CRISPR/Cas9 has emerged in various organisms as a powerful technology for targeted gene knockout; however, no reports of editing the Dictyostelium genome efficiently using this system are available. We describe here the application of CRISPR/Cas9-mediated gene modification in Dictyostelium. The endogenous tRNA-processing system for expressing sgRNA was approximately 10 times more effective than the commonly used U6 promoter. The resulting sgRNA affected the sub-nuclear localisation of Cas9, indicating that the expression level of sgRNA was sufficiently high to form Cas9 and sgRNA complexes within the nucleus. The all-in-one vector containing Cas9 and sgRNA was transiently expressed to generate mutants in five PI3K genes. Mutation detective PCR revealed the mutagenesis frequency of the individual genes to be between 72.9% and 100%. We confirmed that all five targeting loci in the four independent clones had insertion/deletion mutations in their target sites. Thus, we show that the CRISPR/Cas9 system can be used in Dictyostelium cells to enable efficient genome editing of multiple genes. Since this system utilises transient expression of the all-in-one vector, it has the advantage that the drug resistance cassette is not integrated into the genome and simple vector construction, involving annealing two oligo-DNAs.

After a cell divides into two daughter cells, the total cell surface area of the daughter cells should increase to the original size to maintain cell size homeostasis in a single cell cycle. Previously, three models have been proposed to explain the regulation of cell size homeostasis: sizer, timer, and adder models. Here, we precisely measured the total cell surface area of Dictyostelium cells in a whole cell cycle by using the agar-overlay method, which eliminated the influence of surface membrane reservoirs, such as microvilli and membrane wrinkles. The total cell surface area exponentially increased during interphase, slightly decreased at metaphase, and then increased by approximately 20% during cytokinesis. From the analysis of the added surface area, we concluded that the cell size was regulated by the adder or near-adder model in interphase. This adder model is not caused by a simple cell membrane addition, but is more dynamic due to the rapid cell membrane turnover. We propose a 'dynamic adder model' to explain cell size homeostasis in interphase.

Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation.

We examined the mechanism of cell membrane repair in Dictyostelium cells by using a novel laser-based cell poration method. The dynamics of wound pores opening and closing were characterized by live imaging of fluorescent cell membrane proteins, influx of fluorescent dye, and Ca2+ imaging. The wound closed within 2-4 sec, depending on the wound size. Cells could tolerate a wound size of less than 2.0 µm. In the absence of Ca2+ in the external medium, the wound pore did not close and cells ruptured. The release of Ca2+ from intracellular stores also contributed to the elevation of cytoplasmic Ca2+ but not to wound repair. Annexin C1 immediately accumulated at the wound site depending on the external Ca2+ concentration, and annexin C1 knockout cells had a defect in wound repair, but it was not essential. Dictyostelium cells were able to respond to multiple repeated wounds with the same time courses, in contrast to previous reports showing that the first wound accelerates the second wound repair in fibroblasts.

Sporulation in Dictyostelium fruiting bodies evolved from amoebozoan encystation with both being induced by cAMP acting on PKA, but with downstream components still being unknown. Using tagged mutagenesis to find missing pathway components, we identified a sporeless mutant defective in a nuclear protein, SpaA. Expression of prespore genes was strongly reduced in spaA- cells, while expression of many spore stage genes was absent. Chromatin immunoprecipitation (ChIP) of a SpaA-YFP gene fusion showed that (pre)spore gene promoters bind directly to SpaA, identifying SpaA as a transcriptional regulator. SpaA dependent spore gene expression required PKA in vivo and was stimulated in vitro by the membrane-permeant PKA agonist 8Br-cAMP. The PKA agonist also promoted SpaA binding to (pre)spore promoters, placing SpaA downstream of PKA. Sequencing of SpaA-YFP ChIPed DNA fragments revealed that SpaA binds at least 117 (pre)spore promoters, including those of other transcription factors that activate some spore genes. These factors are not in turn required for spaA expression, identifying SpaA as the major trancriptional inducer of sporulation.

