Author response: The wtf4 meiotic driver utilizes controlled protein aggregation to generate selective cell death Article Swipe

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Meiotic drivers are parasitic loci that force their own transmission into greater than half of the offspring of a heterozygote. Many drivers have been identified, but their molecular mechanisms are largely unknown. The wtf4 gene is a meiotic driver in Schizosaccharomyces pombe that uses a poison-antidote mechanism to selectively kill meiotic products (spores) that do not inherit wtf4. Here, we show that the Wtf4 proteins can function outside of gametogenesis and in a distantly related species, Saccharomyces cerevisiae. The Wtf4poison protein forms dispersed, toxic aggregates. The Wtf4antidote can co-assemble with the Wtf4poison and promote its trafficking to vacuoles. We show that neutralization of the Wtf4poison requires both co-assembly with the Wtf4antidote and aggregate trafficking, as mutations that disrupt either of these processes result in cell death in the presence of the Wtf4 proteins. This work reveals that wtf parasites can exploit protein aggregate management pathways to selectively destroy spores. eLife digest Meiotic drivers are genes that break the normal rules of inheritance. Usually, a gene has a 50% chance of passing on to the next generation. Meiotic drivers force their way into the next generation by poisoning the gametes (the sex cells that combine to form a zygote) that do not carry them. Harnessing the power of genetic drivers could allow scientists to spread beneficial genes across populations. One group of meiotic drivers found in fission yeast is called the 'with transposon fission yeast', or 'wtf' gene family. The wtf drivers act during the production of spores, which are the fission yeast equivalent of sperm, and they encode both a poison that can destroy the spores and its antidote. The poison spreads through the sac holding the spores, and can affect all of them, while the antidote only protects the spores that make it. This means that the spores carrying the wtf genes survive, while the rest of the spores are killed. To understand whether it is possible to use the wtf meiotic drivers to spread other genes, perhaps outside of fission yeast, scientists must first establish exactly how the proteins coded for by genes behave. To do this, Nuckolls et al. examined a member of the wtf family called wtf4. Attaching a fluorescent label to the poison and antidote proteins produced by wtf4 made it possible to see what they do. This revealed that the poison clumps, forming toxic aggregates that damage yeast spores. The antidote works by mopping up these aggregates and moving them to the cell's main storage compartment, called the vacuole. Mutations that disrupted the ability of the antidote to interact with the poison or its ability to move the poison into storage stopped the antidote from working. Nuckolls et al. also showed that if genetic engineering was used to introduce wtf4 into a distantly related species of budding yeast the effects of this meiotic driver were the same. This suggests that the wtf genes may be good candidates for future genetic engineering experiments. Engineered systems known as 'gene drives' could spread beneficial genetic traits through populations. This could include disease-resistance genes in crops, or disease-preventing genes in mosquitoes. The wtf genes are small and work independently of other genes, making them promising candidates for this type of system. These experiments also suggest that the wtf genes could be useful for understanding why clumps of proteins are toxic to cells. Future work could explore why clumps of wtf poison kill spores, while clumps of poison plus antidote do not. This could aid research into human ailments caused by protein clumps, such as Huntington’s or Alzheimer’s disease. Introduction Meiotic drivers are selfish DNA sequences that break the traditional rules of sexual reproduction. Whereas most alleles have a 50% chance of being transmitted into a given offspring, meiotic drivers can manipulate gametogenesis to bias their own transmission into most or even all of an individual’s offspring (Burt and Trivers, 2006; Lindholm et al., 2016). This makes meiotic drive a powerful evolutionary force (Sandler and Novitski, 1957). Meiotic drivers are widespread in eukaryotes and the evolutionary pressures they exert are thought to shape major facets