- Email: [email protected]
Small molecule perturbations of septins
CHAPTER
Small molecule perturbations of septins
18
L.R. Heasley, M.A. McMurray1 University of Colorado Anschutz Medical Campus, Aurora, CO, United States 1
Corresponding author: E-mail: [email protected]
CHAPTER OUTLINE 1. Forchlorfenuron.................................................................................................. 311 1.1 Background and Summary of Cellular Effects ........................................ 311 1.2 Properties of Forchlorfenuron ............................................................... 314 1.3 Genetic Manipulation of Septin Dosage in Budding Yeast Alters Forchlorfenuron Sensitivity................................................................... 316 2. 1-Ethyl-3-(4-methoxyphenyl)-6-methylpyrimido[5,4-e][1,2,4]triazine-5,7-dione ..... 316 3. Summary and Perspective................................................................................... 317 References ............................................................................................................. 317
Abstract 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-ethyl3-(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.
1. FORCHLORFENURON 1.1 BACKGROUND AND SUMMARY OF CELLULAR EFFECTS The synthetic plant cytokinin forchlorfenuron (FCF) (also called CPPU) increases fruit size in plants via inhibition of the catabolic cytokinin dehydrogenase CKO (Kopecny´ et al., 2010). Notably, higher plants lack septins. As Methods in Cell Biology, Volume 136, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2016.03.013 © 2016 Elsevier Inc. All rights reserved.
311
312
CHAPTER 18 Small molecule perturbations of septins
described later, FCF addition to various different kinds of nonplant eukaryotes that do express septins drives the reversible formation of ectopic septin structures. In budding yeast cells proliferating vegetatively, all tested septins, rather than localizing exclusively to the bud neck, also appear in additional structures localized somewhat randomly along the cell cortex or as long, straight fibers that often appear to “nucleate” from the normal structures at the bud neck (Heasley, Garcia, & McMurray, 2014; Iwase, Okada, Oguchi, & Toh-e, 2004). Similar ectopic structures form upon addition of FCF at 125 mM to growing cells of the filamentous fungus Ashbya gossypii: septins polymerize into long, stable ectopic fibers (DeMay, Meseroll, Occhipinti, & Gladfelter, 2010). Images of these fibers obtained by electron microscopy reveal striking similarities to septin filaments formed in vitro from purified proteins in the absence of FCF (DeMay et al., 2011). Addition of FCF to cultured mammalian cells has varied effects, depending on the cell type (see Table): in human HeLa, HEK-293 human embryonic kidney, and canine MDCK cells, FCF reversibly increases the length and width of cellular septin structures (Hu, Nelson, & Spiliotis, 2008; Tokhtaeva et al., 2015); in PC-3 human prostate cancer cells, on the other hand, FCF causes a shift from long, structured filaments to shorter filaments, and septin exclusion from the nucleus (Vardi-Oknin, Golan, & Mabjeesh, 2013). In yeast and cultured mammalian cells, these effects of FCF on higher-order septin structures are accompanied by evidence of defects in established septin functions: elongation of bud morphology (Heasley et al., 2014; Iwase et al., 2004) and defects in mitosis and cell migration (Hu et al., 2008), respectively. In vitro, addition of 5 mM FCF to purified mammalian septin complexes promotes lateral association of septin filaments (Hu et al., 2008), and at 25 mM, FCF accelerates polymerization and bundling of a purified Schistosoma mansoni septin complex (Zeraik et al., 2014). Moreover, addition of FCF changes the thermal stabilities of individually purified human septins, but not when excess GDP is also added, consistent with direct binding in the septin GTP-binding pocket (Angelis, Karasmanis, Bai, & Spiliotis, 2014). Indeed, in silico modeling demonstrates that FCF fits well within the septin GTP-binding pocket and binds directly to key residues normally involved in GTP hydrolysis (Angelis et al., 2014). Considering the extensive evidence indicating that GTP binding and hydrolysis are critical for directing normal higher-order septin assembly (Farkasovsky, Herter, Voss, & Wittinghofer, 2005; Nagaraj, Rajendran, Jackson, & Longtine, 2008; Sirajuddin, Farkasovsky, Zent, & Wittinghofer, 2009; Versele & Thorner, 2004; Weems, Johnson, Argueso, & McMurray, 2014), direct FCF-septin interactions likely modify interactions between septin subunits or polymers. Taken together with the in vivo effects described above, these findings suggest that FCF is a useful tool for acutely and reversibly perturbing septin function in living cells. However, there are important caveats in the interpretation of cellular effects accompanying the application of FCF. Addition of FCF to yeast medium inhibits wild-type colony growth (on rich solid medium) or culture growth (in liquid rich medium) to an extent that depends somewhat on the “robustness” of septin function. We
Organism/Cell Type
[Forchlorfenuron]
Septin response (ND, not determined)
Cellular Response
References
Nannochloris bacillaris (green algae) Saccharomyces cerevisiae (baker’s/brewer’s yeast)
100 mM
ND
Inhibition of proliferation
Yamazaki et al. (2013)
1.5 mM 1 mM
2 mM
Formation of long septin fibers Formation of puncta
Schistosoma mansoni (blood fluke) RPE1 cells (human retinal) HIRc cells (rat) Podocytes (mouse)
500 mM
Polymerization (in vitro)
Mitochondrial fragmentation, reduction in mitochondrial membrane potential, inhibition of growth. Inhibition of growth, reduction in cell size Inhibition of growth at hyphal tip, gnarled morphology, thick septa Rounding, loss of cilia, cessation of swimming, disorganization of mitochondria Paralysis