A hallmark of ribosomal RNA (rRNA) are 2'-O-methyl groups that are introduced sequence specifically by box C/D small nucleolar RNAs (snoRNAs) in ribonucleoprotein particles. Most data on this chemical modification and its impact on RNA folding and stability are derived from organisms of the Opisthokonta supergroup. Using bioinformatics and RNA-seq data, we identify 30 novel box C/D snoRNAs in Dictyostelium discoideum, many of which are differentially expressed during the multicellular development of the amoeba. By applying RiboMeth-seq, we find 49 positions in the 17S and 26S rRNA 2'-O-methylated. Several of these nucleotides are substoichiometrically modified, with one displaying dynamic modification levels during development. Using homology-based models for the D. discoideum rRNA secondary structures, we localize many modified nucleotides in the vicinity of the ribosomal A, P and E sites. For most modified positions, a guiding box C/D snoRNA could be identified, allowing to determine idiosyncratic features of the snoRNA/rRNA interactions in the amoeba. Our data from D. discoideum represents the first evidence for ribosome heterogeneity in the Amoebozoa supergroup, allowing to suggest that it is a common feature of all eukaryotes.

The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

Plastins, also known as fimbrins, are highly conserved eukaryotic multidomain proteins that are involved in actin-bundling. They all contain four independently folded Calponin Homology-domains and an N-terminal headpiece that is comprised of two calcium-binding EF-hand motifs. Since calcium-binding has been shown to be integral to regulating the activity of the three mammalian plastin proteins, we decided to study the properties of the headpiece regions of fimbrins from the model plant Arabidopsis thaliana, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the amoeba Dictyostelium discoideum. Of these protein domains only the FimA headpiece from the amoeba protein possesses calcium binding properties. Structural characterization of this protein domain by multidimensional NMR and site-directed mutagenesis studies indicates that this EF-hand region of FimA also contains a regulatory 'switch helix' that is essential to regulating the activity of the human L-plastin protein. Interestingly this regulatory helical region seems to be lacking in the plant and yeast proteins and in fimbrins from all other nonmotile systems. Typical calmodulin antagonists can displace the switch-helix from the FimA headpiece, suggesting that such drugs can deregulate the Ca2+-regulation of the actin-bunding in the amoeba, thereby making it a useful organism for drug screening against mammalian plastins.

The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the α-kinase domain of Dictyostelium myosin-II heavy chain kinase A (MHCK-A) in which nucleotide binding to the catalytic cleft, located at the interface between an N-terminal and C-terminal lobe, is sterically blocked by the side chain of a conserved arginine residue (Arg592). Previous α-kinase structures have shown that an invariant catalytic aspartic acid residue (Asp766) is phosphorylated. Unexpectedly, in the autoinhibited conformation the phosphoryl group is transferred to the adjacent Asp663, creating an interaction network that stabilizes the autoinhibited state. The results suggest that Asp766 phosphorylation may play both catalytic and regulatory roles. The autoinhibited structure also provides the first view of a phosphothreonine residue docked into the phospho-specific allosteric binding site (Pi-pocket) in the C-lobe of the α-kinase domain.

The evolution of multicellularity required novel mechanisms for intercellular communication, but their origin is unclear. Dictyostelium cells exchange signals to position specialized cell types in multicellular spore-bearing structures. These signals activate complex pathways that converge on activation of cAMP-dependent protein kinase (PKA). Genes controlling PKA were detected in the Dictyostelid unicellular ancestors, which like most protists form dormant cysts when experiencing environmental stress. We deleted PKA and the adenylate cyclases AcrA and AcgA, which synthesize cAMP for PKA activation, in the intermediate species Polysphondylium, which can develop into either cysts or into multicellular structures. Loss of PKA prevented multicellular development, but also completely blocked encystation. Loss of AcrA and AcgA, both essential for sporulation in Dictyostelium, did not affect Polysphondylium sporulation, but prevented encystation. We conclude that multicellular cAMP signalling was co-opted from PKA regulation of protist encystation with progressive refunctionalization of pathway components.