of gametogenesis, including recombination landscapes and chromosome structure (Bravo Núñez et al., 2020b; Bravo Núñez et al., 2020a; Crow, 1991; Dyer et al., 2007; Larracuente and Presgraves, 2012; Schimenti, 2000; Pardo-Manuel de Villena and Sapienza, 2001; Hammer et al., 1989; Zanders et al., 2014; Grey et al., 2018). Harnessing and wielding the evolutionary power of meiotic drive has the potential to greatly benefit humanity. Engineered drive systems, known as ‘gene drives,’ are being developed to spread genetic traits in populations (Lindholm et al., 2016; Burt, 2014; Gantz et al., 2015; Esvelt et al., 2014; Burt and Crisanti, 2018). For example, gene drives could be used to spread disease-resistance alleles in crops. Alternatively, gene drives can be used to suppress human disease vectors, such as mosquitoes, or to limit their ability to transmit diseases (Burt, 2014; Burt and Crisanti, 2018; Esvelt et al., 2014; Gantz et al., 2015; Lindholm et al., 2016). While there are many challenges involved in designing effective gene drives, natural meiotic drivers could serve as useful models or components for these systems (Burt, 2014; Lindholm et al., 2016). However, the molecular mechanisms employed by most meiotic drivers are unknown. The recently characterized wtf gene family of Schizosaccharomyces pombe includes several meiotic drivers (Bravo Núñez et al., 2018a; Eickbush et al., 2019; Hu et al., 2017; López Hernández and Zanders, 2018; Nuckolls et al., 2017). The wtf coding sequences are small (~1 kb) and encode autonomous drivers that specifically kill meiotic products (spores) that do not inherit the wtf + allele from wtf+/wtf - heterozygotes. These drivers carry out targeted spore destruction using two proteins: a poison (Wtfpoison) to which all spores are exposed, and an antidote (Wtfantidote) which rescues only the spores that inherit the wtf + allele (Figure 1A and B). The two proteins of a given driver are encoded on largely overlapping coding sequences, but the antidote contains ~45 additional N-terminal amino acids (Figure 1A). The small size and autonomy of the wtf drivers make them promising candidates for use in gene drive systems. It is important, however, to first understand more about the molecular mechanisms of the Wtf proteins and whether they are likely to be functional in other species. Figure 1 with 4 supplements see all Download asset Open asset Wtf4poison and Wtf4antidote protein localization in both S. pombe meiosis and vegetative growth. (A) The wtf4 gene utilizes alternate transcriptional start sites to encode for the Wtf4antidote and Wtf4poison proteins. (B) Model of a tetrad generated from a wtf4+/wtf4- diploid. wtf4+ spores are rescued by the spore-enriched antidote (magenta circles), while the poison (cyan skulls) spreads throughout the ascus. (C) An ascus generated by an mCherry-wtf4/wtf4poison-GFP diploid showing the localization of mCherry-Wtf4antidote (magenta in merged images) and Wtf4poison-GFP (cyan in merged images) (Nuckolls et al., 2017). (D) An ascus generated from a wtf4-GFP/ade6+, +/cpy1 mCherry diploid showing localization of Wtf4-GFP (green in merged images) and Cpy1-mCherry (red in merged images). The arrow highlights the spore that inherited both tagged alleles and thus contains both tagged proteins. (E) A vegetatively growing haploid cell expressing Wtf4poison-GFP (cyan in merged images) and Wtf4antidote-mCherry (magenta in merged images) using the β-estradiol-inducible system. CMAC is a vacuole lumen stain (yellow in merged images). Both Wtf4 proteins colocalize with the vacuole. Cells were imaged 4 hr after induction with 100 nM β-estradiol. (F) A vegetatively growing haploid cell expressing Wtf4antidote-mCherry (magenta in the merged images) using the β-estradiol system and stained with the CMAC vacuole stain (yellow in the merged images) shows Wtf4antidote-mCherry in the vacuole. Cells were induced in the same way as in (E). (G) Asci generated from a wtf4-GFP/ade6+, hht1-RFP/+ diploid. Hht1-RFP (red in merged images) is a histone marker. The nuclei in the spores that do not inherit wtf4-GFP (e.g. lacking GFP signal) can exhibit nuclear condensation and fragmentation. All scale bars represent 2 µm. TL = transmitted light. Here, we investigate the mechanisms of wtf drive using the wtf4 allele as a model. We demonstrate that the Wtf4 proteins are functional outside of gametogenesis and in the budding yeast Saccharomyces cerevisiae, despite over 350 