Specialization of actin isoforms derived from the loss of key interactions with regulatory factors
2022; Springer Nature; Volume: 41; Issue: 5 Linguagem: Inglês
10.15252/embj.2021107982
ISSN1460-2075
AutoresMicaela Boiero Sanders, Christopher P. Toret, Audrey Guillotin, Adrien Antkowiak, Thomas Vannier, Robert Robinson, Alphée Michelot,
Tópico(s)Microtubule and mitosis dynamics
ResumoArticle18 February 2022Open Access Source DataTransparent process Specialization of actin isoforms derived from the loss of key interactions with regulatory factors Micaela Boiero Sanders Micaela Boiero Sanders orcid.org/0000-0003-3513-6779 CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Conceptualization (equal), Data curation (equal), Formal analysis (equal), Validation (equal), Investigation (equal), Visualization (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal) Search for more papers by this author Christopher P Toret Christopher P Toret CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Conceptualization (equal), Formal analysis (equal), Investigation (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Audrey Guillotin Audrey Guillotin CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Formal analysis (equal), Investigation (equal) Search for more papers by this author Adrien Antkowiak Adrien Antkowiak orcid.org/0000-0002-3711-5544 CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Formal analysis (equal), Investigation (equal) Search for more papers by this author Thomas Vannier Thomas Vannier CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Formal analysis (equal), Validation (equal), Investigation (equal), Visualization (equal) Search for more papers by this author Robert C Robinson Robert C Robinson orcid.org/0000-0001-6367-6903 Research Institute for Interdisciplinary Science (RIIS), Okayama University, Okayama, Japan School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand Contribution: Conceptualization (equal), Resources (equal), Data curation (equal), Formal analysis (equal), Funding acquisition (equal), Investigation (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal) Search for more papers by this author Alphée Michelot Corresponding Author Alphée Michelot [email protected] orcid.org/0000-0003-2023-8094 CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Conceptualization (equal), Resources (equal), Data curation (equal), Formal analysis (equal), Supervision (equal), Funding acquisition (equal), Validation (equal), Investigation (equal), Visualization (equal), Methodology (equal), Writing - original draft (equal), Project administration (equal), Writing - review & editing (equal) Search for more papers by this author Micaela Boiero Sanders Micaela Boiero Sanders orcid.org/0000-0003-3513-6779 CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Conceptualization (equal), Data curation (equal), Formal analysis (equal), Validation (equal), Investigation (equal), Visualization (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal) Search for more papers by this author Christopher P Toret Christopher P Toret CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Conceptualization (equal), Formal analysis (equal), Investigation (equal), Methodology (equal), Writing - review & editing (equal) Search for more papers by this author Audrey Guillotin Audrey Guillotin CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Formal analysis (equal), Investigation (equal) Search for more papers by this author Adrien Antkowiak Adrien Antkowiak orcid.org/0000-0002-3711-5544 CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Formal analysis (equal), Investigation (equal) Search for more papers by this author Thomas Vannier Thomas Vannier CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Formal analysis (equal), Validation (equal), Investigation (equal), Visualization (equal) Search for more papers by this author Robert C Robinson Robert C Robinson orcid.org/0000-0001-6367-6903 Research Institute for Interdisciplinary Science (RIIS), Okayama University, Okayama, Japan School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand Contribution: Conceptualization (equal), Resources (equal), Data curation (equal), Formal analysis (equal), Funding acquisition (equal), Investigation (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal) Search for more papers by this author Alphée Michelot Corresponding Author Alphée Michelot [email protected] orcid.org/0000-0003-2023-8094 CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France Contribution: Conceptualization (equal), Resources (equal), Data curation (equal), Formal analysis (equal), Supervision (equal), Funding acquisition (equal), Validation (equal), Investigation (equal), Visualization (equal), Methodology (equal), Writing - original draft (equal), Project administration (equal), Writing - review & editing (equal) Search for more papers by this author Author Information Micaela Boiero Sanders1, Christopher P Toret1,†, Audrey Guillotin1,†, Adrien Antkowiak1, Thomas Vannier1, Robert C Robinson2,3 and Alphée Michelot *,1 1CNRS, IBDM, Turing Centre for Living Systems, Aix Marseille Univ, Marseille, France 2Research Institute for Interdisciplinary Science (RIIS), Okayama University, Okayama, Japan 3School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand † These authors contributed equally to this work *Corresponding author. Tel: +33 4 13 94 94 87; E-mail: [email protected] The EMBO Journal (2022)41:e107982https://doi.org/10.15252/embj.2021107982 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A paradox of eukaryotic cells is that while some species assemble a complex actin cytoskeleton from a single ortholog, other species utilize a greater diversity of actin isoforms. The physiological consequences of using different actin isoforms, and the molecular mechanisms by which highly conserved actin isoforms are segregated into distinct networks, are poorly known. Here, we sought to understand how a simple biological system, composed of a unique actin and a limited set of actin-binding proteins, reacts to a switch to heterologous actin expression. Using yeast as a model system and biomimetic assays, we show that such perturbation causes drastic reorganization of the actin cytoskeleton. Our results indicate that defective interaction of a heterologous actin for important regulators of actin assembly limits certain actin assembly pathways while reinforcing others. Expression of two heterologous actin variants, each specialized in assembling a different network, rescues cytoskeletal organization and confers resistance to external perturbation. Hence, while species using a unique actin have homeostatic actin networks, actin assembly pathways in species using several actin isoforms may act more independently. SYNOPSIS Why assembly of a complex actin cytoskeleton involves a single ortholog in some species and multiple isoforms in others is unclear. Here, actin gene swapping is used to understand how the yeast molecular machinery uses different actin variants, revealing a mechanism for functional segregation of similar actin isoforms into distinct networks. Reducing actin expression by half has negligible effects on budding yeast cell behavior. Minor modifications of the actin protein can disrupt interaction with an actin regulator, and funnel actin preferentially into assembly pathways not promoted by this regulator. Combination of two specialized actin variants that promote different actin organizations restores wild-type actin configuration in yeast. Use of multiple specialized variants hampers efficient redistribution of actin between the different networks of the yeast cell. Introduction A fundamental characteristic of eukaryotic cells is the existence of an organized actin cytoskeleton. Dynamic actin filaments are assembled into diverse architectures which coexist within one cytoplasm, each of which is involved in the exertion of forces for various cellular functions (Blanchoin et al, 2014). Key partners are families of actin-binding proteins (ABPs), which interact with actin monomers and filaments to regulate cytoskeletal organization and dynamics (Moseley & Goode, 2006; Pollard, 2016). Actin sequence is highly conserved across most eukaryotes, but while some cell types only express a single actin (e.g., yeasts), other cell types can express several similar actin isoforms (e.g., nonmuscle mammalian cells express beta- and gamma-actins, which are 99% identical), or even very different actin isoforms (e.g., Chlamydomonas reinhardtii expresses two actins, IDA5 and NAP1, which are only 65% identical) (Gunning et al, 2015; Boiero Sanders et al, 2020). An extreme case is plants, which can express a multitude of actin isoforms (e.g., Zea mays and Arabidopsis thaliana express 21 and 8 actin isoforms, respectively). Adding to this complexity, some actins can undergo partial posttranslational modifications (PTMs), such as arginylation or acetylation, which modify their biochemical properties (Kashina, 2014; A et al, 2020; Boiero Sanders et al, 2020). Hence, while a number of organisms are able to assemble a complex actin cytoskeleton from one (or a limited number) of actin isoforms, other organisms require the presence of multiple actin isoforms to generate such variability. In line with this idea, segregation of actin isoforms is observed in vivo. Results from different mammalian cell lines have found that beta-actin was located mainly in the contractile ring, stress