Heasley et al. (2014), Iwase et al. (2004)
Schizosaccharomyces pombe (fission yeast) Ashbya gossypii (filamentous fungus) Tetrahymena thermophila (pond scum)
Ectopic polymerization, mislocalization to but tip, cell cortex, bud shaft, cytosol ND
50 mM 50 mM 50 mM
Mitochondrial fragmentation Increased glucose uptake Increase glucose uptake
Heasley et al. (2014) Wasik et al. (2012) Wasik et al. (2012)
HeLa cells
100e200 mM
ND ND Sept7 bundling at cell periphery ND
Sharma et al. (2013)
HeLa cells
5 mM
ND
PC-3 (human cancer cells)
200 mM
HEK-293 (human kidney cells) LNCaP, HCT-116, MCF-7 (human cancer cells)
20e100 mM
Formation of short, disordered filaments, loss of nuclear localization Increase in length of septin filaments ND
Reduction in store-operated Ca2þ uptake, Inhibition of Golgi reorganization during “wound healing” Reduction in proliferation, migration, transformation, HIF-1a expression, HIF-1 transcriptional activity Inhibition of constitutive and stimulated exocytosis and acetycholine secretion Reduction in HIF-1a expression
125 mM
200 mM
Heasley et al. (2014) DeMay et al. (2011), DeMay et al. (2010) Heasley et al. (2014)
Zeraik et al. (2014)
Lee et al. (2014) Vardi-Oknin et al. (2013) Tokhtaeva et al. (2015) Vardi-Oknin et al. (2013)
314
CHAPTER 18 Small molecule perturbations of septins
showed that the W303 strain background, which carries a truncation in the anillinlike protein Bud4 known to contribute to septin dynamics and function, is particularly sensitive to FCF inhibition and introducing a bud4D mutation sensitized the otherwise “robust” BY4743 background (Heasley et al., 2014). Moreover, in BUD4þ yeast cells, colony and culture growth are noticeably slower in the presence of 1 mM and 0.5 mM FCF, respectively (we suspect that the differences between liquid and solid media reflect differences in FCF solubility; see below), and at these concentrations there is little effect on ectopic septin polymerization (Heasley et al., 2014). Instead, mitochondria rapidly and reversibly fragment (Heasley et al., 2014), indicative of an overall change in metabolism and/or a systemic stress response. We described similar mitochondrial responses in Tetrahymena thermophila and human retinal pigmented epithelial cells, which did not correspond to known effects of septin dysfunction (Heasley et al., 2014). In Schizosaccharomyces pombe, we found that, at relatively low concentrations (0.1 mM), FCF inhibits culture growth and perturbs cellular morphology (Heasley et al., 2014), whereas even null septin mutations do not share these phenotypes (An, Morrell, Jennings, Link, & Gould, 2004). In A. gossypii, as well, multiple cellular phenotypes that accompanied FCF treatment were consistent with a stress response, rather than septin dysfunction (DeMay et al., 2010) (see Table). These demonstrations of nonseptin (ie, off-target) cellular effects by no means discount the ability of FCF to modulate the septin cytoskeleton, but they do suggest caution in the use of FCF in vivo. Among other processes and cell types (see Table for a summary), published studies in mammalian cells have interpreted cellular effects of FCF to indicate septin involvement in exocytosis (Tokhtaeva et al., 2015), hypoxic response (Vardi-Oknin et al., 2013), calcium signaling (Sharma et al., 2013), and glucose uptake (Wasik et al., 2012). Septin depletion via RNAi was in most cases also used to corroborate these roles; some sort of independent genetic perturbation of septins is highly recommended as a prerequisite to arriving at a firm conclusion.
1.2 PROPERTIES OF FORCHLORFENURON FCF is a synthetic compound derived from urea and containing a phenol ring and chlorinated pyridine (Fig. 1A). We have purchased FCF from two sources, SigmaAldrich (#C2791) and Santa Cruz Biotechnology (#KT-30). These powders differ noticeably in their appearance, but their cellular effects are indistinguishable, in our hands. FCF is scarcely soluble in water and far more soluble in ethanol. The poor solubility of FCF becomes problematic in fungal studies, where tough cell walls (and, presumably, potent efflux pumps) necessitate much higher concentrations of FCF compared to other organisms. Two millimolar is the highest concentration we have been able to reliably achieve in solution; the concentration needed to induce ectopic polymerization of septins in a sensitized background is 1 mM. We typically prepare stock solutions of FCF at 250 mM in 95% ethanol, with rigorous vortexing. This means that, as an appropriate “solvent” control, when using high
1. Forchlorfenuron
FIGURE 1 Structures of small molecules that perturb septin function, and alterations in such perturbations upon subtle manipulations of yeast septins. (A) Chemical structures of forchlorfenuron (FCF), 1-ethyl-3-(4-methoxyphenyl)-6-methylpyrimido[5,4-e][1,2,4] triazine-5,7-dione (PCID 906558), and, for comparison, guanosine. (B) Yeast cells of the BY4743 background and the indicated genotypes were exposed to 1 mM FCF in rich liquid medium for 18 h, spotted on an agarose pad, and imaged by transmitted light microscopy with a 60X objective. Brackets indicate extrachromosomal low-copy plasmids carried by wild-type haploid cells.
315
316
CHAPTER 18 Small molecule perturbations of septins
FCF concentrations, one should probably also expose cells to the appropriate concentration of ethanol alone (eg, 0.38% for 1 mM FCF). FCF cannot be “top-spread” on solid media plates; it must be dissolved into molten agar, which still does not guarantee prolonged solubility. We have found that at concentrations 1 mM, FCF eventually crystalizes on top of and within the solid media, which seems to decrease the local effective concentration of the compound. Stock solutions of FCF appear quite stable during long-term storage in ethanol, in terms of activity (if not solubility).