million years since the two yeasts shared a common ancestor (Hoffman et al., 2015). We also show that the two Wtf4 proteins assemble into distinct aggregated forms. Wtf4poison forms toxic aggregates that are dispersed throughout the cytoplasm. The Wtf4antidote forms aggregates that are recruited to the vacuole and vacuole-associated inclusions and are largely non-toxic. When the two Wtf4 proteins are expressed together, the Wtf4antidote and Wtf4poison co-assemble and are trafficked to the vacuole. This work adds to our understanding of how wtf meiotic drivers work. In addition, the conserved function of Wtf4poison’s toxicity and the fact that the Wtf4antidote exploits conserved aggregate management processes suggests that wtf genes represent good candidates for gene drive systems. Results Wtf4 proteins localize to the vacuole and endoplasmic reticulum within S. pombe spores The wtf4 meiotic driver used in this work is from S. kambucha, an isolate that is almost identical (99.5% DNA sequence identity) to the commonly studied lab isolate of S. pombe (Rhind et al., 2011; Singh and Klar, 2002). Our previous work demonstrated that the Wtf4antidote localizes to a region within the spores that inherit the wtf4 gene. The Wtf4poison protein, however, is found in all four spores and throughout the sac (ascus) that holds them (Nuckolls et al., 2017). Here, we explored the localization of these proteins in greater depth to gain insight into their mechanisms. We used fluorescently tagged alleles of wtf4 to visualize the proteins. The two Wtf4 proteins have different translational start sites and thus different N-termini (Figure 1A, Figure 1—figure supplement 1A). We took advantage of this feature to visualize the proteins separately. For the Wtf4antidote, we used an allele with an mCherry tag immediately upstream of the first start codon. This mCherry-wtf4 allele tags only the Wtf4antidote (mCherry-Wtf4antidote) but still encodes an untagged Wtf4poison. We previously demonstrated that this allele is fully functional (Nuckolls et al., 2017). To visualize Wtf4poison, we used the wtf4poison-GFP allele. This separation-of-function allele encodes only a C-terminally tagged poison but no Wtf4antidote protein. We previously demonstrated that this tagged allele is functional but has a slightly weaker phenotype than an untagged wtf4poison separation-of-function allele (Nuckolls et al., 2017). We integrated the tagged alleles at the ade6 locus in separate haploid S. pombe strains. We then crossed those two haploid strains to create heterozygous mCherry-wtf4/wtf4poison-GFP diploids and induced these diploids to undergo meiosis. We imaged the asci using both standard and time-lapse fluorescence microscopy (Figure 1C, Figure 1—figure supplement 1B). We confirmed our previous observations that the mCherry-Wtf4antidote was enriched in two spores, whereas Wtf4poison-GFP was found throughout the ascus and often formed puncta of various sizes. In the spores that did not inherit the antidote, Wtf4poison-GFP also appeared dispersed throughout the spores. In the spores that inherited and thus expressed mCherry-wtf4, however, the localization of Wtf4poison-GFP was more restricted. Specifically, we observed that the Wtf4poison-GFP largely colocalized with mCherry-Wtf4antidote in a limited region of the spore (Figure 1C, Figure 1—figure supplement 1B). In time-lapse microscopy, it was evident that the two Wtf4 proteins consistently colocalized in a defined region of the spore, even as this region changed shape over time. This co-diffusion suggests the two proteins are either physically interacting or are present in the same compartment (Figure 1—figure supplement 1B). It also appeared that the level of Wtf4poison-GFP protein is reduced in spores containing the antidote. We did not distinguish if this was due to technical reasons (i.e. quenching of the GFP molecules) or biological reasons such as degradation of Wtf4poison-GFP in spores with mCherry-Wtf4antidote and/or due to a higher expression of Wtf4poison-GFP in the spores that inherit it (non-antidote spores) (Figure 1C). We completed Pearson correlation analysis (Adler and Parmryd, 2010) of mCherry-Wtf4antidote and Wtf4poison-GFP in the spores (where a result of >0 is positive correlation; 0, no correlation; <0 anti correlation) and obtained a coefficient of 0.61, indicating strong colocalization between the two Wtf4 proteins (Figure 1—figure supplement 1C). The limited distribution of