fibers, filopodia, and cell–cell contacts while gamma-actin was localized primarily in the cortex and lamellipodia (Dugina et al, 2009; Chen et al, 2017). In Arabidopsis thaliana, the main vegetative actin isoforms organize into different structures in epidermal cells (Kijima et al, 2018). However, it should be noted that expression in mice of a beta-coded gamma-actin, where the nucleotide sequence of beta-actin is modified minimally to express gamma-actin, led to viable mice with no detectable change in behavior (Vedula et al, 2017). This result indicates that at least in some cases, the absence of an actin isoform can be compensated by the expression of a similar isoform. A particular challenge for the field is to understand how small differences at the molecular level lead to a major segregation of actin isoforms at the cellular level. To decipher the underlying mechanisms, it is natural to postulate that actin isoforms bear small yet significant biochemical differences. Our knowledge of the distinctions between actins is limited to a small number of actin orthologs (mainly S. cerevisiae Act1p, rabbit muscle actin, to a lesser extent beta- and gamma-actins, S. pombe Act1p and plant actins). Nonetheless, these studies reveal notable differences in their biochemical properties (Nefsky & Bretscher, 1992; Buzan & Frieden, 1996; Kim et al, 1996; Bryan & Rubenstein, 2005; Takaine & Mabuchi, 2007; Kijima et al, 2016), in their mechanical properties (Orlova et al, 2001; McCullough et al, 2011), and their ability to interact with the different actin-binding proteins (Nefsky & Bretscher, 1992; Eads et al, 1998; Takaine & Mabuchi, 2007; Ezezika et al, 2009; McCullough et al, 2011; Kang et al, 2014; Kijima et al, 2016), including nucleation factors of actin assembly (Ti & Pollard, 2011; Chen et al, 2017). How such differences account for spatial segregation of actin isoforms on a cellular scale remains unclear. In this work, we investigated, from a general perspective, the molecular principles by which actin isoforms can be addressed to different networks. Analysis in a model system, that exploits at least two actins to perform various actin functions, would explain a particular mechanism in a relevant physiological context. However, the importance of actin renders genetic manipulations difficult, and the inter-connection of actin networks in such models complicates cellular analysis. Mammalian systems in particular express many ABP isoforms, which makes interpretation of molecular mechanisms combinatorially challenging. Furthermore, coexpression of multiple actin isoforms makes endogenous purification as a single species difficult, although new powerful protocols have been developed in recent years for their expression and purification (Hatano et al, 2018, 2020). To overcome these limitations, we decided to adopt an alternative strategy, by determining the consequences of heterologous actin expression in a system normally using a single actin. With this approach, we aimed at measuring the consequences of a perturbation caused by the use of a different actin at the level of the cell and its cytoskeleton. We decided to use the well-studied organism, budding yeast, for the simplicity of its genetics. Another advantage of budding yeast is that actin assembles predominantly into two well-defined structures. These structures are actin patches, which are sites of endocytosis and where actin filaments are short and branched by the Arp2/3 complex, and actin cables, which are central for maintenance of cell polarity and intra-cellular trafficking, and where actin filaments are nucleated by the formin family of proteins (Moseley & Goode, 2006). Lastly, budding yeast allows for clean purification of ABPs in a defined organismal context. Our results demonstrate that actin functions are regulated both at the nucleotide level where defects in actin expression leads to cell growth defects, and at the amino acid level where expression of heterologous actins induces a massive reorganization of the actin cytoskeleton. We demonstrate that actin isoforms are used with different efficiencies by the distinct actin assembly pathways, resulting in their targeting to particular actin structures. Finally, dissection of the underlying molecular mechanisms allows us to propose an explanation of our results, and a general model of the molecular mechanisms enabling segregation of actin isoforms in cells. Results Generation of a library of yeast strains expressing a variety of actin orthologs We created a library of S. cerevisiae strains that express different actin orthologs to evaluate the consequences of actin variation on yeast actin cytoskeleton assembly. In order to ensure that defects were not due to potential