Specialization of actin isoforms derived from the loss of key interactions with regulatory factors

• DOI: 10.15252/embj.2021107982
• Publication Date: 2022-02-18T09:21:17Z
• Authors: Micaela Boiero Sa...; Christopher P Toret; Audrey Guillotin; Adrien Antkowiak; Thomas Vannier; Robert C Robinson; Alphée Michelot
• Keywords: Actin Cytoskeleton; 0303 health sciences; 03 medical and health sciences; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; [SDV]Life Sciences [q-bio]; Microfilament Proteins; Protein Isoforms; Articles; Amino Acid Sequence; Saccharomyces cerevisiae; Actins

Article18 February 2022Open Access Source DataTransparent process Specialization of actin isoforms derived from the loss key interactions with regulatory factors 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 Formal analysis Validation ​Investigation Visualization Methodology Writing - original draft review & editing (equal) Search more papers by this author Christopher P Toret Audrey Guillotin Adrien Antkowiak orcid.org/0000-0002-3711-5544 Thomas Vannier Robert C Robinson orcid.org/0000-0001-6367-6903 Research Institute Interdisciplinary Science (RIIS), Okayama University, Okayama, Japan School Biomolecular and Engineering (BSE), Vidyasirimedhi Technology (VISTEC), Rayong, Thailand Resources Funding acquisition Alphée Michelot Corresponding Author [email protected] orcid.org/0000-0003-2023-8094 Supervision Project administration Information Sanders1, Toret1,†, Guillotin1,†, Antkowiak1, Vannier1, Robinson2,3 *,1 1CNRS, 2Research 3School † These authors contributed equally to work *Corresponding author. Tel: [phone]; E-mail: The EMBO Journal (2022)41:e107982https://doi.org/10.15252/embj.2021107982 PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures Info Abstract A paradox eukaryotic cells is that while some species assemble complex cytoskeleton single ortholog, other utilize greater diversity isoforms. physiological consequences using different isoforms, molecular mechanisms which highly conserved are segregated into distinct networks, poorly known. Here, we sought understand how simple biological system, composed unique limited set actin-binding proteins, reacts switch heterologous expression. Using yeast as model system biomimetic assays, show such perturbation causes drastic reorganization cytoskeleton. Our results indicate defective interaction important regulators assembly limits certain pathways reinforcing others. Expression two variants, each specialized in assembling network, rescues cytoskeletal organization confers resistance external perturbation. Hence, have homeostatic several may act independently. SYNOPSIS Why involves ortholog multiple others unclear. gene swapping used machinery uses revealing mechanism functional segregation similar networks. Reducing expression half has negligible effects on budding cell behavior. Minor modifications protein can disrupt an regulator, funnel preferentially not promoted regulator. Combination variants promote organizations restores wild-type configuration yeast. Use hampers efficient redistribution between networks cell. Introduction fundamental characteristic existence organized Dynamic filaments assembled diverse architectures coexist within one cytoplasm, involved exertion forces various cellular functions (Blanchoin et al, 2014). Key partners families proteins (ABPs), interact monomers regulate dynamics (Moseley Goode, 2006; Pollard, 2016). Actin sequence across most eukaryotes, but types only express (e.g., yeasts), nonmuscle mammalian beta- gamma-actins, 99% identical), or even very Chlamydomonas reinhardtii expresses actins, IDA5 NAP1, 65% identical) (Gunning 2015; 2020). An extreme case plants, multitude Zea mays Arabidopsis thaliana 21 8 respectively). Adding complexity, actins undergo partial posttranslational (PTMs), arginylation acetylation, modify their biochemical properties (Kashina, 2014; 2020; number organisms able (or number) require presence generate variability. In line idea, observed vivo. Results lines found beta-actin was located mainly contractile ring, stress fibers, filopodia, cell–cell contacts gamma-actin localized primarily cortex lamellipodia (Dugina 2009; Chen 2017). thaliana, vegetative organize structures epidermal (Kijima 2018). However, it should be noted mice beta-coded gamma-actin, where nucleotide modified minimally led viable no detectable change behavior (Vedula This result indicates at least cases, absence isoform compensated isoform. particular challenge field small differences level lead major level. To decipher underlying mechanisms, natural postulate bear yet significant differences. knowledge distinctions orthologs (mainly S. cerevisiae Act1p, rabbit muscle actin, lesser extent pombe Act1p plant actins). Nonetheless, these studies reveal notable (Nefsky Bretscher, 1992; Buzan Frieden, 1996; Kim Bryan Rubenstein, 2005; Takaine Mabuchi, 2007; Kijima 2016), mechanical (Orlova 2001; McCullough 2011), ability Eads 1998; Ezezika 2011; Kang nucleation (Ti How account spatial scale remains work, investigated, general perspective, principles addressed Analysis exploits perform functions, would explain relevant context. importance renders genetic manipulations difficult, inter-connection models complicates analysis. Mammalian systems many ABP makes interpretation combinatorially challenging. Furthermore, coexpression endogenous purification although new powerful protocols been developed recent years (Hatano 2018, overcome limitations, decided adopt alternative strategy, determining normally actin. With approach, aimed measuring caused use its We well-studied organism, yeast, simplicity