the Wtf4 poison and antidote proteins within wtf4+ spores suggested they may be confined to a specific cellular compartment. To test this idea, we looked for colocalization of Wtf4 proteins with the vacuole, endoplasmic reticulum (ER) and nucleus (see below). For these experiments, we used the fully functional wtf4-GFP allele, which tags both the poison and antidote proteins (Nuckolls et al., 2017). To assay the localization of the Wtf4 proteins relative to the vacuole, we imaged asci produced by diploids that were heterozygous for both wtf4-GFP and cpy1-mCherry. Cpy1-mCherry localizes to the lumen of the vacuole in vegetative cells (Sun et al., 2013) but has not, to our knowledge, been imaged in spores. We could observe mCherry in two of the four spores – presumably the two that inherited the cpy1-mCherry allele (Figure 1D). This 2:2 spore localization pattern has been previously observed in budding yeast for vacuolar proteins and several other organelles (Neiman, 2011; Roeder and Shaw, 1996; Suda et al., 2007). We found that the Wtf4-GFP and Cpy1-mCherry proteins colocalized within the spores that inherited both tagged alleles, suggesting the Wtf4 proteins are found within the vacuole (Pearson coefficient of 0.89, Figure 1D, Figure 1—figure supplement 2A–B). Interestingly, we also saw colocalization of Wtf4-GFP proteins with an ER marker, pbip1-mCherry-AHDL (Zhang et al., 2012; Figure 1—figure supplement 2C–D). We speculate this colocalization with the ER is due to nitrogen starvation which is required to induce meiosis and promotes organelle autophagy in S. pombe (Kohda et al., 2007; Zhao et al., 2016). Wtf4antidote localizes to the vacuole when its expression is induced in vegetatively growing S. pombe cells Because we could not distinguish the vacuole and ER within spores, we assayed the localization of the Wtf4 proteins in the absence of nitrogen starvation. To do this, we fluorescently tagged the coding sequence of wtf4poison (wtf4poison-GFP) and wtf4antidote (wtf4antidote-mCherry) separation-of-function alleles under the control of β-estradiol-inducible promoters (Ohira et al., 2017). We then integrated the wtf4poison-GFP allele at the ura4 locus and the wtf4antidote-mCherry allele at the lys4 locus of the same haploid strain. Next, we observed the localization of the Wtf proteins relative to the vacuole (visualized using the CellTracker Blue CMAC lumen stain) or the ER (using Sec63-YFP) following β-estradiol induction. Similar to our observations in spores, we saw that the Wtf4poison-GFP and Wtf4antidote-mCherry proteins largely colocalized, with a Pearson coefficient of 0.68 (Figure 1—figure supplement 3D and E). We also found that the Wtf4 proteins colocalized with the CMAC stain (Figure 1E), which suggests that the Wtf4 poison and antidote proteins are largely within the vacuole. However, there were Wtf4poison-GFP puncta that lined the periphery of the cell and a circle in the middle of the cell, reminiscent of ER localization. These puncta were devoid of Wtf4antidote-mCherry (Figure 1—figure supplement 3D). We also attempted to assay the localization of the Wtf4 antidote and poison proteins individually to test if the localization of the Wtf4poison was altered in the presence of the Wtf4antidote, as we observed in spores (Figure 1C). We found that the localization of the Wtf4antidote-mCherry to the vacuole was similar in the absence of the Wtf4poison (Figure 1F, Figure 1—figure supplement 3B), with a Pearson coefficient of 0.69 (Figure 1—figure supplement 3C). This is analogous to previous observations of the localization of the slightly different Wtf4antidote protein (82.2% amino acid identity) found in the S. pombe lab strain (Matsuyama et al., 2006). We failed, however, to generate cells carrying the wtf4poison-GFP allele without the wtf4antidote-mCherry allele by transformation, or by crossing the strain carrying both wtf4poison-GFP and wtf4antidote-mCherry to a wild-type strain (Figure 1—figure supplement 3A). This is likely due to leaky expression of the wtf4poison-GFP from the inducible promoter even without addition of β-estradiol. Overall, our results suggest that the Wtf4poison protein is toxic in vegetative cells, but the antidote is still capable of neutralizing the poison, as we could obtain cells carrying both the Wtf4 poison and antidote proteins. S. pombe spores destroyed by wtf4 display nuclear condensation followed by nuclear fragmentation In the process of trying to