misfolding or nonfunctional actin, we selected a diversity of actins from other species rather than using directed mutations. This approach guarantees that the actin orthologs are functional in a biologically relevant context, and maintain key physiological properties such as polymerization, depolymerization, nucleotide binding, and hydrolysis. We chose 126 different actins from species covering the entire eukaryotic and archeal phylogenetic tree for analysis (Appendix Table S1 and Appendix Fig S1A). We also computationally predicted ancestral sequences to extend the range of actin variant possibilities. Because the actin protein sequence is highly conserved across species, ancestral sequence reconstructions score with high confidence (Appendix Fig S1B). We obtained in total 227 actin sequences (including 101 ancestral actins), from which we selected 19 for analysis. These actin orthologs were chosen to cover a spectrum from the most similar to wild-type S. cerevisiae's actin (Act1p, called here Act_Sc) to very divergent actin orthologs, which represent a wide range of identities (from 99 to 60%) (Fig 1A, Table EV1 and Appendix Fig S1B and C), and to display differences across all domains of the actin fold (Fig 1B and C and Appendix Fig S1C). Figure 1. Variety of actins selected for this study and analysis strategies Simplified phylogenetic tree showing mainly the Dikarya subkingdom and including the external branches Homo sapiens (Hs) and Arabidopsis thaliana (At). The Id. column indicates amino acid sequences percentage identities, ranging from 100% (green) to 84% (magenta) identity for eukaryotic actins to S. cerevisiae's actin, and 60–62% (black) for archaeal actins. Squares' outlines are solid or dotted for sequences deriving from existing species or ancestral reconstruction, respectively. The "coded by" column indicates, which coding sequences were originally used to code genes of interest. Nucleotide sequence identities are ranging from 100% (blue) to 76% (orange) compared to S. cerevisiae's actin coding sequence. Amino acid sequence of Saccharomyces cerevisiae actin. Arrows denote all the positions that are mutated in at least one of the actin variants tested in this study. Schematic representation of S. cerevisiae actin 3D structure (1YAG; Vorobiev et al, 2003), showing that mutations cover all regions of the protein. Dots indicate where mutations are located, using a different color code for all actins studied here. Schematic showing the mutagenesis strategies applied in this study, enabling to question respectively the importance of actin's intron, the nucleotide sequence, the amino acid sequence, and the effect of expressing copolymers. Green color indicates whether modifications are brought in the coding sequence (leading to expression of wild-type Act1 protein (pink) or in the amino acid sequence (leading to expression of an Act1* actin ortholog). Download figure Download PowerPoint We synthesized the actin nucleotide sequences and subcloned them in a plasmid created specifically for rapid and robust actin gene replacement under endogenous promoter control in S. cerevisiae (Appendix Fig S1D). Homologous recombination was performed on diploid cells so that the presence of a wild-type actin copy would favor viability of the sickest strains. Despite this, we were unable to generate viable strains that could express four highly divergent archeal actins, corresponding to those of phyla Lokiarchaeota, Odinarchaeota, Thorarchaeota, and Heimdallarchaeota, which all share 60–62% of sequence identity with budding yeast actin. All mutants expressing eukaryotic actins could be generated, and were sporulated in order to study haploid cells expressing only the new actin variant. With this strategy, we created an extended library of yeast strains, from which we systematically studied the effect of deleting the actin intron in haploid cells, changing the nucleotide sequence without modifying the final actin protein in haploid cells, switching actin protein variants in haploid cells, and expressing copolymers of actin in diploid cells (Fig 1D). Previous studies have demonstrated that the yeast actin intron is not essential for actin gene transcription and for normal cell growth (Ng et al, 1985). Indeed, our analysis found that an act1 gene construct without the intron in S. cerevisiae S288C (ScNI) does affect neither cell growth (Appendix Fig S2A and B) nor actin expression (Appendix Fig S2C and D). Fixation and phalloidin-labeling of the actin cytoskeleton reveals that the two main structures of actin filaments in yeast, actin patches and actin cables, are well-organized in yeast strains expressing actin in the absence of the intron and indistinguishable from wild-type cells (Sc) (Appendix Fig S2E–G). Therefore, all experiments presented in the following sections of this study were conducted on actins expressed in the absence of an intron. Cell fitness tolerates reduced wild-type actin expression above a threshold We were concerned that small changes to the actin nucleotide sequence might have consequences on actin expression levels and cell viability (Hoekema et al, 1987; Zhou et al, 2016). In mammals, for instance, nucleotide sequence was shown to differentiate beta and gamma actin functions (Vedula et al, 2017). Therefore, we expressed wild-type actin from a range of different nucleotide sequences. We used coding sequences from other organisms, which we modified minimally so that the final product remained S. cerevisiae's actin at the protein level (Table EV1 and Appendix Fig S2H). Western blot analysis showed that silent mutations affect wild-type actin's expression level to various extents (Fig 2A and B), with correlation between actin expression and the level of conservation of the nucleotide sequence (Fig EV1A). RNAseq analysis showed, on the contrary, that genes encoding actin regulators are not significantly differentially expressed (Fig EV1B). These data also revealed that a sizeable drop of actin expression (e.g., Act_Sc[Ca], derived from C. albicans' actin gene, is expressed at 46% of normal level) has no effect on cell viability (Figs 2C and D, and EV1C and Appendix Fig S2I–J). We analyzed the organization of the cytoskeleton of phalloidin-labeled cells by measuring the total intensity of patches and cables (Appendix Fig S2K), their numbers (Appendix Fig S2L), as well as the overall balance between these two structures whose assembly is interdependent (Burke et al, 2014) by calculating a deviation index (Antkowiak et al, 2019) (Fig 2F). Sc[Ca] cells expressing less actin also have a less bright cytoskeleton, but keep a normal distribution between actin patches and cables, and normal cell polarity (Fig 2H). However, a more drastic drop of actin expression (e.g., Act_Sc[At], derived from A. thaliana's ACT8 gene, is expressed at 24% of normal level) affects visibly cell viability (Fig 2C and D and Appendix Fig S2I–J), the organization (Fig 2E–G and Appendix Fig S2K–L), and the polarization (Fig 2H) of the actin cytoskeleton. Unexpectedly, none of these strains expressing wild-type actin showed a significant change in monomeric-to-filamentous (G/F) actin ratios (Fig EV1D). Expressing actin from a gene derived from the nucleotide sequence of H. sapiens ActB (Act_Sc[Hs]), whose nucleotide sequence is even less conserved, is lethal for cells. From these observations, we concluded that expression levels of actin orthologs should be controlled carefully in this study, but that half variations in actin expression have negligible effect on cell behavior. Figure 2. Effects of silent mutations on actin expression levels, cell viability, and cytoskeletal organization In this figure, the shape of the dots allows to identify the strains on the different graphs (circles for Sc, squares for ScNI, triangles for Sc[Ca], inversed triangles for Sc[Sp] and diamonds for Sc[At]). The color of the dots indicates the percentage of identity of the nucleotide sequences to the actin gene of S. cerevisiae, ranging from 100% (blue) to 76% (orange). Actin expression levels shown by western blotting for strains expressing S. cerevisiae's actin protein from various coding sequences, with tubulin (Tub1p) as a loading control. Quantification of actin expression levels, showing a decrease when more silent mutations are present. Data are presented as mean ± SD (n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp], and Sc[At]; 2 biological replicates with n/2 technical replicates each). *P < 0.05 (Brown–Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). Doubling times of yeast strains cultures, grown at 25°C in YPD medium. Data are presented as mean ± SD (n = 6 for Sc, n = 3 for ScNI, Sc[Ca], Sc[Sp] and Sc[At]; technical replicates). *P < 0.05 (Brown–Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). Level of actin expression as a function of growth constant does not show any clear correlation. Rather, there is an apparent level of actin expression (0.25 < expression < 0.35) below which growth rates drastically reduce. Data are presented as mean ± SD (for actin expression values, n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each; for growth constants, n = 6 for Sc, n = 3 for ScNI, Sc[Ca], Sc[Sp] and Sc[At]; technical replicates). r is a Pearson correlation coefficient considered nonsignificant if its two-tailed P-value is > 0.05. Phalloidin staining depicting F-actin organization. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. In vivo actin network deviation indexes, defined to evaluate the patch-cable balance compared to S. cerevisiae haploid cells (value is 0 in S. cerevisiae's cells, 1 when cells contain only actin patches and −1 when cells contain only cables). Data are presented as mean ± SD (n = 30 for all conditions). ***P < 0.001 (Brown–Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). In vivo actin network deviation indexes as a function of actin expression levels does not show any clear correlation. Rather, we observe a threshold of actin expression levels (0.25 < expression < 0.35) below which actin cytoskeleton organization is affected. Data are presented as mean ± SD (for actin expression values, n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each; for indexes, n = 30 for all conditions). Pearson correlation coefficient r is considered nonsignificant if P > 0.05. Polarity indexes, defined to assess whether cell polarity is normal or affected (value is 1 when all patches of medium to large budded cells are present in the bud, and −1 refers when all patches are in the mother cell). Data are presented as mean ± SD (n = 30 for all conditions). **P < 0.01, ***P < 0.001 (Brown–Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). Data information: Abbreviations: ns - nonsignificant, Sc - wild-type S. cerevisiae cells, ScNI – S. cerevisiae cells where the actin gene has been replaced with the wild-type gene but without the intron, Sc[X] – S. cerevisiae cells where the actin gene has been replaced with a gene carrying silent mutations based on the sequences from species X (for the list of species and coding, see Table EV1 or Fig 1). Source data are available online for this figure. Source Data for Figure 2 [embj2021107982-sup-0005-SDataFig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Effects of silent mutations on gene expression, cell growth, and monomeric-to-filamentous actin ratio, related to Fig 2In this expanded view figure, the shape of the dots is conserved from Fig 2 and allows to identify the strains on the different graphs (circles for Sc, squares for ScNI, triangles for Sc[Ca], inversed triangles for Sc[Sp], diamonds for Sc[At] and crosses for nonviable strains). The color of the dots indicates the percentage of identity of the nucleotide sequences to the actin gene of S. cerevisiae, ranging from 100% (blue) to 75% (orange). Actin expression levels, relative to wild-type, as a function of nucleotide conservation, showing that increased number of silent mutations lowers actin expression. Data are presented as mean ± SD (n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each). Pearson correlation coefficient r is considered nonsignificant if P > 0.05. Differential gene expression of Sc[Ca], Sc[Sp] and Sc[At] strains compared to ScNI strain. Y-axis represents the adjusted P-value of FDR (False Discovery Rate) calculated with Benjamini and Hochberg method, and X-axis fold-changes. Red dots highlight proteins of interest (actin and 32 regulatory proteins) and grey dots represent all the other proteins identified by RNA-seq. Growth constant as a function of nucleotide identity, showing a threshold of nucleotide conservation (78% < id < 82%) below which growth rates drastically reduce. Data are presented as mean ± SD (n = 6 for Sc, n = 3 for ScNI, Sc[Ca], Sc[Sp] and Sc[At]; technical replicates). Pearson correlation coefficient r is considered nonsignificant if P > 0.05. Evaluation of monomeric-to-filamentous actin ratios. Data are presented as mean ± SD (n = 12 for Sc and ScNI and 4 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each). (Brown–Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). Source data are available online for this figure. Download figure Download PowerPoint Actin amino acid sequence variations affect cell fitness and imbalance the linear-to-branched actin network ratio We next focused our attention on the consequences of expressing 15 heterologous eukaryotic actin orthologs in yeast haploid cells. Actin genes were designed based on S. cerevisiae's act1 sequence by making point mutations using yeast codon usage. Overall, all coding sequences used in this section are more than 90% identical to that of S. cerevisiae, which, according to the previous section, lowers the risk that actin expression is reduced excessively. Only 8 actin orthologs led to viable conditions. Their expression level varied, and appeared not to be correlated with the evolutionary relationship (Fig 3A and B). For example, Act_N1 was only expressed at 39% despite having a 98.4% identity to wild-type actin and showed normal viability and cytoskeletal organization (Fig 3A–G). For two strains studied in detail in this article, N2 and Ca, we
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