Dictyostelids, or social amoebae, have a unique life style in forming multicellular fruiting bodies from unicellular amoeboids upon starvation. Recently, dictyostelids were found to contain terpene synthase (TPS) genes, a gene type of secondary metabolism previously known to occur only in plants, fungi and bacteria. Here we report an evolutionary functional study of dictyostelid TPS genes. The number of TPS genes in six species of dictyostelids examined ranges from 1 to 19; and the model species Dictyostelium purpureum contains 12 genes. Using in vitro enzyme assays, the 12 TPS genes from D. purpureum were shown to encode functional enzymes with distinct product profiles. The expression of the 12 TPS genes in D. purpureum is developmentally regulated. During multicellular development, D. purpureum releases a mixture of volatile terpenes dominated by sesquiterpenes that are the in vitro products of a subset of the 12 TPS genes. The quality and quantity of the terpenes released from D. purpureum, however, bear little resemblance to those of D. discoideum, a closely related dictyostelid. Despite these variations, the conserved clade of dictyostelid TPSs, which have an evolutionary distance of more than 600 million years, has the same biochemical function, catalyzing the formation of a sesquiterpene protoillud-7-ene. Taken together, our results indicate that the dynamic evolution of dictyostelid TPS genes includes both purifying selection of an orthologous group and species-specific expansion with functional divergence. Consequently, the terpenes produced by these TPSs most likely have conserved as well as species-adaptive biological functions as chemical languages in dictyostelids.

Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment.

Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems.

Autophagy, the lysosome-mediated self-degradation process, is implicated in survival during starvation in yeast, Dictyostelium and animals. In these eukaryotic taxa (collectively called Unikonts), autophagy is induced primarily through the Atg1/ULK1 complex in response to nutrient depletion. Autophagy has also been well-studied in non-unikont parasites, such as Trypanosoma and Plasmodium, and found important in their life-cycle transitions. However, how autophagy is induced in non-unikonts remains largely unrevealed. Using a bioinformatics approach, we examined the presence of Atg1 and of its complex in the genomes of 40 non-unikonts. We found that these genomes do not encode typical Atg1 proteins: BLAST and HMMER queries matched only with the kinase domain of Atg1, while other segments responsible for regulation and protein-binding were missing. Non-unikonts also lacked other components of the Atg1-inducing complex. Orthologs of an alternative autophagy inducer, Atg6 were found only in the half of the species, indicating that the other half may possess other inducing mechanisms. As key autophagy genes have differential expression patterns during life-cycle, we raise the possibility that autophagy in these protists is induced mainly at the post-transcriptional level. Understanding Atg1-independent autophagy induction mechanisms in these parasites may lead to novel pharmacological interventions, not affecting human Atg1-dependent autophagy.

Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.

Living organisms employ various mechanisms to escape harm. At the cellular level, mobile cells employ movement to avoid harmful chemicals or repellents. The present study is the first to report that cells move away from the site of injury in response to local wounding. When a migrating Dictyostelium cell was locally wounded at its anterior region by laserporation, the cell retracted its anterior pseudopods, extended a new pseudopod at the posterior region, and migrated in the opposite direction with increasing velocity. When wounded in the posterior region, the cell did not change its polarity and moved away from the site of wounding. Since the cells repair wounds within a short period, we successfully manipulated cell migration by applying multiple wounds. Herein, we discussed the signals that contributed to the wound-induced escape behavior of Dictyostelium cells. Our findings provide important insights into the mechanisms by which cells establish their polarity.