understand the localization patterns of Wtf4 proteins, we assayed the localization of the Wtf4 proteins relative to the nucleus. For this experiment, we imaged asci produced by wtf4-GFP/ade6+ heterozygotes also carrying a tagged histone allele, hht1-RFP (Tomita and Cooper, 2007). Although we did not observe colocalization of Wtf4 proteins and the nucleus, we frequently (24/38 asci) observed that the nuclei in the wtf4- spores appeared more condensed (Figure 1G, younger ascus). Additionally, in 11 out of 38 asci, one or both of the nuclei in the wtf4- spores were disrupted and the nuclear contents were dispersed throughout the spores (Figure 1G, older ascus). To address the timing of these nuclear phenotypes, we imaged diploids undergoing gametogenesis using time-lapse microscopy. We saw that all four nuclei tended to look similar shortly after the second meiotic division. As spores matured, however, we observed nuclear condensation sometimes followed by fragmentation in the spores that did not inherit wtf4 (i.e. in spores lacking the enriched GFP expression and antidote function) (Figure 1—figure supplement 4A and B, see Materials and methods). This nuclear condensation and fragmentation are reminiscent of apoptotic cell death (Carmona-Gutierrez et al., 2010; Kerr et al., 1972). Wtf4 proteins function in the budding yeast, Saccharomyces cerevisiae Our experiments in S. pombe suggest that the Wtf4 proteins can act when expressed outside of gametogenesis. However, our inability to induce expression of the Wtf4poison in the absence of the Wtf4antidote limited our ability to explore their mechanisms of action in this system. We, therefore, tested if the Wtf4 proteins functioned in the budding yeast Saccharomyces cerevisiae. To do this, we cloned the coding sequences of wtf4poison-GFP and wtf4antidote-mCherry under the control of β-estradiol inducible promoters on separate plasmids (Ottoz et al., 2014). We then introduced these plasmids into S. cerevisiae individually and together. We found that cells carrying the wtf4poison-GFP plasmid were largely inviable when wtf4poison-GFP expression was induced, indicating the poison is also toxic to S. cerevisiae (Figure 2A). However, cells expressing Wtf4antidote-mCherry had only a slight growth defect relative to control cells carrying empty plasmids (Figure 2A). Importantly, expression of the Wtf4antidote-mCherry plasmid largely ameliorated the toxicity of Wtf4poison-GFP (Figure 2A). Given that S. pombe and S. cerevisiae diverged >350 million years ago (Hoffman et al., 2015), our results suggest that the target(s) of Wtf4poison toxicity are conserved and the Wtf4antidote does not require cofactors that are specific to S. pombe or gametogenesis to neutralize Wtf4poison’s toxicity. Figure 2 with 5 supplements see all Download asset Open asset Wtf4poison and Wtf4antidote proteins physically interact and are functional in vegetative S. cerevisiae cells. (A) Spot assay of serial dilutions on non-inducing (SC -His -Trp -Ura) and inducing (SC -His -Trp -Ura + 500 nM β-estradiol) media. Each strain contains [TRP1] and [URA3] ARS CEN plasmids that are either empty (EV) or carry the indicated β-estradiol-inducible wtf4 alleles. (B) A cell carrying an empty [TRP1] vector and a [URA3] vector with a β-estradiol inducible wtf4poison-GFP allele (cyan). (C) A haploid cell carrying an empty [URA3] vector and a [TRP1] plasmid with a β-estradiol inducible wtf4antidote-mCherry allele (magenta). (D) A haploid cell carrying a [URA3] plasmid with a β-estradiol inducible wtf4poison-GFP allele (cyan in merged images) and a [TRP1] plasmid with a β-estradiol inducible wtf4antidote-mCherry allele (magenta in merged images). The vacuole is marked with ‘v’. (E) Cartoon of acceptor photobleaching Fluorescence Resonance Energy Transfer (FRET). If the two proteins interact, Wtf4poison-GFP (the donor) transfers energy to Wtf4antidote-mCherry (the acceptor). After photobleaching of the acceptor, the donor emission will increase. (F) Quantification of FRET values measured in cells carrying β-estradiol inducible Wtf4antidote-mCherry and β-estradiol inducible Wtf4poison-GFP. The cells showed an average of 40% FRET. These data are also presented in Figure 5C. In all experiments, the cells were imaged approximately 4 hr after induction in 500 nM β-estradiol. All scale bars represent 4 µm. TL = transmitted light. Wtf4 poison and antidote proteins assemble into aggregates individually and in budding yeast