1.3 GENETIC MANIPULATION OF SEPTIN DOSAGE IN BUDDING YEAST ALTERS FORCHLORFENURON SENSITIVITY Here we report a few unpublished observations that may be of interest to the field regarding the use of FCF. Our finding that deletion of BUD4-sensitized budding yeast cells to FCF prompted us to ask if other perturbations of the septin system could sensitize cells. First, we hypothesized that, if FCF binds preferentially to a single septin subunit, eliminating or reducing the amount of that subunit might confer FCF resistance. Yeast cells tolerate deletion of CDC10, CDC11, SHS1, or both CDC11 and SHS1 (Flescher, Madden, & Snyder, 1993; Frazier et al., 1998; Iwase, Luo, Bi, & Toh-e, 2007; McMurray et al., 2011). Loss of one copy of any one of the five mitotic septins is also well tolerated in diploids. We compared the morphology of BY4743-background (BUD4þ) haploid homozygous mutants (cdc10D and shs1D) and diploid heterozygous mutants (CDC3/cdc3D, CDC10/cdc10D, CDC11/cdc11D, CDC12/cdc12D, SHS1/shs1D) grown for 18 h in rich medium in the presence or absence of 1 mM FCF. While the morphology of wild-type diploids was generally unaffected, all of the septin mutants exhibited variable morphological defects (Fig. 1B). These findings suggest that the septin response to FCF is highly contingent on the stoichiometry of certain septin proteins. In separate experiments, we also noticed that tagging a single septin with a C-terminal fluorophore fusion sensitizes cells to the effects of FCF in a manner that depends on the identity of the septin and of the fluorophore (see Table 5.1). Wild-type diploid cells expressing from plasmids different fluorescently tagged septins (Cdc3GFP, Cdc10-mCherry, Cdc10-GFP, Cdc12-YFP, Shs1-GFP) were grown for 18 h in rich medium in the presence or absence of 1 mM FCF. Again, we observed differential morphological responses to FCF (Fig. 1B). These preliminary studies confirm the conclusion from the bud4 studies that, while off-target effects of FCF are a major concern, even slight perturbations of septin assembly/function sensitize cells to FCF. Hence, the “on-target” effects of FCF are significant and are strongly influenced by even subtle changes in individual septins.
2. 1-ETHYL-3-(4-METHOXYPHENYL)-6-METHYLPYRIMIDO [5,4-E][1,2,4]TRIAZINE-5,7-DIONE This compound badly needs a shorter name. We will refer to it hereafter as PCID 906558 (for its PubChem ID number). Until it is available in large quantities from
References
a reliable commercial source, there is not much to be said about the use of PCID 906558, other than what has been already reported. Namely, of all w1100 tested diploid yeast strains harboring a deletion of one copy of an essential gene, the CDC12/cdc12D heterozygote is uniquely sensitive to PCID 906558, with an IC50 of 1 mM (Lee et al., 2014). Upon exposure to 1 mM PCID 906558, HeLa cells displayed defects in Golgi reorientation in an in vitro model of wound healing that were equivalent to effects seen with 5 mM FCF (Lee et al., 2014); such defects are consistent with septin dysfunction (Chacko et al., 2005). Changes in septin appearance upon cellular exposure to PCID 906558 have not yet been reported. PCID 906558 superficially resembles guanosine (Fig. 1A), to which purified human SEPT7 can bind in vitro in the context of a nonnative dimer (Zent & Wittinghofer, 2014). It thus requires no great stretch of the imagination to suppose that PCID 906558 binds directly to septins and induces or inhibits conformational changes that perturb septin assembly or important interactions with nonseptin molecules.
3. SUMMARY AND PERSPECTIVE Improved techniques for chemicogenetic screening have already yielded a compound (PCID 906558) that represents an improvement over FCF in terms of dosage required to exhibit an effect on septin function. However, little is known regarding the off-target effects of PCID 906558 and whether or not it binds directly to one or more septin proteins. Future work will be required to determine both for FCF and PCID 906558 if and how these molecules influence septin folding or postfolding conformations. Toward this goal, it would seem straightforward enough to solve a crystal structure of a septin in the presence of either (or both) of these compounds, and hopefully such experiments will be reported in the near future. Until then, septin researchers are encouraged to use FCF (with caution) and PCID 906558 (if they can get their hands on some) and to undertake screens to discover other useful compounds.
REFERENCES An, H., Morrell, J. L., Jennings, J. L., Link, A. J., & Gould, K. L. (2004). Requirements of fission yeast septins for complex formation, localization, and function. Molecular Biology of the Cell, 15(12), 5551e5564. http://dx.doi.org/10.1091/mbc.E04-07-0640. Angelis, D., Karasmanis, E. P., Bai, X., & Spiliotis, E. T. (2014). In silico docking of forchlorfenuron (FCF) to septins suggests that FCF interferes with GTP binding. PLoS One, 9(5), e96390. http://dx.doi.org/10.1371/journal.pone.0096390. Chacko, A. D., Hyland, P. L., McDade, S. S., Hamilton, P. W., Russell, S. H., & Hall, P. A. (2005). SEPT9_v4 expression induces morphological change, increased motility and disturbed polarity. The Journal of Pathology, 206(4), 458e465. http://dx.doi.org/ 10.1002/path.1794. DeMay, B. S., Bai, X., Howard, L., Occhipinti, P., Meseroll, R. A., Spiliotis, E. T., … Gladfelter, A. S. (2011). Septin filaments exhibit a dynamic, paired organization that is
317