genetics. Another advantage assembles predominantly well-defined structures. patches, sites endocytosis short branched Arp2/3 complex, cables, central maintenance polarity intra-cellular trafficking, nucleated formin family 2006). Lastly, allows clean ABPs defined organismal demonstrate regulated both defects leads growth defects, amino acid induces massive efficiencies pathways, resulting targeting Finally, dissection us propose explanation our results, enabling cells. Generation library strains expressing variety created evaluate variation assembly. order ensure were due potential misfolding nonfunctional selected rather than directed mutations. approach guarantees biologically context, maintain polymerization, depolymerization, binding, hydrolysis. chose 126 covering entire archeal phylogenetic tree (Appendix Table S1 Appendix Fig S1A). also computationally predicted ancestral sequences extend range variant possibilities. Because species, reconstructions score high confidence S1B). obtained total 227 (including 101 actins), 19 chosen cover spectrum cerevisiae's (Act1p, called here Act_Sc) divergent orthologs, represent wide identities (from 99 60%) (Fig 1A, EV1 S1B C), display all domains fold 1B S1C). Figure 1. Variety study strategies Simplified showing Dikarya subkingdom branches Homo sapiens (Hs) (At). Id. column percentage identities, ranging 100% (green) 84% (magenta) identity 60–62% (black) archaeal actins. Squares' outlines solid dotted deriving existing reconstruction, respectively. "coded by" indicates, coding originally code genes interest. Nucleotide (blue) 76% (orange) compared sequence. Amino Saccharomyces Arrows denote positions mutated tested study. Schematic representation 3D structure (1YAG; Vorobiev 2003), mutations regions protein. Dots located, color studied here. mutagenesis applied study, question respectively actin's intron, sequence, effect copolymers. Green whether brought (leading Act1 (pink) Act1* ortholog). Download figure PowerPoint synthesized subcloned them plasmid specifically rapid robust replacement under promoter control S1D). Homologous recombination performed diploid so copy favor viability sickest strains. Despite this, unable could four corresponding those phyla Lokiarchaeota, Odinarchaeota, Thorarchaeota, Heimdallarchaeota, share All mutants generated, sporulated haploid variant. extended strains, systematically deleting intron cells, changing without modifying final switching copolymers 1D). Previous demonstrated essential transcription normal (Ng 1985). Indeed, act1 construct S288C (ScNI) does affect neither S2A B) nor S2C D). Fixation phalloidin-labeling reveals patches well-organized indistinguishable (Sc) S2E–G). Therefore, experiments presented following sections conducted expressed intron. Cell fitness tolerates reduced above threshold concerned changes might levels (Hoekema 1987; Zhou mammals, instance, shown differentiate beta gamma sequences. organisms, product remained (Table S2H). Western blot showed silent extents 2A B), correlation conservation EV1A). RNAseq showed, contrary, encoding significantly differentially EV1B). data revealed sizeable drop Act_Sc[Ca], C. albicans' gene, 46% level) (Figs 2C D, EV1C S2I–J). analyzed phalloidin-labeled intensity cables S2K), numbers S2L), well overall balance whose interdependent (Burke 2014) calculating deviation index (Antkowiak 2019) 2F). Sc[Ca] less bright cytoskeleton, keep distribution 2H). Act_Sc[At], A. thaliana's ACT8 24% affects visibly D S2I–J), 2E–G S2K–L), polarization 2H) Unexpectedly, none monomeric-to-filamentous (G/F) ratios EV1D). Expressing H. ActB (Act_Sc[Hs]), conserved, lethal From observations, concluded controlled carefully variations 2. Effects levels, viability, figure, shape dots identify graphs (circles Sc, squares ScNI, triangles Sc[Ca], inversed Sc[Sp] diamonds Sc[At]). cerevisiae, (orange). western blotting sequences, tubulin (Tub1p) loading control. Quantification decrease when present. mean ± SD (n = n ScNI; 12 Sc[Sp], Sc[At]; 2 replicates n/2 technical each). *P < 0.05 (Brown–Forsythe Welch ANOVA tests, Dunnett's T3 comparisons tests). Doubling times cultures, grown 25°C YPD medium. 6 3 replicates). Level function constant any clear correlation. Rather, there apparent (0.25 0.35) below rates drastically reduce. (for values, each; constants, r Pearson coefficient considered nonsignificant if two-tailed P-value > 0.05. Phalloidin staining depicting F-actin organization. Images maximum projections stacks. Scale bar: µm. vivo network indexes, patch-cable (value 0 1 contain −1 cables). 30 conditions). ***P 0.001 indexes observe affected. Polarity assess affected medium large budded present bud, refers mother cell). **P 0.01, information: Abbreviations: ns nonsignificant, Sc ScNI – replaced Sc[X] carrying based X list coding, see 1). available online figure. [embj2021107982-sup-0005-SDataFig2.xlsx] Click expand EV1. expression, growth, ratio, related 2In expanded view Sc[At] crosses nonviable strains). 75% relative wild-type, conservation, increased lowers Differential strain. Y-axis represents adjusted FDR (False Discovery Rate) calculated Benjamini Hochberg method, X-axis fold-changes. Red highlight interest (actin 32 proteins) grey identified RNA-seq. Growth identity, (78% id 82%) Evaluation ratios. imbalance linear-to-branched ratio next focused attention 15 designed making point codon usage. Overall, section 90% identical which, according previous section, risk excessively. Only conditions. Their varied, appeared correlated evolutionary relationship 3A B). For example, Act_N1 39% despite having 98.4% 3A–G). detail article, N2 Ca,