We assayed the localization of the Wtf4 proteins in S. cerevisiae using the inducible wtf4poison-GFP and wtf4antidote-mCherry alleles Similar to our observations in S. pombe gametogenesis, we saw that Wtf4poison-GFP as puncta of throughout the (Figure We also observed of the Wtf4poison-GFP protein to the ER (Figure supplement et al., to our in S. pombe spores, we saw nuclear condensation in cells expressing Wtf4poison-GFP relative to wild-type cells (Figure supplement on the other to one or two to the vacuole (Figure When Wtf4poison-GFP and Wtf4antidote-mCherry to this region next to the vacuole (Figure In cells, a circle of Wtf4poison-GFP could also be observed ER however, the colocalized with the antidote in the vacuole-associated region (Figure supplement This localization was similar but not identical to our observations in S. pombe cells, the Wtf4 proteins localize than the vacuole. To the in localization to a and cell of the Wtf4poison-GFP protein observed in the cells Wtf4antidote-mCherry was not due to the mCherry we also confirmed these results with an untagged Wtf4antidote (Figure supplement Given that we to the Wtf proteins the vacuole, as we saw in S. we were in if the Wtf proteins were the vacuole but being in S. cerevisiae. To test this, we protein from cells expressing Wtf4antidote-mCherry and Wtf4poison-GFP and using and We protein from both the and of our protein the proteins are likely to be with (Figure 1—figure supplement and their is et al., we observed a at that showed with protein This was not observed in from cells not expressing Wtf proteins, suggesting it is likely Wtf4antidote-mCherry protein size The was more in the than the with (Figure supplement we observed from the size of Wtf4poison-GFP is The were not observed in control from cells not expressing Wtf proteins, suggesting the is specific to Wtf4poison-GFP. The size of of the Wtf4poison-GFP suggest the protein may have In addition, the small size of of the and the of the is with degradation of the protein. the a of the Wtf4poison-GFP was found in the suggesting both proteins have (Figure supplement Given that the Wtf4poison and Wtf4antidote proteins colocalize and are both found in the of protein we tested if the proteins physically interact by using acceptor photobleaching Fluorescence Resonance Energy Transfer and in cells expressing both Wtf4poison-GFP and Wtf4antidote-mCherry proteins. This process the fluorescence of a tagged protein (the and for a in fluorescence of tagged protein (the If an in fluorescence of the donor is the proteins are to be physically as they are in than to energy to other and When we we saw a in Wtf4poison-GFP the that the two proteins physically interact (Figure and Figure supplement The Wtf4 proteins localize as puncta of we that the proteins assemble into aggregates. To explore the of the Wtf4 protein we the recently developed FRET assay et al., 2018). This for FRET between and of the same in a of proteins as a of the to (Figure supplement We generated and alleles, both under β-estradiol inducible promoters on ARS CEN Both tagged encoded functional proteins in S. cerevisiae, but the wtf4poison allele was not as toxic as the allele (Figure supplement We then out on cells either or on cells both proteins We observed FRET between proteins and between proteins. In all cells expressing and/or proteins FRET as to of the expression level of the proteins (Figure supplement The level of did not when both proteins were expressed these experiments show that the Wtf4 proteins and that the proteins do not Interestingly, we did not cells Wtf4poison in our indicating that does not require a that is of proteins et al., 2018). with the shape of the GFP puncta in our suggests that the toxic species of Wtf4poison is a of the protein. To explore this idea, we tested if of that promote protein could neutralize Wtf4poison aggregates. we independently plasmids carrying various into a strain carrying the allele (Figure supplement et al., of of these to the toxicity of even when we reduced the of induction of the Wtf4poison (Figure supplement This suggests that neutralization of Wtf4poison may require the induction of or response pathways at one time. promote co-assembly of Wtf4 proteins and neutralization of the Wtf4poison The Wtf4poison and Wtf4antidote proteins the same amino acids (Figure 1A, Figure 1—figure supplement 1A). All the known proteins are similar to the they neutralize (Bravo Núñez et al., 2020a;