red-free DMEM supplemented with 10% FBS, mM Hepes (Invitrogen) using the Marianas system (Intelligent Imaging Innovations) equipped with the MicroPoint fluorescence recovery after photobleaching (FRAP) laser system (Photonic Instruments). For wound-healing assays, HeLa cells (2 ϫ 106) were grown on 35-mm dishes overnight in DMEM and 10% FBS.
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