318
CHAPTER 18 Small molecule perturbations of septins
conserved from yeast to mammals. The Journal of Cell Biology, 193(6), 1065e1081. http://dx.doi.org/10.1083/jcb.201012143. DeMay, B. S., Meseroll, R. A., Occhipinti, P., & Gladfelter, A. S. (2010). Cellular requirements for the small molecule forchlorfenuron to stabilize the septin cytoskeleton. Cytoskeleton (Hoboken, NJ), 67(6), 383e399. http://dx.doi.org/10.1002/cm.20452. Farkasovsky, M., Herter, P., Voss, B., & Wittinghofer, A. (2005). Nucleotide binding and filament assembly of recombinant yeast septin complexes. Biological Chemistry, 386(7), 643e656. http://dx.doi.org/10.1515/BC.2005.075. Flescher, E. G., Madden, K., & Snyder, M. (1993). Components required for cytokinesis are important for bud site selection in yeast. The Journal of Cell Biology, 122(2), 373e386. Frazier, J. A., Wong, M. L., Longtine, M. S., Pringle, J. R., Mann, M., Mitchison, T. J., & Field, C. (1998). Polymerization of purified yeast septins: evidence that organized filament arrays may not be required for septin function. The Journal of Cell Biology, 143(3), 737e749. Heasley, L. R., Garcia, G., & McMurray, M. A. (2014). Off-target effects of the septin drug forchlorfenuron on nonplant eukaryotes. Eukaryotic Cell, 13(11), 1411e1420. http:// dx.doi.org/10.1128/EC.00191-14. Hu, Q., Nelson, W. J., & Spiliotis, E. T. (2008). Forchlorfenuron alters mammalian septin assembly, organization, and dynamics. The Journal of Biological Chemistry, 283(43), 29563e29571. http://dx.doi.org/10.1074/jbc.M804962200. Iwase, M., Luo, J., Bi, E., & Toh-e, A. (2007). Shs1 plays separable roles in septin organization and cytokinesis in Saccharomyces cerevisiae. Genetics, 177(1), 215e229. http:// dx.doi.org/10.1534/genetics.107.073007. Iwase, M., Okada, S., Oguchi, T., & Toh-e, A. (2004). Forchlorfenuron, a phenylurea cytokinin, disturbs septin organization in Saccharomyces cerevisiae. Genes & Genetic Systems, 79(4), 199e206. Kopecny´, D., Briozzo, P., Popelkova´, H., Sebela, M., Koncitı´kova´, R., Spı´chal, L., … HoubaHe´rin, N. (2010). Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study. Biochimie, 92(8), 1052e1062. http://dx.doi.org/ 10.1016/j.biochi.2010.05.006. Lee, A. Y., St Onge, R. P., Proctor, M. J., Wallace, I. M., Nile, A. H., Spagnuolo, P. A., … Giaever, G. (2014). Mapping the cellular response to small molecules using chemogenomic fitness signatures. Science (New York, NY), 344(6180), 208e211. http://dx.doi.org/10.1126/ science.1250217. McMurray, M. A., Bertin, A., Garcia, G., 3rd, Lam, L., Nogales, E., & Thorner, J. (2011). Septin filament formation is essential in budding yeast. Developmental Cell, 20(4), 540e549. http://dx.doi.org/10.1016/j.devcel.2011.02.004. Nagaraj, S., Rajendran, A., Jackson, C. E., & Longtine, M. S. (2008). Role of nucleotide binding in septin-septin interactions and septin localization in Saccharomyces cerevisiae. Molecular and Cellular Biology, 28(16), 5120e5137. http://dx.doi.org/10.1128/MCB.00786-08. Sharma, S., Quintana, A., Findlay, G. M., Mettlen, M., Baust, B., Jain, M., … Hogan, P. G. (2013). An siRNA screen for NFAT activation identifies septins as coordinators of storeoperated Ca2þ entry. Nature, 499(7457), 238e242. http://dx.doi.org/10.1038/nature12229. Sirajuddin, M., Farkasovsky, M., Zent, E., & Wittinghofer, A. (2009). GTP-induced conformational changes in septins and implications for function. Proceedings of the National Academy of Sciences of the United States of America, 106(39), 16592e16597. http:// dx.doi.org/10.1073/pnas.0902858106.
References
Tokhtaeva, E., Capri, J., Marcus, E. A., Whitelegge, J. P., Khuzakhmetova, V., Bukharaeva, E., … Vagin, O. (2015). Septin dynamics are essential for exocytosis. The Journal of Biological Chemistry, 290(9), 5280e5297. http://dx.doi.org/10.1074/jbc. M114.616201. Vardi-Oknin, D., Golan, M., & Mabjeesh, N. J. (2013). Forchlorfenuron disrupts SEPT9_i1 filaments and inhibits HIF-1. PLoS One, 8(8), e73179. http://dx.doi.org/10.1371/ journal.pone.0073179. Versele, M., & Thorner, J. (2004). Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4. The Journal of Cell Biology, 164(5), 701e715. http://dx.doi.org/10.1083/jcb.200312070. Wasik, A. A., Polianskyte-Prause, Z., Dong, M.-Q., Shaw, A. S., Yates, J. R., Farquhar, M. G., & Lehtonen, S. (2012). Septin 7 forms a complex with CD2AP and nephrin and regulates glucose transporter trafficking. Molecular Biology of the Cell, 23(17), 3370e3379. http:// dx.doi.org/10.1091/mbc.E11-12-1010. Weems, A. D., Johnson, C. R., Argueso, J. L., & McMurray, M. A. (2014). Higher-order septin assembly is driven by GTP-promoted conformational changes: evidence from unbiased mutational analysis in Saccharomyces cerevisiae. Genetics, 196(3), 711e727. http:// dx.doi.org/10.1534/genetics.114.161182. Yamazaki, T., Owari, S., Ota, S., Sumiya, N., Yamamoto, M., Watanabe, K., … Plant, J. (2013 May). Localization and evolution of septins in algae, 74(4), 605e614. http://dx.doi.org/ 10.1111/tpj.12147. Zent, E., & Wittinghofer, A. (2014). Human septin isoforms and the GDP-GTP cycle. Biological Chemistry, 395(2), 169e180. http://dx.doi.org/10.1515/hsz-2013-0268. Zeraik, A. E., Galkin, V. E., Rinaldi, G., Garratt, R. C., Smout, M. J., Loukas, A., … Brindley, P. J. (2014). Reversible paralysis of Schistosoma mansoni by forchlorfenuron, a phenylurea cytokinin that affects septins. International Journal for Parasitology, 44(8), 523e531. http://dx.doi.org/10.1016/j.ijpara.2014.03.010.