ADP-ribosyltransferases (ARTs) modify proteins with single units or polymers of ADP-ribose to regulate DNA repair. However, the substrates for these enzymes are ill-defined. For example, although histones are modified by ARTs, the sites on these proteins ADP-ribosylated following DNA damage and the ARTs that catalyse these events are unknown. This, in part, is due to the lack of a eukaryotic model that contains ARTs, in addition to histone genes that can be manipulated to assess ADP-ribosylation events in vivo. Here we exploit the model Dictyostelium to identify site-specific histone ADP-ribosylation events in vivo and define the ARTs that mediate these modifications. Dictyostelium histones are modified in response to DNA double strand breaks (DSBs) in vivo by the ARTs Adprt1a and Adprt2. Adprt1a is a mono-ART that modifies H2BE18 in vitro, although disruption of this site allows ADP-ribosylation at H2BE19. Although redundancy between H2BE18 and H2BE19 ADP-ribosylation is also apparent following DSBs in vivo, by generating a strain with mutations at E18/E19 in the h2b locus we demonstrate these are the principal sites modified by Adprt1a/Adprt2. This identifies DNA damage induced histone mono-ADP-ribosylation sites by specific ARTs in vivo, providing a unique platform to assess how histone ADP-ribosylation regulates DNA repair.

O-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria. The O-GlcNAc modification can affect protein cellular localization, function, and signaling interactions. The specific impact of O-GlcNAcylation on mitochondrial morphology and function has been elusive. In this manuscript, the role of O-GlcNAcylation on mitochondrial fission, oxidative phosphorylation (Oxphos), and the activity of electron transport chain (ETC) complexes were evaluated. In a cellular environment with hyper O-GlcNAcylation due to the deletion of O-GlcNAcase (OGA), mitochondria showed a dramatic reduction in size and a corresponding increase in number and total mitochondrial mass. Because of the increased mitochondrial content, OGA knockout cells exhibited comparable coupled mitochondrial Oxphos and ATP levels when compared to WT cells. However, we observed reduced protein levels for complex I and II when comparing normalized mitochondrial content and reduced linked activity for complexes I and III when examining individual ETC complex activities. In assessing mitochondrial fission, we observed increased amounts of O-GlcNAcylated dynamin-related protein 1 (Drp1) in cells genetically null for OGA and in glioblastoma cells. Individual regions of Drp1 were evaluated for O-GlcNAc modifications, and we found that this post-translational modification (PTM) was not limited to the previously characterized residues in the variable domain (VD). Additional modification sites are predicted in the GTPase domain, which may influence enzyme activity. Collectively, these results highlight the impact of O-GlcNAcylation on mitochondrial dynamics and ETC function and mimic the changes that may occur during glucose toxicity from hyperglycemia.

Cell permeating peptides (CPPs) are attracting great interest for use as molecular delivery vehicles for the transport of biologically active cargo across the cell membrane. The sequence of a novel CPP sequence, termed 'Cupid', was identified from the genome of Dictyostelium discoideum. A Cupid-Green Fluorescent Protein (Cupid-GFP) fusion protein was tested on mammalian, whole plant cells, plant leaf protoplast and fungal cell cultures and observed using confocal microscopy. GFP fluorescence builds up within the cell cytosol in 60 min, demonstrating Cupid-GFP has permeated them and folded correctly into its fluorescent form. Our combined data suggest Cupid can act as a molecular vehicle capable of delivering proteins, such as GFP, into the cytosol of a variety of cells.

Entamoeba histolytica is an anaerobic parasitic protist and possesses mitosomes, one of the most highly divergent mitochondrion-related organelles (MROs). Although unique metabolism and protein/metabolite transport machinery have been demonstrated in Entamoeba mitosomes, the mechanism of mitosomal fusion and fission remains to be elucidated. In this study, we demonstrate that two dynamin-related proteins (DRPs) are cooperatively involved in the fission of Entamoeba mitosomes. Expression of a dominant negative form of EhDrpA and EhDrpB, and alternatively, repression of gene expression of EhDrpA and EhDrpB genes, caused elongation of mitosomes, reflecting inhibition of mitosomal fission. Moreover, EhDrpA and EhDrpB formed an unprecedented hetero-oligomeric complex with an approximate 1:2 to 1:3 ratio, suggesting that the observed elongation of mitosomes is likely caused by the disruption and instability of the complex caused by an imbalance in the two DRPs. Altogether, this is the first report of a hetero-oligomeric DRP complex which participates in the fission of mitochondria and MROs.