319
Small molecule perturbations of septins
18
L.R. Heasley, M.A. McMurray1 University of Colorado Anschutz Medical Campus, Aurora, CO, United States 1
Corresponding author: E-mail: [email protected]
CHAPTER OUTLINE 1. Forchlorfenuron.................................................................................................. 311 1.1 Background and Summary of Cellular Effects ........................................ 311 1.2 Properties of Forchlorfenuron ............................................................... 314 1.3 Genetic Manipulation of Septin Dosage in Budding Yeast Alters Forchlorfenuron Sensitivity................................................................... 316 2. 1-Ethyl-3-(4-methoxyphenyl)-6-methylpyrimido[5,4-e][1,2,4]triazine-5,7-dione ..... 316 3. Summary and Perspective................................................................................... 317 References ............................................................................................................. 317
Abstract 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-ethyl3-(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.
1. FORCHLORFENURON 1.1 BACKGROUND AND SUMMARY OF CELLULAR EFFECTS The synthetic plant cytokinin forchlorfenuron (FCF) (also called CPPU) increases fruit size in plants via inhibition of the catabolic cytokinin dehydrogenase CKO (Kopecny´ et al., 2010). Notably, higher plants lack septins. As Methods in Cell Biology, Volume 136, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2016.03.013 © 2016 Elsevier Inc. All rights reserved.
311
312
CHAPTER 18 Small molecule perturbations of septins
described later, FCF addition to various different kinds of nonplant eukaryotes that do express septins drives the reversible formation of ectopic septin structures. In budding yeast cells proliferating vegetatively, all tested septins, rather than localizing exclusively to the bud neck, also appear in additional structures localized somewhat randomly along the cell cortex or as long, straight fibers that often appear to “nucleate” from the normal structures at the bud neck (Heasley, Garcia, & McMurray, 2014; Iwase, Okada, Oguchi, & Toh-e, 2004). Similar ectopic structures form upon addition of FCF at 125 mM to growing cells of the filamentous fungus Ashbya gossypii: septins polymerize into long, stable ectopic fibers (DeMay, Meseroll, Occhipinti, & Gladfelter, 2010). Images of these fibers obtained by electron microscopy reveal striking similarities to septin filaments formed in vitro from purified proteins in the absence of FCF (DeMay et al., 2011). Addition of FCF to cultured mammalian cells has varied effects, depending on the cell type (see Table): in human HeLa, HEK-293 human embryonic kidney, and canine MDCK cells, FCF reversibly increases the length and width of cellular septin structures (Hu, Nelson, & Spiliotis, 2008; Tokhtaeva et al., 2015); in PC-3 human prostate cancer cells, on the other hand, FCF causes a shift from long, structured filaments to shorter filaments, and septin exclusion from the nucleus (Vardi-Oknin, Golan, & Mabjeesh, 2013). In yeast and cultured mammalian cells, these effects of FCF on higher-order septin structures are accompanied by evidence of defects in established septin functions: elongation of bud morphology (Heasley et al., 2014; Iwase et al., 2004) and defects in mitosis and cell migration (Hu et al., 2008), respectively. In vitro, addition of 5 mM FCF to purified mammalian septin complexes promotes lateral association of septin filaments (Hu et al., 2008), and at 25 mM, FCF accelerates polymerization and bundling of a purified Schistosoma mansoni septin complex (Zeraik et al., 2014). Moreover, addition of FCF changes the thermal stabilities of individually purified human septins, but not when excess GDP is also added, consistent with direct binding in the septin GTP-binding pocket (Angelis, Karasmanis, Bai, & Spiliotis, 2014). Indeed, in silico modeling demonstrates that FCF fits well within the septin GTP-binding pocket and binds directly to key residues normally involved in GTP hydrolysis (Angelis et al., 2014). Considering the extensive evidence indicating that GTP binding and hydrolysis are critical for directing normal higher-order septin assembly (Farkasovsky, Herter, Voss, & Wittinghofer, 2005; Nagaraj, Rajendran, Jackson, & Longtine, 2008; Sirajuddin, Farkasovsky, Zent, & Wittinghofer, 2009; Versele & Thorner, 2004; Weems, Johnson, Argueso, & McMurray, 2014), direct FCF-septin interactions likely modify interactions between septin subunits or polymers. Taken together with the in vivo effects described above, these findings suggest that FCF is a useful tool for acutely and reversibly perturbing septin function in living cells. However, there are important caveats in the interpretation of cellular effects accompanying the application of FCF. Addition of FCF to yeast medium inhibits wild-type colony growth (on rich solid medium) or culture growth (in liquid rich medium) to an extent that depends somewhat on the “robustness” of septin function. We
Organism/Cell Type
[Forchlorfenuron]
Septin response (ND, not determined)
Cellular Response
References
Nannochloris bacillaris (green algae) Saccharomyces cerevisiae (baker’s/brewer’s yeast)
100 mM
ND
Inhibition of proliferation
Yamazaki et al. (2013)
1.5 mM 1 mM
2 mM
Formation of long septin fibers Formation of puncta
Schistosoma mansoni (blood fluke) RPE1 cells (human retinal) HIRc cells (rat) Podocytes (mouse)
500 mM
Polymerization (in vitro)
Mitochondrial fragmentation, reduction in mitochondrial membrane potential, inhibition of growth. Inhibition of growth, reduction in cell size Inhibition of growth at hyphal tip, gnarled morphology, thick septa Rounding, loss of cilia, cessation of swimming, disorganization of mitochondria Paralysis
Heasley et al. (2014), Iwase et al. (2004)
Schizosaccharomyces pombe (fission yeast) Ashbya gossypii (filamentous fungus) Tetrahymena thermophila (pond scum)
Ectopic polymerization, mislocalization to but tip, cell cortex, bud shaft, cytosol ND
50 mM 50 mM 50 mM
Mitochondrial fragmentation Increased glucose uptake Increase glucose uptake
Heasley et al. (2014) Wasik et al. (2012) Wasik et al. (2012)
HeLa cells
100e200 mM
ND ND Sept7 bundling at cell periphery ND
Sharma et al. (2013)
HeLa cells
5 mM
ND
PC-3 (human cancer cells)
200 mM
HEK-293 (human kidney cells) LNCaP, HCT-116, MCF-7 (human cancer cells)
20e100 mM
Formation of short, disordered filaments, loss of nuclear localization Increase in length of septin filaments ND
Reduction in store-operated Ca2þ uptake, Inhibition of Golgi reorganization during “wound healing” Reduction in proliferation, migration, transformation, HIF-1a expression, HIF-1 transcriptional activity Inhibition of constitutive and stimulated exocytosis and acetycholine secretion Reduction in HIF-1a expression
125 mM
200 mM
Heasley et al. (2014) DeMay et al. (2011), DeMay et al. (2010) Heasley et al. (2014)
Zeraik et al. (2014)
Lee et al. (2014) Vardi-Oknin et al. (2013) Tokhtaeva et al. (2015) Vardi-Oknin et al. (2013)
314
CHAPTER 18 Small molecule perturbations of septins
showed that the W303 strain background, which carries a truncation in the anillinlike protein Bud4 known to contribute to septin dynamics and function, is particularly sensitive to FCF inhibition and introducing a bud4D mutation sensitized the otherwise “robust” BY4743 background (Heasley et al., 2014). Moreover, in BUD4þ yeast cells, colony and culture growth are noticeably slower in the presence of 1 mM and 0.5 mM FCF, respectively (we suspect that the differences between liquid and solid media reflect differences in FCF solubility; see below), and at these concentrations there is little effect on ectopic septin polymerization (Heasley et al., 2014). Instead, mitochondria rapidly and reversibly fragment (Heasley et al., 2014), indicative of an overall change in metabolism and/or a systemic stress response. We described similar mitochondrial responses in Tetrahymena thermophila and human retinal pigmented epithelial cells, which did not correspond to known effects of septin dysfunction (Heasley et al., 2014). In Schizosaccharomyces pombe, we found that, at relatively low concentrations (0.1 mM), FCF inhibits culture growth and perturbs cellular morphology (Heasley et al., 2014), whereas even null septin mutations do not share these phenotypes (An, Morrell, Jennings, Link, & Gould, 2004). In A. gossypii, as well, multiple cellular phenotypes that accompanied FCF treatment were consistent with a stress response, rather than septin dysfunction (DeMay et al., 2010) (see Table). These demonstrations of nonseptin (ie, off-target) cellular effects by no means discount the ability of FCF to modulate the septin cytoskeleton, but they do suggest caution in the use of FCF in vivo. Among other processes and cell types (see Table for a summary), published studies in mammalian cells have interpreted cellular effects of FCF to indicate septin involvement in exocytosis (Tokhtaeva et al., 2015), hypoxic response (Vardi-Oknin et al., 2013), calcium signaling (Sharma et al., 2013), and glucose uptake (Wasik et al., 2012). Septin depletion via RNAi was in most cases also used to corroborate these roles; some sort of independent genetic perturbation of septins is highly recommended as a prerequisite to arriving at a firm conclusion.
1.2 PROPERTIES OF FORCHLORFENURON FCF is a synthetic compound derived from urea and containing a phenol ring and chlorinated pyridine (Fig. 1A). We have purchased FCF from two sources, SigmaAldrich (#C2791) and Santa Cruz Biotechnology (#KT-30). These powders differ noticeably in their appearance, but their cellular effects are indistinguishable, in our hands. FCF is scarcely soluble in water and far more soluble in ethanol. The poor solubility of FCF becomes problematic in fungal studies, where tough cell walls (and, presumably, potent efflux pumps) necessitate much higher concentrations of FCF compared to other organisms. Two millimolar is the highest concentration we have been able to reliably achieve in solution; the concentration needed to induce ectopic polymerization of septins in a sensitized background is 1 mM. We typically prepare stock solutions of FCF at 250 mM in 95% ethanol, with rigorous vortexing. This means that, as an appropriate “solvent” control, when using high
1. Forchlorfenuron
FIGURE 1 Structures of small molecules that perturb septin function, and alterations in such perturbations upon subtle manipulations of yeast septins. (A) Chemical structures of forchlorfenuron (FCF), 1-ethyl-3-(4-methoxyphenyl)-6-methylpyrimido[5,4-e][1,2,4] triazine-5,7-dione (PCID 906558), and, for comparison, guanosine. (B) Yeast cells of the BY4743 background and the indicated genotypes were exposed to 1 mM FCF in rich liquid medium for 18 h, spotted on an agarose pad, and imaged by transmitted light microscopy with a 60X objective. Brackets indicate extrachromosomal low-copy plasmids carried by wild-type haploid cells.
315
316
CHAPTER 18 Small molecule perturbations of septins
FCF concentrations, one should probably also expose cells to the appropriate concentration of ethanol alone (eg, 0.38% for 1 mM FCF). FCF cannot be “top-spread” on solid media plates; it must be dissolved into molten agar, which still does not guarantee prolonged solubility. We have found that at concentrations 1 mM, FCF eventually crystalizes on top of and within the solid media, which seems to decrease the local effective concentration of the compound. Stock solutions of FCF appear quite stable during long-term storage in ethanol, in terms of activity (if not solubility).
1.3 GENETIC MANIPULATION OF SEPTIN DOSAGE IN BUDDING YEAST ALTERS FORCHLORFENURON SENSITIVITY Here we report a few unpublished observations that may be of interest to the field regarding the use of FCF. Our finding that deletion of BUD4-sensitized budding yeast cells to FCF prompted us to ask if other perturbations of the septin system could sensitize cells. First, we hypothesized that, if FCF binds preferentially to a single septin subunit, eliminating or reducing the amount of that subunit might confer FCF resistance. Yeast cells tolerate deletion of CDC10, CDC11, SHS1, or both CDC11 and SHS1 (Flescher, Madden, & Snyder, 1993; Frazier et al., 1998; Iwase, Luo, Bi, & Toh-e, 2007; McMurray et al., 2011). Loss of one copy of any one of the five mitotic septins is also well tolerated in diploids. We compared the morphology of BY4743-background (BUD4þ) haploid homozygous mutants (cdc10D and shs1D) and diploid heterozygous mutants (CDC3/cdc3D, CDC10/cdc10D, CDC11/cdc11D, CDC12/cdc12D, SHS1/shs1D) grown for 18 h in rich medium in the presence or absence of 1 mM FCF. While the morphology of wild-type diploids was generally unaffected, all of the septin mutants exhibited variable morphological defects (Fig. 1B). These findings suggest that the septin response to FCF is highly contingent on the stoichiometry of certain septin proteins. In separate experiments, we also noticed that tagging a single septin with a C-terminal fluorophore fusion sensitizes cells to the effects of FCF in a manner that depends on the identity of the septin and of the fluorophore (see Table 5.1). Wild-type diploid cells expressing from plasmids different fluorescently tagged septins (Cdc3GFP, Cdc10-mCherry, Cdc10-GFP, Cdc12-YFP, Shs1-GFP) were grown for 18 h in rich medium in the presence or absence of 1 mM FCF. Again, we observed differential morphological responses to FCF (Fig. 1B). These preliminary studies confirm the conclusion from the bud4 studies that, while off-target effects of FCF are a major concern, even slight perturbations of septin assembly/function sensitize cells to FCF. Hence, the “on-target” effects of FCF are significant and are strongly influenced by even subtle changes in individual septins.
2. 1-ETHYL-3-(4-METHOXYPHENYL)-6-METHYLPYRIMIDO [5,4-E][1,2,4]TRIAZINE-5,7-DIONE This compound badly needs a shorter name. We will refer to it hereafter as PCID 906558 (for its PubChem ID number). Until it is available in large quantities from
References
a reliable commercial source, there is not much to be said about the use of PCID 906558, other than what has been already reported. Namely, of all w1100 tested diploid yeast strains harboring a deletion of one copy of an essential gene, the CDC12/cdc12D heterozygote is uniquely sensitive to PCID 906558, with an IC50 of 1 mM (Lee et al., 2014). Upon exposure to 1 mM PCID 906558, HeLa cells displayed defects in Golgi reorientation in an in vitro model of wound healing that were equivalent to effects seen with 5 mM FCF (Lee et al., 2014); such defects are consistent with septin dysfunction (Chacko et al., 2005). Changes in septin appearance upon cellular exposure to PCID 906558 have not yet been reported. PCID 906558 superficially resembles guanosine (Fig. 1A), to which purified human SEPT7 can bind in vitro in the context of a nonnative dimer (Zent & Wittinghofer, 2014). It thus requires no great stretch of the imagination to suppose that PCID 906558 binds directly to septins and induces or inhibits conformational changes that perturb septin assembly or important interactions with nonseptin molecules.
3. SUMMARY AND PERSPECTIVE Improved techniques for chemicogenetic screening have already yielded a compound (PCID 906558) that represents an improvement over FCF in terms of dosage required to exhibit an effect on septin function. However, little is known regarding the off-target effects of PCID 906558 and whether or not it binds directly to one or more septin proteins. Future work will be required to determine both for FCF and PCID 906558 if and how these molecules influence septin folding or postfolding conformations. Toward this goal, it would seem straightforward enough to solve a crystal structure of a septin in the presence of either (or both) of these compounds, and hopefully such experiments will be reported in the near future. Until then, septin researchers are encouraged to use FCF (with caution) and PCID 906558 (if they can get their hands on some) and to undertake screens to discover other useful compounds.
REFERENCES An, H., Morrell, J. L., Jennings, J. L., Link, A. J., & Gould, K. L. (2004). Requirements of fission yeast septins for complex formation, localization, and function. Molecular Biology of the Cell, 15(12), 5551e5564. http://dx.doi.org/10.1091/mbc.E04-07-0640. Angelis, D., Karasmanis, E. P., Bai, X., & Spiliotis, E. T. (2014). In silico docking of forchlorfenuron (FCF) to septins suggests that FCF interferes with GTP binding. PLoS One, 9(5), e96390. http://dx.doi.org/10.1371/journal.pone.0096390. Chacko, A. D., Hyland, P. L., McDade, S. S., Hamilton, P. W., Russell, S. H., & Hall, P. A. (2005). SEPT9_v4 expression induces morphological change, increased motility and disturbed polarity. The Journal of Pathology, 206(4), 458e465. http://dx.doi.org/ 10.1002/path.1794. DeMay, B. S., Bai, X., Howard, L., Occhipinti, P., Meseroll, R. A., Spiliotis, E. T., … Gladfelter, A. S. (2011). Septin filaments exhibit a dynamic, paired organization that is
317
318
CHAPTER 18 Small molecule perturbations of septins
conserved from yeast to mammals. The Journal of Cell Biology, 193(6), 1065e1081. http://dx.doi.org/10.1083/jcb.201012143. DeMay, B. S., Meseroll, R. A., Occhipinti, P., & Gladfelter, A. S. (2010). Cellular requirements for the small molecule forchlorfenuron to stabilize the septin cytoskeleton. Cytoskeleton (Hoboken, NJ), 67(6), 383e399. http://dx.doi.org/10.1002/cm.20452. Farkasovsky, M., Herter, P., Voss, B., & Wittinghofer, A. (2005). Nucleotide binding and filament assembly of recombinant yeast septin complexes. Biological Chemistry, 386(7), 643e656. http://dx.doi.org/10.1515/BC.2005.075. Flescher, E. G., Madden, K., & Snyder, M. (1993). Components required for cytokinesis are important for bud site selection in yeast. The Journal of Cell Biology, 122(2), 373e386. Frazier, J. A., Wong, M. L., Longtine, M. S., Pringle, J. R., Mann, M., Mitchison, T. J., & Field, C. (1998). Polymerization of purified yeast septins: evidence that organized filament arrays may not be required for septin function. The Journal of Cell Biology, 143(3), 737e749. Heasley, L. R., Garcia, G., & McMurray, M. A. (2014). Off-target effects of the septin drug forchlorfenuron on nonplant eukaryotes. Eukaryotic Cell, 13(11), 1411e1420. http:// dx.doi.org/10.1128/EC.00191-14. Hu, Q., Nelson, W. J., & Spiliotis, E. T. (2008). Forchlorfenuron alters mammalian septin assembly, organization, and dynamics. The Journal of Biological Chemistry, 283(43), 29563e29571. http://dx.doi.org/10.1074/jbc.M804962200. Iwase, M., Luo, J., Bi, E., & Toh-e, A. (2007). Shs1 plays separable roles in septin organization and cytokinesis in Saccharomyces cerevisiae. Genetics, 177(1), 215e229. http:// dx.doi.org/10.1534/genetics.107.073007. Iwase, M., Okada, S., Oguchi, T., & Toh-e, A. (2004). Forchlorfenuron, a phenylurea cytokinin, disturbs septin organization in Saccharomyces cerevisiae. Genes & Genetic Systems, 79(4), 199e206. Kopecny´, D., Briozzo, P., Popelkova´, H., Sebela, M., Koncitı´kova´, R., Spı´chal, L., … HoubaHe´rin, N. (2010). Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study. Biochimie, 92(8), 1052e1062. http://dx.doi.org/ 10.1016/j.biochi.2010.05.006. Lee, A. Y., St Onge, R. P., Proctor, M. J., Wallace, I. M., Nile, A. H., Spagnuolo, P. A., … Giaever, G. (2014). Mapping the cellular response to small molecules using chemogenomic fitness signatures. Science (New York, NY), 344(6180), 208e211. http://dx.doi.org/10.1126/ science.1250217. McMurray, M. A., Bertin, A., Garcia, G., 3rd, Lam, L., Nogales, E., & Thorner, J. (2011). Septin filament formation is essential in budding yeast. Developmental Cell, 20(4), 540e549. http://dx.doi.org/10.1016/j.devcel.2011.02.004. Nagaraj, S., Rajendran, A., Jackson, C. E., & Longtine, M. S. (2008). Role of nucleotide binding in septin-septin interactions and septin localization in Saccharomyces cerevisiae. Molecular and Cellular Biology, 28(16), 5120e5137. http://dx.doi.org/10.1128/MCB.00786-08. Sharma, S., Quintana, A., Findlay, G. M., Mettlen, M., Baust, B., Jain, M., … Hogan, P. G. (2013). An siRNA screen for NFAT activation identifies septins as coordinators of storeoperated Ca2þ entry. Nature, 499(7457), 238e242. http://dx.doi.org/10.1038/nature12229. Sirajuddin, M., Farkasovsky, M., Zent, E., & Wittinghofer, A. (2009). GTP-induced conformational changes in septins and implications for function. Proceedings of the National Academy of Sciences of the United States of America, 106(39), 16592e16597. http:// dx.doi.org/10.1073/pnas.0902858106.
References
Tokhtaeva, E., Capri, J., Marcus, E. A., Whitelegge, J. P., Khuzakhmetova, V., Bukharaeva, E., … Vagin, O. (2015). Septin dynamics are essential for exocytosis. The Journal of Biological Chemistry, 290(9), 5280e5297. http://dx.doi.org/10.1074/jbc. M114.616201. Vardi-Oknin, D., Golan, M., & Mabjeesh, N. J. (2013). Forchlorfenuron disrupts SEPT9_i1 filaments and inhibits HIF-1. PLoS One, 8(8), e73179. http://dx.doi.org/10.1371/ journal.pone.0073179. Versele, M., & Thorner, J. (2004). Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4. The Journal of Cell Biology, 164(5), 701e715. http://dx.doi.org/10.1083/jcb.200312070. Wasik, A. A., Polianskyte-Prause, Z., Dong, M.-Q., Shaw, A. S., Yates, J. R., Farquhar, M. G., & Lehtonen, S. (2012). Septin 7 forms a complex with CD2AP and nephrin and regulates glucose transporter trafficking. Molecular Biology of the Cell, 23(17), 3370e3379. http:// dx.doi.org/10.1091/mbc.E11-12-1010. Weems, A. D., Johnson, C. R., Argueso, J. L., & McMurray, M. A. (2014). Higher-order septin assembly is driven by GTP-promoted conformational changes: evidence from unbiased mutational analysis in Saccharomyces cerevisiae. Genetics, 196(3), 711e727. http:// dx.doi.org/10.1534/genetics.114.161182. Yamazaki, T., Owari, S., Ota, S., Sumiya, N., Yamamoto, M., Watanabe, K., … Plant, J. (2013 May). Localization and evolution of septins in algae, 74(4), 605e614. http://dx.doi.org/ 10.1111/tpj.12147. Zent, E., & Wittinghofer, A. (2014). Human septin isoforms and the GDP-GTP cycle. Biological Chemistry, 395(2), 169e180. http://dx.doi.org/10.1515/hsz-2013-0268. Zeraik, A. E., Galkin, V. E., Rinaldi, G., Garratt, R. C., Smout, M. J., Loukas, A., … Brindley, P. J. (2014). Reversible paralysis of Schistosoma mansoni by forchlorfenuron, a phenylurea cytokinin that affects septins. International Journal for Parasitology, 44(8), 523e531. http://dx.doi.org/10.1016/j.ijpara.2014.03.010.
319